Copyright: © Zha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the National Natural Science Foundation of China (30800171), the Ministry of Science and Technology of China (2011CB966301 and 2011CB911102), and the Science and Technology Commission of Shanghai Municipality (10JC1416500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Intracellular calcium is an important secondary messenger, of which the concentration ranges from a basal value of about 50 nM to stimulated levels of 1–10 µM in response to signals such as growth factors and neurotransmitters . One of the key proteins that sense the increased calcium concentration is calmodulin (CaM), which consists of four EF hands . When activated, CaM binds to and stimulates the activities of a family of Ca2+/CaM-dependent serine/threonine protein kinases (CaMKs), thereby regulating their functions. The CaMKs regulated by Ca2+/CaM include mono-functional kinases myosin light-chain kinase and phosphorylase kinase, and multi-functional enzymes CaMKI, CaMKII, CaMKIV, and CaMK kinase (CaMKK).
CaMKI plays pivotal roles in the nervous system. It is critical for long-term potentiation via activation of ERK  and recruitment of synaptic Ca2+-permeable AMPARs . It also promotes dendritic arborization , neurite outgrowth , and formation of spines, synapses and axons in hippocampal neurons , . Besides exerting important functions in the nervous system, CaMKI might also be involved in osteoclast differentiation and bone resorption . The kinase recognizes a consensus sequence Hyd-X-Arg-X-X-Ser/Thr-X-X-X-Hyd, in which Hyd is a hydrophobic residue , and its substrates include the synaptic vesicle-associated proteins, namely synapsin 1 and 2 , the cAMP response element-binding protein (CREB) , and the recently identified target glial cell missing 1 (GCM1) .
It has been shown that the kinase activity of CaMKI is regulated by a C-terminal regulatory region which contains an autoinhibitory segment (residues 286–307) and an overlapping CaM-binding segment (residues 303–316) , . In particular, three truncation forms of CaMKI exhibit distinct characteristics in basal kinase activity and responsiveness to Ca2+/CaM: the truncate encompassing residues 1–321 which contains both the autoinhibitory segment and the CaM-binding segment displays properties similar to those of the full-length protein; the truncate encompassing residues 1–314 which contains the autoinhibitory segment has a substantially lower kinase activity than the full-length kinase although it can bind Ca2+/CaM; and the truncate encompassing residues 1–293 which excludes the regulatory region is constitutively active and exhibits an activity comparable to that of the full-length kinase .
The crystal structure of the apo rat CaMKI320 (residues 1–320) has been determined, leading to the proposal that the regulatory region of CaMKI inhibits the kinase activity by interacting with the N-terminal lobe and hence occluding the nucleotide-binding site and restraining the kinase in an inactive conformation . However, the regulation mechanism of CaMKI remains elusive. Here we report four crystal structures of three truncation variants of human CaMKIα, namely CaMKI320 (residues 1–320), CaMKI315 (residues 1–315), and CaMKI293 (residues 1–293), which correspond to the aforementioned three truncation forms of CaMKI , respectively. The structural data reveal new insights into the regulation mechanism of CaMKI.
The cDNA fragments encoding different human CaMKIα truncates were inserted into the BamHI and SalI restriction sites of the pGEX4T-1 expression plasmid (Novagen) which attaches a GST tag at the N-terminus of the protein. The plasmids were transformed into E. coli BL21(DE3) strain (Novagen), and the transformed cells were grown in LB medium at 37°C in the presence of 50 µg/ml ampicillin until OD600 reached 0.8 and then induced with 0.5 mM IPTG for 12 hours at 16°C. The cells were harvested and lysed by sonication in a lysis buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.3). Protein purification was carried out by affinity chromatography using a GSTrap FF 5 ml column (GE healthcare). Cleavage of the fusion proteins with thrombin protease on column was conducted at 16°C for 24 hours. The elution fractions were further purified by gel filtration using a Superdex 200 16/60 column (GE healthcare). The target proteins were concentrated to about 10 mg/ml with Centricon-10 (Millipore) for structural studies.
Crystallization was performed using the hanging drop vapor diffusion method. The drop consisted of equal volumes of the protein solution and the reservoir solution. The protein solution contained 10 mg/ml CaMKI protein in the lysis buffer alone or supplemented with 20 mM MgCl2 and 5 mM ATP. For the apo CaMKI320, the crystals were grown at 18°C and the reservoir solution contained 2.0 M (NH4)2SO4 and 100 mM bicine, pH 8.0. For the CaMKI320-ATP complex, the crystals were grown at 18°C and the reservoir solution contained 0.5 M (NH4)2SO4, 0.8 M Li2SO4, and 100 mM tri-sodium citrate, pH 5.6. For the CaMKI315-ATP complex, the crystals were grown at 4°C and the reservoir solution contained 1.5 M (NH4)2SO4, 3% dioxane, and 100 mM MES, pH 6.0. For the CaMKI293-ATP complex, the crystals were grown at 4°C and the reservoir solution contained 25% polyethylene glycol 8,000, 200 mM NH4Ac, and 100 mM (CH3)2AsO2Na, pH 6.0. The diffraction data of the CaMKI293-ATP complex were collected at beamline BL-6A of Photon Factory, Japan; those of the CaMKI320-ATP complex at beamline 1W2B of Beijing Synchrotron Radiation Facility, China; and those of the apo CaMKI320 and the CaMKI315-ATP complex at beamline 17U of Shanghai Synchrotron Radiation Facility, China. The data were processed, integrated, and scaled together using the HKL2000 suite . For apo CaMKI320, the crystal used for data collection had a little bit ice, and hence during data processing the data in the resolution shell of 3.5–3.8 Å were largely excluded which yielded a relatively low overall completeness of the whole dataset. The statistics of the diffraction data are summarized in Table 1.
The structure of the CaMKI293-ATP complex was solved with the molecular replacement method implemented in program CNS  using the apo rat CaMKI320 structure (PDB code 1A06) as the search model. The other structures of CaMKI were solved with the molecular replacement method using the structure of the CaMKI293-ATP complex as the search model. The model building was performed using Coot , and the structure refinement using programs CNS  and REFMAC5 . The statistics of the structure refinement and the structure models are also summarized in Table 1.
To explore the molecular mechanism underlying the regulation of CaMKI activity, we obtained the crystal structures of CaMKI320, CaMKI315, and CaMKI293 which diverge in the regulatory region. The structures of CaMKI320 were determined in apo form and in complex with ATP, CaMKI315 in complex with ATP, and CaMKI293 in complex with ATP, respectively (Table 1). These structures are similar in the overall conformation with root mean square deviations of 0.6–1.6 Å for 244 Cα atoms. The structure of CaMKI320-ATP is shown in Figure 1A as a representative, and the nomenclature of the secondary structure elements are named after that of cAPK  (Figure 1B). The catalytic core of CaMKI takes a canonical bilobate architecture, with the N-terminal lobe (N lobe) consisting of five antiparallel β-strands and a prominent helix named helix αC and the C-terminal lobe (C lobe) comprised primarily of α-helices. The active site is formed at the interface of the two lobes where ATP is bound. The C-terminal region (residues 276–315) containing the autoinhibitory and CaM-binding sequences assumes a helix-loop-helix structure. Although the overall conformation is similar, detailed analyses of these structures reveal their distinctive features in the activation segment, the nucleotide-binding site, and the regulatory region, which together provide new insights into the regulation mechanism of CaMKI.
Figure 1. Structure of CaMKI.
A A stereoview of the overall structure of CaMKI320-ATP. The various CaMKI truncation forms have similar overall structure and the CaMKI320-ATP complex is selected as a representative. CaMKI consists of a catalytic core containing the N lobe (green), the C lobe (yellow) and a regulatory region (red). The bound ATP is shown with a ball-and-stick model (orange). The activation segment is primarily disordered and denoted with a dashed curve in orange. B Sequence alignment of human CaMKI (isoforms α, β, δ, and γ), rat CaMKIα, human CaMKIIα, human CaMKIV, human Akt/PKB, and rat cAPK. The highly conserved residues are indicated with closed red boxes, and the conserved residues with open red boxes. The secondary structure elements of human CaMKIα are indicated on the top of the alignment.
Surprisingly, structural comparison shows that the apo human CaMKI320 structure obtained in this study is substantially different from the apo rat CaMKI320 structure , particularly in the activation segment, the nucleotide-binding site, and the regulatory region (Figure 2A). In the rat CaMKI320, the activation segment is disordered and the phosphate-binding loop (P-loop, the β1-β2 hairpin)  takes an unusual conformation with residues 29–34 forming a distorted 310 helix. In addition, the regulatory region runs across the catalytic core with its C-terminus interacting with the N lobe, leading to an open conformation of the overall structure. It was proposed that the aberrant conformation of the P-loop and the obstruction of the nucleotide-binding site by helix αR2 and the αR1-αR2 loop of the regulatory region contribute to autoinhibition of the enzyme . In contrast, in our CaMKI320, the P-loop takes a canonical conformation, the C-terminus of the regulatory region (residues 300–320) is undetected, and the nucleotide-binding site is amenable to nucleotide binding. More intriguingly, the C-terminal part of the activation segment encompassing residues Pro171 to Thr181 assumes a unique helical conformation (designated as helix αT) (Figure 2A) with well-defined electron density (an average B factor of 39.8 Å2) and the DFG motif at the N-terminal part of the activation segment takes an "in" conformation. The rare helical conformation of αT is stabilized by the hydrophilic and hydrophobic interactions within the activation segment and with the surrounding secondary structure elements including helices αEF and αG and the αEF-αF loop (Figure 2B). Specifically, the hydroxyl of Thr177 (the phosphorylation site of the activation segment) forms hydrogen-bonding interactions indirectly with the hydroxyl of Tyr195 (a strictly conserved residue on the αEF-αF loop) and the main-chain carbonyl of Met168 via a water molecule, and the hydroxyl of Ser173 forms a hydrogen bond with the main-chain carbonyl of Lys167. In addition, Pro182 of the activation segment C-terminal to helix αT forms hydrophobic interactions with Leu190 of helix αEF and Leu226, Phe227, and Ile230 of helix αG, further stabilizing the specific conformation of the activation segment (Figure 2B).
Figure 2. A distinctive autoinhibited state of CaMKI.
A Comparison of the apo human CaMKI320 and the apo rat CaMKI320 (PDB code 1A06) . The apo human CaMKI320 is colored as in Figure 1A. The apo rat CaMKI320 is colored in light blue. Substantial conformational differences are observed between the two structures in the regulatory region, the activation segment, and the nucleotide-binding site which are indicated with ovals. In particular, the C-terminal part of the activation segment encompassing residues Pro171 to Thr181 assumes a rare helical conformation (helix αT). B Hydrophilic and hydrophobic interactions of helixαT with the surrounding residues in the apo CaMKI320. The involved residues are shown with ball-and-stick models, and the hydrogen-bonding interactions are indicated with dashed lines. C Interactions of the N-terminal part of the activation segment with the N lobe in the apo CaMKI320. The N-terminal part of the activation segment including the DFG motif and the following Leu165 form both hydrophilic and hydrophobic interactions with helix αC and strand β3 of the N lobe. The involved residues and additionally Glu66 on helix αC are shown with ball-and-stick models, and the hydrogen-bonding interactions are indicated with dashed lines. D Comparison of the apo CaMKI320 and the Akt/PKB-GSK3β complex in the substrate-binding site. In the Akt/PKB-GSK3β complex (gray), the bound GSK3β peptide is shown with a magenta ribbon, and the bound AMPPNP is shown with a ball-and-stick model in orange. Residues at P(-6), P(-3), P0, and P+3 positions of the GSK3β peptide, Glu236 of Akt/PKB which forms a salt bridge with Arg at P(-3) of the peptide, and the equivalent Glu102 in the apo CaMKI320 are shown with ball-and-stick models.
On the other hand, helix αT also forms a part of the interface of two two-fold symmetry-related molecules, and we performed crystal packing analysis to examine whether the conformation of helix αT is constrained by the crystal lattice. In the apo CaMKI320 belonging to space group C2, two-symmetry related molecules are juxtaposed head to tail, and the interface is formed mainly between helix αT of one molecule and helices αG and αEF of the other (Figure S1A). The interface is stabilized mainly by hydrophobic interactions. Particularly, the side chains of Pro171, Leu175, and Cys179 of helix αT of one molecule make hydrophobic contacts with the side chains of Phe227 of helix αG and Leu190 of helix αEF of the other, forming a hydrophobic cluster (Figure S1A). Interestingly, the unique conformation of helixαT captured in the apo CaMKI320 is also observed in the structure of CaMKI293 in complex with an inhibitor which is crystallized in a different space group (I222), and in that structure the interface is formed by similar structure elements of two two-fold symmetry-related molecules but involves different interacting residues (unpublished data). In addition, in the CaMKI320-ATP and CaMKI315-ATP structures which are crystallized in another space group (P63), two pseudo two-fold symmetry-related molecules are juxtaposed head to tail in a similar way as in the apo CaMKI320. Consistently, the interface is comparable to that in the apo CaMKI320 and particularly, helices αG and αEF occupy similar positions as those in the apo CaMKI320 structure (Figure S1B). However, helix αT is largely disordered in one molecule and completely disordered in the other, although the hydrophobic cluster at the interface is partially maintained (Figure S1B). In contrast, in the CaMKI293-ATP structure which is crystallized in space group I222, the C-terminal part of the activation segment is completely disordered, and the two symmetry-related molecules are juxtaposed head to head. Correspondingly, the interface is substantially different from those in the CaMKI320, CaMKI320-ATP, and CaMKI315-ATP structures. Taken together, these results indicate that the activation segment has a high flexibility and the crystal packing interactions do not contribute to the formation of helix αT. Thus, the apo CaMKI320 structure with a helix αT-containing activation segment should represent a biologically relevant state of CaMKI.
To explore the biological relevance of the apo CaMKI320, we compared it with the structure of the Akt/PKB-GSK3β complex . Akt/PKB belongs to the AGC family which is the most closely related to the CaMK family , and the Akt/PKB-GSK3β complex represents an active state of the enzyme . The comparison reveals substantial conformational differences especially at helix αC and the activation segment. It was shown previously that the conformation of helix αC is critical for the maintenance of the nucleotide-binding site and the coordination of the conformational changes at the catalytic site , . In the active Akt/PKB-GSK3β complex, helix αC is coupled to the nucleotide-binding site and the activation segment via a salt-bridging interaction between two conserved residues in the N lobe, an invariant Glu (Glu66 in CaMKI) of helix αC and a highly conserved Lys (Lys49 in CaMKI) of strand β3, which is a hallmark of the active kinase . In the apo CaMKI320, Lys49 interacts with Asp162 and Phe163 of the DFG motif of the activation segment (Figure 2C). Concurrently, helix αC is rotated outward and Glu66 points its side chain away from the catalytic site, making it impossible to interact with Lys49. This inactive conformation of helix αC is maintained largely by the hydrophobic contacts between Phe163 and Leu165 of the activation segment and Met63, Ile67, Leu70, and Ile73 of the hydrophobic surface of helix αC (Figure 2C).
In many kinases, phosphorylation of the activation segment can enhance the kinase activity . In Akt/PKB-GSK3β, Thr309 of the activation segment is phosphorylated and the activation segment takes an extended loop conformation which is stabilized by electrostatic interactions via the phosphate group of phosphorylated Thr309, leaving the catalytic site open for substrate binding. Whereas in CaMKI320, the activation segment including helix αT and the β8-αT loop occupies part of the catalytic site. Although unphosphorylated, the side chain of Thr177 (equivalent to Thr309 of Akt/PKB) is oriented inwards and forms interactions with the surrounding residues to stabilize the specific conformation of the activation segment, shielding it from surface exposure and hence from CaMKK phosphorylation (Figure 2A). It was reported that Thr177 of the full-length CaMKI cannot be phosphorylated in the absence of Ca2+/CaM, and that an inactive form of CaMKI (CaMKI306) which contains the autoinhibitory segment but not the CaM-binding segment cannot be phosphorylated either in the presence or absence of Ca2+/CaM . The previously reported apo rat CaMKI320 structure could not explain the unresponsiveness of the inactive CaMKI306 to CaMKK as the activation segment is completely disordered . In contrast, the unusual helical conformation of the activation segment observed in our apo CaMKI320 structure and the resulted sequestration of Thr177 provide a reasonable explanation for the biochemical data .
The consensus sequence of the substrates of CaMKI (Hyd-X-Arg-X-X-Ser/Thr-X-X-X-Hyd) is related to that of Akt/PKB (Arg-X-X-Arg-X-X-Ser/Thr-Hyd), both of which contain an Arg at the P(-3) position. The conservation of Akt/PKB and CaMKI and their substrates suggest that CaMKI might bind its substrate in a way similar to that of Akt/PKB with the GSK3β peptide. In the Akt/PKB-GSK3β complex, Glu236 of the hinge region (the β5-αD loop) forms a salt bridge with Arg at P(-3) of the substrate peptide . In the apo CaMKI320, the equivalent Glu102 occupies the same position as Glu236 of Akt/PKB, suggesting that the interaction between Glu102 and Arg at P(-3) of a CaMKI substrate might be conserved (Figure 2D). On the other hand, there are obvious steric conflicts between the residues at P+1 to P+3 of the GSK3β peptide and the N-terminus of helix αT and between the residues at P(-5) and P(-6) of the GSK3β peptide and the C-terminus of helix αR1 of the autoinhibitory segment (see details later) (Figure 2D), suggesting that helix αT and helix αR1 in the apo CaMKI320 might interfere with substrate binding due to potential steric blockage of the substrate-binding site. These results together suggest that the unique helix αT of the activation segment together with the autoinhibitory segment observed in the apo CaMKI320 restrains helix αC in an inactive conformation, sequesters Thr177 of the activation segment from being phosphorylated, and blocks the substrate-binding site, leading to the autoinhibition of CaMKI, and thus the apo CaMKI320 represents a distinctive autoinhibited state of CaMKI.
As described above, unlike the rat apo CaMKI320, in the apo CaMKI320 the nucleotide-binding site is not blocked by the regulatory region and thus is amenable to nucleotide binding. Therefore, we co-crystallized CaMKI320 with ATP and successfully obtained the complex structure (Figure 3A), in which ATP is observed with evident electron density; however, no Mg2+ was visible despite the presence of 20 mM MgCl2 in the protein solution (Figure 3B). Upon ATP binding, CaMKI320 undergoes significant conformational changes characterized by a different N lobe orientation, the disordering of helix αT of the activation segment, and the formation of helix αR2 of the CaM-binding segment compared with the apo CaMKI320 (Figure 3A). In particular, the residues on strand β1 of the P-loop exhibit positional displacements of about 5–7 Å, and ATP bound at the nucleotide-binding site interacts with the surrounding residues via a network of hydrogen bonds (Figure 3C). Specifically, the N1 and N6 atoms of the adenosine form hydrogen bonds with the main-chain amide of Val98 and the main-chain carbonyl of Gln96, respectively. The 2′- and 3′-hydroxyls of the ribose moiety form hydrogen bonds with the side-chain carboxyl of Glu102, and additionally, the 3′-hydroxyl forms a hydrogen bond with the main-chain carbonyl of Glu145. The α- phosphate forms hydrophilic interactions with the side-chain amine of Lys49 of strand β3, the side-chain carboxyl of Asp162 of the DFG motif, and the side-chain hydroxyl of Ser32 of the P-loop. The β-phosphate forms a hydrogen bond with the main-chain carbonyl of Thr28 of the P-loop. The γ-phosphate interacts with the side-chain carboxyl of Asp141 and the side-chain amine of Lys143 of the catalytic loop (the αE-β7 loop) and the side-chain carboxyl of Asp162. The CaMKI320-ATP complex displays structural features of an inactive conformation. Compared with the apo CaMKI320, although the structure elements forming the nucleotide-binding site show significant positional displacements up to 7 Å and helix αC is rotated towards strands β3-β5, Glu66 and Lys49 does not form a salt bridge which is critical for the maintenance of the active kinase.
Figure 3. Structures of the ATP-bound CaMKI.
A Comparison of the CaMKI320-ATP complex with the apo CaMKI320. The CaMKI320-ATP complex is colored as in Figure 1A, and the apo CaMKI320 is colored in gray. The three regions where the CaMKI320-ATP complex shows substantial conformational differences from the apo CaMKI are indicated with ovals. B The 2Fo-Fc maps of the bound ATP molecules (contoured at 1σ level) in the CaMKI320-ATP, CaMKI315-ATP, and CaMKI293-ATP structures. C Hydrophilic interactions of ATP with the surrounding residues in CaMKI320-ATP. ATP and the interacting residues are shown with ball-and-stick models, and the hydrogen-bonding interactions are indicated with dashed lines. D Comparison of the CaMKI293-ATP complex and the Akt/PKB-GSK3β complex at the catalytic site. The color coding is the same as in Figures 1A and 2D. The bound ATP is shown with a ball-and-stick model. Glu66 on helix αC and Lys49 on strand β3 in CaMKI293-ATP and their equivalents (Glu200 and Lys181) in Akt/PKB-GSK3β are shown with ball-and-stick models.
Additionally, we also obtained the structures of CaMKI315 and CaMKI293 in complexes with ATP. CaMKI315 containing the autoinhibition segment cannot be activated by CaM as the CaM-binding segment is incomplete . The CaMKI315-ATP structure is very similar to the CaMKI320-ATP structure and also adopts an inactive conformation. The bound ATP shows evident electron density (Figure 3B) and maintains similar interactions with the enzyme as in the CaMKI320-ATP complex; however, the phosphate groups of ATP have a much higher average B factor and no Mg2+ was observed in the electron density.
On the other hand, the CaMKI293-ATP structure shows notable differences from the CaMKI320-ATP structure in the overall conformation and at the nucleotide-binding site (Figure 3A). CaMKI293 lacks the autoinhibitory segment and exhibits a constitutive activity comparable to that of the full-length enzyme . Consistently, the CaMKI293-ATP structure resembles the Akt/PKB-GSK3β structure and displays the structural features of an active conformation (Figure 3D). Specifically, helix αC assumes a position similar to that in the active Akt/PKB-GSK3β complex, and concomitantly Glu66 of helix αC adopts the same conformation as that of Glu200 of Akt/PKB and forms a salt bridge with Lys49. The bound ATP maintains similar interactions with the enzyme as in the CaMKI320-ATP and CaMKI315-ATP complexes but the phosphate groups of ATP also have a high average B factor, indicating a high flexibility that probably allows conformational changes to occur readily when the metal ion and/or the substrate bind to the catalytic site.
Although the CaMKI320-ATP and CaMKI315-ATP complexes show substantial differences from the apo CaMKI320 in the activation segment and the nucleotide-binding site, in all three structures helix αR1 (residues 287–297) of the autoinhibitory segment interlocks with helices αD and αF, and the surrounding structure elements including the αD-αE loop and helix αE adopt essentially identical conformations (Figure 4A). Superposition of these structures with Akt/PKB-GSK3β shows that the residues at the P(-5) and P(-6) positions of the substrate peptide would collide with the C-terminal part of helix αR1, suggesting that helix αR1 might exert the inhibitory function via occlusion of part of the substrate-binding site (Figure 2D). In contrast, in the CaMKI293-ATP, the C-terminus (residues 278–293) is disordered, and consequently helix αD and the αD-αE loop show evident conformational changes. Particularly, Tyr113 of the αD-αE loop moves about 4.5 Å to insert into a narrow space surrounded by helices αD, αE, and αF, and its side chain is re-oriented to form hydrophobic interactions with Leu103 and Ile107 of helix αD, Leu121 of helix αE, and Leu211 of αF, stabilizing helix αD in the new conformation and preventing the re-binding of the autoinhibitory segment (Figure 4B). Concurrently, the space originally occupied by the C-terminal part of helix αR1 is vacant for substrate binding. The conformations of helix αD and the αD-αE loop particularly Tyr113 in CaMKI293-ATP are similar to those of the equivalents in Akt/PKB-GSK3β. In addition, Tyr113 is strictly conserved across the CaMK family (Figure 1B), underscoring the importance of this residue and supporting our notion that the conformational changes of helix αD and the αD-αE loop are critical for CaMKI activation. It was reported that mutation of Ile294 and Phe298 to Ala resulted in a modest level of CaM-independent activity, and additional mutation of Ile286 and Val290 to Ala resulted in significant increase of the basal activity . It is likely that mutation of these hydrophobic residues of the autoinhibitory segment might dislodge helix αR1 from the substrate-binding site, leading to conformational changes of helix αD and the αD-αE loop and hence partial activation of the kinase.
Figure 4. Structures of the regulatory region.
A Comparison of the regulatory region in the apo CaMKI320 (gray), and the CaMKI320-ATP (red) and CaMKI315-ATP (yellow) complexes. In all three structures, the autoinhibitory segment (helix αR1) interlocks with helices αD and αF. B Comparison of the conformations of helix αD and the αD-αE loop in the CaMKI320-ATP (gray) and CaMKI293-ATP (red) complexes. The involved residues are shown with ball-and-stick models and colored in cyan. C Structure of the regulatory region in the CaMKI320-ATP complex showing the functional roles of helix αR2. CaMKI320 is colored the same as in Figure 1A. Residues Lys300 and Glu305 of helix αR2 and the interacting residues including Glu102 of the hinge region and Ser291 and Lys295 of helix αR1 are shown with ball-and-stick models and colored in cyan. The hydrogen-bonding interactions are indicated with dashed lines. D Comparison of helix αR2 (residues 299–314) in the CaMKI320-ATP complex (red) with the corresponding region in the CaM-CaMKI peptide complex (blue, PDB code 1MXE)  and the CaMKIIδ-CaM complex (gray, PDB code 2WEL) . For simplicity, only CaM and the CaM-binding segment of CaMKII in the CaMKIIδ-CaM complex are shown.
It was also reported that CaMKI297 is constitutively active albeit with a relatively low activity . CaMKI297 contains all the residues that form helix αR1 in the apo CaMKI320 and the CaMKI320-ATP and CaMKI315-ATP complexes but its activity is not completely inhibited, suggesting that the CaM-binding segment might play some role in facilitating the autoinhibitory segment in the inhibition of the activity. In the rat CaMKI320, the CaM-binding segment forms a long loop that curves into the entry of the ATP-binding site followed by a short αR2 helix that interacts with the N lobe of the kinase  (Figure 2A). Particularly, Lys300 of the αR1-αR2 loop forms a salt bridge with the strictly conserved Glu102 of the hinge region, which might prohibit Glu102 from binding ATP or the substrate . Intriguingly, in the CaMKI320-ATP complex, the CaM-binding segment mainly forms a long αR2 helix which protrudes away from the catalytic core (Figure 2A). A detailed analysis indicates that helix αR2 of this conformation plays an important role in the maintenance of an inactive state of the enzyme through interaction with Glu102 and stabilization of the inactive conformations of helices αR1 and αD. Specifically, Lys300 on helix αR2 also forms a salt bridge with Glu102; this interaction does not abrogate the ability of Glu102 to bind ATP as Glu102 still makes hydrogen-binding interactions with the 2′- and 3′-hydroxyls of the ribose moiety of ATP (Figure 4C), however, it could have an impact on its ability to bind the substrate as Glu102 is also suggested to play a role in the recognition and binding of Arg at P(-3) of the substrate (see discussion above) (Figure 2D). In addition, the N-terminal part of helix αR2 would have steric conflicts with the C-terminal part of helix αD in the CaMKI293-ATP complex, preventing helix αD from adopting an active conformation. Furthermore, the side chain of Gln305 of αR2 forms two hydrogen-bonding interactions with the side chains of Ser291 and Lys295, and thus helix αR2 also contributes to stabilization of helix αR1 in the inactive conformation (Figure 4C).
To better understand how CaM binds to and activated CaMKI, we superposed the available structures of kinases with the CaM-binding and/or autoinhibitory segments including other CaMK members and the death-associated protein kinase (Figure 4D and S2A). In the crystal structure of CaM in complex with a peptide corresponding to the CaM-binding segment of CaMKI (residues 294–318), the peptide forms a long α-helix . The NMR spectra of CaM bound to either CaMKI320 or a similar peptide (residues 299–320) were virtually identical , indicating that the binding mode observed in the CaM-CaMKI peptide complex might be retained in the binding of CaM with CaMKI. Superposition of the CaMKI320-ATP structure with the CaM-CaMKI peptide structure  and the recently reported CaMKIIδ-CaM structure  based on the CaM-binding segment (corresponding to residues 299–314 of CaMKI) demonstrates that CaM binds to CaMKI and CaMKII in a similar mode, and helix αR2 in CaMKI320-ATP encompasses almost all the residues required for direct interaction with CaM (Figure 4D). Therefore, the position and conformation of the CaM-binding segment in CaMKI320-ATP correspond to a biologically relevant state of CaMKI ready for CaM binding. On the other hand, a short region at the N-terminus of CaM appears to have steric conflicts with helix αD in the CaMKI320-ATP complex, indicating that proper conformational change or dissociation of the N-terminal part of helix αR2 and the autoinhibitory segment is required prior to the binding of the complete αR2 helix by CaM (Figure S2B).
In the structure of CaMKIV in complex with an inhibitor (PDB code 2W4O), the residues corresponding to helices αR1 and αR2 form a unified long α-helix which folds along the catalytic core to interact with both N lobe and C lobe. Superposition of residues 299–314 (helix αR2) of the CaMKI peptide in the CaM-CaMKI peptide structure with the corresponding region (residues 322–337) in the CaMKIV structure shows severe steric conflicts between CaM and the N lobe of CaMKIV (Figure S2C), indicating that the regulatory region with such a long helical conformation is unsuitable for CaM binding. Given the conservation of the regulatory region across the CaMK family (Figure 1B), the CaM-binding segment in CaMKIV is likely to assume a conformation as in CaMKI320-ATP prior to CaM binding. The regulatory scheme of the CaMKs by CaM seems to be different from that of death-associated protein kinase, in which the autoregulatory domain forms a long helix and the bound CaM takes an extended conformation .
Our structural data suggest that the binding of helix αR2 by CaM would require conformational change or dissociation of the autoinhibitory segment from the catalytic core. Consistently, structural studies of CaMKII show that with respect to the autoinhibited kinase the binding of CaM to CaMKIIδ leads to dissociation of the autoinhibitory and CaM-binding segments from the catalytic core, resulting in the rearrangement of helix αD . Given that the conformation of helix αD in the active CaMKI293-ATP complex is essentially the same as that in the CaMKIIδ-CaM complex  and the Akt/PKB-GSK3β complex , these studies together support the notion that the binding of CaM to CaMKs leads to dissociation of the regulatory region from the catalytic core and the consequent rearrangement of helix αD into an active conformation, which is critical for the activation of CaMKs.
Now the question is how the activation of the kinase takes place after the removal of the restraint of the regulatory region on helix αD. Comparisons of the apo CaMKI320, CaMKI320-ATP, and CaMKI293-ATP structures provide some clues. In CaMKI320-ATP, helix αD takes an inactive conformation, and the side chain of Glu102 of the hinge region forms hydrogen-binding interactions with both 2′- and 3′-hydroxyls of the ribose moiety of ATP, pulling the ribose and phosphate groups deep into the nucleotide-binding site (Figure 5A). In CaMKI293-ATP, with the rearrangement of helix αR1 and the consequent relaxation of helix αD, the constraint on Glu102 by Lys300 of helix αR2 is removed. The side chain of Glu102 displays a positional displacement of 1.5 Å towards the catalytic site and binds to the 3′-hydroxyl rather than both hydroxyls of the ribose of ATP (Figure 5B). Concomitantly, ATP shifts upwards by about 1.5 Å, and several residues at the N-terminal part of the activation segment show notable conformational changes and form different interactions. Specifically, Asp162 interacts with the γ-phosphate group of ATP, and the side chain of the following Phe163 rotates about 30° towards helix αE, leaving space for the rotation of helix αC towards strand β3. In addition, the side chain of Leu165, which is disordered in CaMKI320-ATP and CaMK315-ATP, becomes evident and takes a position opposite to that in the apo CaMKI320 (Figures 2C, 5A and 5B), and therefore no longer participates in the interactions between the activation segment and helix αC. Accompanying these changes, several structure elements of the N lobe, in particular strand β3 and helix αC, also undergo substantial conformational changes. Lys49 of strand β3 moves upwards along with ATP by 2.5 Å and forms a salt bridge with Glu66 of helix αC. Concurrently, the side chain of Asn65 on helix αC is hydrogen-bonded to the side chain of Ser166 on the activation segment, stabilizing the N-terminal part of the activation segment. With the salt bridge between Lys49 and Glu66 established and helix αC rotated to the active position, the catalytic site is assembled.
Figure 5. Model of the regulation of CaMKI.
A Structure of the catalytic site in the CaMKI320-ATP complex. B Structure of the catalytic site in the CaMKI293-ATP complex. Comparison of these two structures reveals the conformational changes upon activation. CaMKI is colored in yellow and the bound ATP in orange. In B, the residues that play important roles in the conformational changes are shown with ball-and-stick models in cyan. C A schematic diagram showing the proposed model of the regulation of CaMKI. The conversion of CaMKI from an autoinhibited state to an inactive, pre-CaM binding state, and subsequently an active state is illustrated. The α-helices that undergo conformational changes during the conversion and the key residues are indicated with cylinders and stick models, respectively. ATP is represented with an oval.
The structures of CaMKI presented in this study represent three different conformational states of the kinase. Analyses of these structures reveal conformational differences particularly in the regulatory region, the activation segment, and the nucleotide-binding site. The structural data are in consistency with the previous biochemical and structural data including the unresponsiveness of the inactive CaMKI to CaMKK , the constitutive albeit relatively low activity of CaMKI297 , and the adoption of a helical conformation of the CaM-binding segment for CaM binding . Based on these results, we can propose a model of the regulation mechanism of CaMKI (Figure 5C). In the apo form, CaMKI exists in an autoinhibited state which is represented by the apo CaMKI320 structure (Figure 5C, autoinhibited state). In this state, the autoinhibitory segment (helix αR1) together with the CaM-binding segment (helix αR2) places strong constraints on helix αD of the C lobe to stabilize it in an inactive conformation, which blocks the binding of the substrate residues N-terminal to the P0 position. Meanwhile, the activation segment assumes a loop conformation at the N-terminus and a helical conformation at the C-terminus (helix αT). This unique conformation of the activation segment also contributes to the inhibition of CaMKI in three possible ways: (1) it interacts with the hydrophobic surface of helix αC via the highly conserved Phe163 and Leu165 to restrain helix αC in an inactive position; (2) it sequesters Thr177 and hence prevents its phosphorylation by CaMKK; (3) it prevents the binding of the substrate residues C-terminal to the P0 position. The apo CaMKI in the autoinhibited state is amenable to nucleotide binding. ATP binding can induce conformational changes at the nucleotide-binding site and the activation segment, leading to the formation of an inactive, pre-CaM binding state which is represented by the CaMKI320-ATP and CaMKI315-ATP structures (Figure 5C, pre-CaM binding state). In this state, the activation segment is largely disordered and its restraint on helix αC is alleviated. Nevertheless, helix αC remains in an inactive conformation and the catalytic site is only partially assembled. On the other hand, the CaM-binding segment adopts a long helical conformation that is ready for CaM binding. Upon CaM binding with the CaM-binding segment, the regulatory region is dissociated from the catalytic core, leading to the formation of an active state which is represented by the CaMKI293-ATP structure (Figure 5C, active state). The dissociation of the regulatory region releases the constraints on helix αD, resulting in rearrangements of helix αD, the preceding β5-αD loop (hinge region), and the following αD-αE loop. These conformational changes in the C lobe are propagated in long range to the N lobe via Glu102 of the hinge region, the bound ATP, and Asp162, Phe163, and Leu165 of the activation segment, which leads to the formation of the salt-bridging interaction between Lys49 of strand β3 and Glu66 of helix αC and hence completes the assembly of the catalytic site. In summary, the regulatory region, the activation segment, and the nucleotide-binding site of CaMKI are correlated together, and upon the binding of ATP and CaM these regions interplay in a concerted way to regulate the activity states of the kinase.
As written by Noble et al. in paraphrase of the opening sentence of Anna Karenina, "all active kinases are alike but an inactive kinase is inactive after its own fashion" . The CaMKI structures presented here unexpectedly reveal a rare helical conformation of the activation segment. Among >5000 kinase structures in Protein Data Bank, there are only very few in which the activation segment contains an α-helix, including serine/threonine kinases CDKs ,  and Nek2 ,  and tyrosine kinases Src/Hck (reviewed in ) and EGFR , . In most of these structures, the N-terminal part of the activation segment folds into a short α-helix which interacts with helix αC to constrain it in an inactive position , , , , . Hydrophilic interactions of this part with helix αC, particularly with an invariant Glu, have been suggested to be critical for maintenance of the inactive conformation of the kinase . In addition, hydrophobic interactions between this part and helix αC have also been demonstrated to be important. For example, in the inactive EGFR, a Leu immediately following the DFG motif (Leu834) and an adjacent Leu (Leu837) on the short α-helix of the activation segment pack against the hydrophobic side of helix αC to stabilize its inactive conformation , . Mutations of these two residues to polar ones (L834R and L837E) have been found in lung cancer patients, and the EGFR kinase domain carrying the L834R mutation displays a substantially increased activity, underscoring the importance of the two residues in the inhibition of the kinase activity .
In the CaMK family, only the Leu residue equivalent to Leu834 of EGFR is conserved, corresponding to Leu165 of CaMKI. In the apo CaMKI320, although the N-terminal part of the activation segment takes a loop conformation rather than a helical conformation, the side chain of Leu165 is also oriented towards helix αC and forms hydrophobic interactions with residues of helix αC, contributing to the stabilization of helix αC in the inactive conformation. The specific conformation of Leu165 is apparently associated with the αT-containing activation segment, as in the rat CaMKI320 and CaMKI320-ATP and CaMKI315-ATP structures, Leu165 is disordered, while in the CaMK293-ATP structure, the side chain of Leu165 points to an opposite direction towards the catalytic site.
In some Nek2 structures, the C-terminal part of the activation segment adopts a helical conformation and occupies a position similar to that of helix αT in CaMKI320, including the wild-type Nek2 in complex with ADP, and the T175A mutant Nek2 in apo form and in complex with an ATP analog , indicating that the helical conformation is formed independent of Thr175 and nucleotide binding. Consistently, in the wild-type Nek2, Thr175 (equivalent to Thr177 of CaMKI) is exposed to the solvent; whereas in CaMKI320, Thr177 points to the catalytic site to interact with Tyr195 and hence is inaccessible to CaMKK. Considering the previous report that Thr177 of the full-length CaMKI cannot be phosphorylated in the absence of CaM , the helical conformation of the activation segment that is characterized by the sequestration of Thr177 is likely to be adopted by the autoinhibited full-length CaMKI.
Drugs that specifically target a specific inactive conformation of kinases can achieve high selectivity, which has been exemplified by Gleevec , . Therefore, it is noteworthy that the newly identified helical conformation of the activation segment in CaMKI combined with the differences between CaMKI and other kinases in this region could be exploited in the development of inhibitors with high selectivity that can specifically lock CaMKI in this unique inactive conformation.
The crystal structures of the apo CaMKI320, the CaMKI320-ATP complex, the CaMKI315-ATP complex, and the CaMKI293-ATP complex have been deposited with the RCSB Protein Data Bank under accession codes 4FG7, 4FG8, 4FG9, and 4FGB, respectively.
Analysis of the interface between two two-fold symmetry-related molecules in the CaMKI structures. A The interface in the apo CaMKI320 structure. The interface is formed mainly between helix αT of one molecule (in green) and helices αG and αEF of the other (in cyan, denoted with apostrophes), and is stabilized mainly by hydrophobic interactions. The involved residues in the hydrophobic interactions are shown with ball-and-stick models and colored accordingly. B The interface in the CaMKI320-ATP structure. The interface is comparable to that in the apo CaMKI320 structure, and helices αG and αEF occupy similar positions as those in the apo CaMKI320 structure; however, helix αT is largely disordered in one molecule and completely disordered in the other.
Regulatory segments of CaMKs. A Comparison of the overall structures of kinases with the CaM-binding and/or autoinhibitory segments including human CaMKIα (the CaMKI315-ATP structure determined in this study, green), rat CaMKIα (PDB code 1A06, cyan), human CaMK1δ (2JC6, magenta), human CaMK1γ (2JAM, yellow), human CaMK2α (2VZ6, wheat), human CaMK2β (3BHH, grey), human CaMK2δ (2VN9, sky-blue), human CaMK2γ (2V7O, orange), human CaMKIV (2W4O, blue), and human death-associated kinase 1 (2X0G, red). B Docking of CaM to the CaMKI320-ATP complex based on the CaM-binding segment (helix αR2, residues 299–314). CaM is shown with a ribbon-and-surface representation in gray. CaMKI320 is shown with a ribbon representation in magenta. C Superposition of the structure of the CaM-CaMKI peptide complex and the structure of CaMKIV in complex with an inhibitor (blue, PDB code 2W4O) based on the CaM-binding segment (residues 299–314 of CaMKI and residues 322–337 of CaMKIV) shows severe steric conflicts between CaM and the N lobe of CaMKIV.
We thank the staff members at the Shanghai Synchrotron Radiation Facility of China, Beijing Synchrotron Radiation Facility of China, and the Photon Factory of Japan for technical support in diffraction data collection, and other members of our group for helpful discussion.
Conceived and designed the experiments: MZ CZ JD. Performed the experiments: MZ YO LH. Analyzed the data: MZ CZ JW JD. Wrote the paper: MZ CZ JD.
1. Wayman GA, Tokumitsu H, Davare MA, Soderling TR (2011) Analysis of CaM-kinase signaling in cells. Cell Calcium 50: 1–8.
2. Babu YS, Bugg CE, Cook WJ (1988) Structure of calmodulin refined at 2.2 A resolution. J Mol Biol 204: 191–204.
3. Schmitt JM, Guire ES, Saneyoshi T, Soderling TR (2005) Calmodulin-dependent kinase kinase/calmodulin kinase I activity gates extracellular-regulated kinase-dependent long-term potentiation. J Neurosci 25: 1281–1290.
4. Guire ES, Oh MC, Soderling TR, Derkach VA (2008) Recruitment of calcium-permeable AMPA receptors during synaptic potentiation is regulated by CaM-kinase I. J Neurosci 28: 6000–6009.
5. Wayman GA, Impey S, Marks D, Saneyoshi T, Grant WF, et al. (2006) Activity-dependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2. Neuron 50: 897–909.
6. Uboha NV, Flajolet M, Nairn AC, Picciotto MR (2007) A calcium- and calmodulin-dependent kinase Ialpha/microtubule affinity regulating kinase 2 signaling cascade mediates calcium-dependent neurite outgrowth. J Neurosci 27: 4413–4423.
7. Saneyoshi T, Wayman G, Fortin D, Davare M, Hoshi N, et al. (2008) Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex. Neuron 57: 94–107.
8. Davare MA, Fortin DA, Saneyoshi T, Nygaard S, Kaech S, et al. (2009) Transient receptor potential canonical 5 channels activate Ca2+/calmodulin kinase Igamma to promote axon formation in hippocampal neurons. J Neurosci 29: 9794–9808.
9. Ang ES, Zhang P, Steer JH, Tan JW, Yip K, et al. (2007) Calcium/calmodulin-dependent kinase activity is required for efficient induction of osteoclast differentiation and bone resorption by receptor activator of nuclear factor kappa B ligand (RANKL). J Cell Physiol 212: 787–795.
10. Lee JC, Kwon YG, Lawrence DS, Edelman AM (1994) A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia. Proc Natl Acad Sci U S A 91: 6413–6417.
11. Nairn AC, Greengard P (1987) Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain. J Biol Chem 262: 7273–7281.
12. Sheng M, Thompson MA, Greenberg ME (1991) CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252: 1427–1430.
13. Chang CW, Chang GD, Chen H (2011) A novel cyclic AMP/Epac1/CaMKI signaling cascade promotes GCM1 desumoylation and placental cell fusion. Mol Cell Biol 31: 3820–3831.
14. Yokokura H, Picciotto MR, Nairn AC, Hidaka H (1995) The regulatory region of calcium/calmodulin-dependent protein kinase I contains closely associated autoinhibitory and calmodulin-binding domains. J Biol Chem 270: 23851–23859.
15. Matsushita M, Nairn AC (1998) Characterization of the mechanism of regulation of Ca2+/ calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase. J Biol Chem 273: 21473–21481.
16. Goldberg J, Nairn AC, Kuriyan J (1996) Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I. Cell 84: 875–887.
17. Otwinowski Z, Minor W (1997) Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology 276: 307–326.
18. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D54: 905–921.
19. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D60: 2126–2132.
20. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D53: 240–255.
21. Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, et al. (1991) Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science 253: 407–414.
22. Saraste M, Sibbald PR, Wittinghofer A (1990) The P-loop–a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15: 430–434.
23. Yang J, Cron P, Good VM, Thompson V, Hemmings BA, et al. (2002) Crystal structure of an activated Akt/protein kinase B ternary complex with GSK3-peptide and AMP-PNP. Nat Struct Biol 9: 940–944.
24. Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298: 1912–1934.
25. Huse M, Kuriyan J (2002) The conformational plasticity of protein kinases. Cell 109: 275–282.
26. Haribabu B, Hook SS, Selbert MA, Goldstein EG, Tomhave ED, et al. (1995) Human calcium-calmodulin dependent protein kinase I: cDNA cloning, domain structure and activation by phosphorylation at threonine-177 by calcium-calmodulin dependent protein kinase I kinase. EMBO J 14: 3679–3686.
27. Clapperton JA, Martin SR, Smerdon SJ, Gamblin SJ, Bayley PM (2002) Structure of the complex of calmodulin with the target sequence of calmodulin-dependent protein kinase I: studies of the kinase activation mechanism. Biochemistry 41: 14669–14679.
28. Kranz JK, Lee EK, Nairn AC, Wand AJ (2002) A direct test of the reductionist approach to structural studies of calmodulin activity: relevance of peptide models of target proteins. J Biol Chem 277: 16351–16354.
29. Rellos P, Pike AC, Niesen FH, Salah E, Lee WH, et al. (2010) Structure of the CaMKIIdelta/calmodulin complex reveals the molecular mechanism of CaMKII kinase activation. PLoS Biol 8: e1000426.
30. de Diego I, Kuper J, Bakalova N, Kursula P, Wilmanns M (2010) Molecular basis of the death-associated protein kinase-calcium/calmodulin regulator complex. Sci Signal 3: ra6.
31. Noble ME, Endicott JA, Johnson LN (2004) Protein kinase inhibitors: insights into drug design from structure. Science 303: 1800–1805.
32. Brown NR, Noble ME, Lawrie AM, Morris MC, Tunnah P, et al. (1999) Effects of phosphorylation of threonine 160 on cyclin-dependent kinase 2 structure and activity. J Biol Chem 274: 8746–8756.
33. Day PJ, Cleasby A, Tickle IJ, O'Reilly M, Coyle JE, et al. (2009) Crystal structure of human CDK4 in complex with a D-type cyclin. Proc Natl Acad Sci U S A 106: 4166–4170.
34. Rellos P, Ivins FJ, Baxter JE, Pike A, Nott TJ, et al. (2007) Structure and regulation of the human Nek2 centrosomal kinase. J Biol Chem 282: 6833–6842.
35. Westwood I, Cheary DM, Baxter JE, Richards MW, van Montfort RL, et al. (2009) Insights into the conformational variability and regulation of human Nek2 kinase. J Mol Biol 386: 476–485.
36. Sicheri F, Kuriyan J (1997) Structures of Src-family tyrosine kinases. Curr Opin Struct Biol 7: 777–785.
37. Zhang X, Gureasko J, Shen K, Cole PA, Kuriyan J (2006) An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor. Cell 125: 1137–1149.
38. Wood ER, Truesdale AT, McDonald OB, Yuan D, Hassell A, et al. (2004) A unique structure for epidermal growth factor receptor bound to GW572016 (Lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res 64: 6652–6659.
39. Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, et al. (2000) Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 289: 1938–1942.


text:
Farmer Mike Excels at Kenda Mohican 100
John Gorrila sent me a link from www.singletrack.com regarding an article on the Kenda Mohican 100, held last weekend. Thought you all would be interested:
On its anniversary year, The Kenda Mohican 100 also made history by recording the oldest racer to ever complete the race. Mike Dietlin, (Kenda) will celebrate his seventieth birthday but he is not letting that slow him down. Out of 27 masters who started, eighteen finished, with Dietlin placing fourteenth, despite having twenty years on this field of young bucks in their fifties. At the awards, BikeSource of Columbus recognized Dietlin's achievement with a $75 gift certificate. As he picked up his award, Dietlin, who has completed three Mohican 100's in a row, including the wet year when many dropped out, yelled out to the crowd, "I think I may be done with the 100 mile and may enter the sissy race next year!" he said referring to the Mohican 100k, sticking out his tongue.
Mike is a class act. Kudos to him.
Congratulations to Mike, he doesn't do the computer thing, so won't be able to read this. He is truly an inspiration.

redpajama_set_name: RedPajamaCommonCrawl


text:
Electronic Yellow Pages are online versions of traditional printed business directories produced by telephone companies around the world. Typical functionalities of online yellow pages include the alphabetical listings of businesses and search functionality of the business database by name, business or location. Since Electronic Yellow Pages are not limited by space considerations, they often contain far more comprehensive business information such as vicinity maps, company profiles, product information, and more.

An advantage of Electronic Yellow Pages is that they can be updated in real time; therefore, listed businesses are not constrained by once-a-year publishing of the printed version which leads to greater accuracy of the listings since contact information may change at any time.

Before the popularity of the internet, business telephone numbers in the United Kingdom could be searched by accessing a remote computer terminal by modem. The initial prototype of this was superseded in 1990 with a commercial service. This service allowed searches via Name, Business classification and locality for business listings and a free text field was provided to allow "unstructured text" searching of Adverts. This dialup service was available via Prestel and "BT Gold" services. The service Electronic Yellow Pages was superseded in the mid-1990s by the internet service www.yell.com. A similar system called Phonebase for published residential phone numbers was discontinued in the 1990s, being superseded by a web-based search interface.

History
The first true online Yellow Pages, was a creation based on the independent YP publisher in Seattle, Washington called Banana Pages. This was the first print directory which was registered with both YPPA (the Yellow Pages Publishers Association), and the ADP (Association of Directory Publishers) to place their listings online. The Yellow Pages product was the brain child of the co-owner brothers of the company, Peter and John Richards. Many RBOCS (Regional Bell Operating Companies) would follow. Banana Pages eventually became YPI (Yellow Pages on the Internet, Inc) which was sold to InfoSpace.com and became their Yellow Pages offering to cobrands.

Among the first to place their listings on the Web was Pacific Bell's now-defunct "At Hand" online yellow pages that was officially launched on August 30, 1996. At Hand debuted with approximately 1.2 million merchant listings from across California. At the time, the service was competing with BigBook.com, a nationwide Web directory that was since absorbed by SuperPages.

Although it is "old hat" now, At Hand allowed an online visitor to look for a French restaurant in a given town and get an interactive map. If the restaurant has paid for an enhanced listing, the visitor could also find out other information, such as whether it took American Express or if it served bouillabaisse. At Hand showcased editorial content from 14 publishers, including HarperCollins, the Hearst Corporation, the New York Times Company and American Express.

Another early entrant into the then-$11 billion yellow pages industry occurred in 1997 when four Baby Bells banded together to create a co-branded Web site and initiated a $1 million marketing campaign to drive more traffic to their regional yellow pages sites. That "original yellow pages" site was simply a map of the United States, where users could click on any state and be linked to the appropriate online directory, as published by Ameritech, BellSouth, Pacific Bell, or US West.  The site experienced stiff competition from Big Yellow (now part of SuperPages) and SuperPages itself.

On Nov. 4, 2004 SBC Communications Incorporated (now AT&T) and BellSouth announced a joint venture to acquire Yellowpages.com. The venture utilized the highly recognized brand name to become the market leader in local search (Internet). At the time of acquisitions, it was expected that the YellowPages.com site would receive more than 50 million consumer searches per month.

In 2005, Amazon.com elbowed its way into the online yellow pages business, with a new service from its A9.com search engine, featuring photo-rich listings that allow an online visitor to "wander around" near a given destination.

According to market research company the Kelsey Group, advertising in online yellow pages is rapidly expanding but still only retain less than 7% of the total market, including online and print. The reason for this is that traditional yellow pages publishers, such as the phone companies, have big sales forces to approach local businesses.

MarketWatch, an online financial newsletter, carried an article on Dec. 18, 2006 detailing how mobile online yellow pages were enhancing small business lead generation. The article noted that Internet-based yellow pages, local search and wireless was growing by an estimated 30.5% versus print advertising, and that a company could only benefit from this online growth by being listed in an Internet-based directory.

Search Engines
Search engines such as Google Maps carry a significant number of business listings, often rivaling and sometimes exceeding the number of listings found in the traditional online yellow pages. These search engine results are a very effective way to reach new customers.

Search engine listings can often be edited by users, not just business owners. It is therefore important for business owners to verify and register their business address with all the major search engines.

Some of the problems associated with search engine advertising is click fraud. This is when ads are clicked on with no intention of purchasing anything. The search engines still charge the advertiser for these clicks, if the advertiser does not discover the fraud. Many online advertisers have discovered that clicks do not equal calls.

Many small businesses find the search engine keyword model very complicated. The average category in an Internet Yellow Pages would take an average of 20different keywords/search terms to "cover" on a search engine. For example, a plumber might need to buy leaky sink, leaky pipe, Toilet repair, sink repair, drain cleaning and on and on. It is easier for many businesses to buy an ad under "plumbers" in their local IYP's.

Migration of Physical Publishers to Electronic Yellow Pages
Electronic Yellow Pages or Online Yellow Pages use software to quickly edit and change online content for display on the internet.  Printed Yellow Pages or business directories often discontinue publication to limit printing costs and instead publish directly to the internet as a web directory.  Online yellow pages or directories continue publishing online because advertisers pay for the visual exposure from those who read or use these online publications on the internet. Publishers or people that create these online directories create software with their own resources or use opensource or Off-the shelf software to build their website or online directory.  Banana Pages was an online directory function built with their own resources.  The cost of online yellow pages or online directory software is limited compared to the large cost of physical printing and distribution of a phone book or directory of information.

See also
 Yellow Pages
 List of Yellow Pages
 YellowPagesGoesGreen.Org
 White pages - Residential (as opposed to commercial) counterpart of the Yellow Pages
 Blue pages - Government related counterpart
 Yellowikis

References

External links
 EADP - European Association of Directory Publishers

Telephone numbers
Yellow pages

redpajama_set_name: RedPajamaWikipedia


Instead of finishing last, nice guys actually come out on top. Earning a reputation as a cooperative, hard worker pays off in community support, which can in turn lead to healthier families, report anthropologists in a recent paper in Proceedings of the National Academy of Sciences.
For decades scientists have wondered how cooperation is maintained within a group. Judged by standard evolutionary and economic models, an individual has the most to gain by free riding–benefiting from the cooperation of others but giving nothing in return. But when free-riding spreads this behavior soon causes a cooperative system to unravel.
In the Nuñoa District of southeastern Peru there is a small collective of 24 Quechua households. This group of indigenous families jointly owns herds, gardens, irrigation canals and buildings. Life in the mountains is hard for these people. Their livelihood depends, in large part, upon group-owned resources. Maintaining the resources often requires significant time, energy and expertise.
"At this point I knew that high contributors must be gaining some benefit to offset the costs of their hard work and make them more tolerant of free riding.
Lyle and his colleague Eric Smith found that community members who contributed the most time, energy and risk into group projects gained valuable reputations as reliable, hard workers. They were known as the most generous, influential and respected people in the community. But the benefits of a good reputation may extend beyond the merely social. High contributors attracted large social support networks and this aid, in turn, is associated with healthier families. The researchers think this link is causal, though it cannot be conclusively proven.
Some critics may contend that sicker families simply have less time and energy to contribute to community projects. But the author's present several lines of evidence that this isn't so. Morbidity and time invested in group tasks were not correlated. And after controlling for morbidity in a model, they still find investment in group tasks can predict a person's reputation.
Other societies maintain cooperation by punishing free-riders. In Nuñoa the free riders themselves get a free pass. The value of a good reputation is enough, in seems, to encourage people to work hard.


text:
Q: Explain Matlab ode45 output. Is ode45 an iterative algorithm? I tried to use ode45 to solve an equation, and get output like the following. I get the idea it is trying to estimate using nearby points (as explained here https://www.mathworks.com/videos/solving-odes-in-matlab-6-ode45-117537.html). By my understanding, it should solve the equation in one round of computation? but the output looks like ode45 is an iterative algorithm (so that it generates output that repeat the '... steps ... failed attempt ... function evaluations' over and over again)? If it is iterative, could you help give some detail or references? Thanks!

A: ode45 is an iterative adaptive ODE solver. That is, it uses a 5th order (FSAL) method to propose the an update using some stepsize h. Then it does the same again, but now with a 4th order method, then it compared those two updates to one another, if the difference is less than some local tolerance, it accepts the proposed update. If the difference is larger than some local tolerance, the update is rejected and the stepsize is lowered (in some smart way).
To reduce the cost of using both a 4th and 5th order method, those two methods uses (roughly) the same function evaluations.
As for your output, it is, as also noted by @LutzL, not the standard output, which might point to an error in your code.

redpajama_set_name: RedPajamaStackExchange


text:
8 of the best private island escapes you can book with points | CNN Underscored
CNN Underscored reviews financial products such as credit cards and bank accounts based on their overall value, but does not review all financial companies or all available financial offers. We may receive compensation for sending traffic to partner sites, such as CreditCards.com, and this compensation may impact how and where links appear on this site. Terms apply to American Express benefits and offers. Enrollment may be required for select American Express benefits and offers. Visit americanexpress.com to learn more.
Private islands are the playgrounds of the very rich and very famous. They're the ultimate status symbol to announce that you've "made it."
But what if we told you that you could have a private island experience of your own and for a fraction of the cost thanks to credit card and hotel loyalty points and miles?
Well, that's exactly what we're here to tell you. Private islands are no longer just for the ultra-wealthy. These days, they're accessible to anyone with a stash of the correct credit card or hotel loyalty points.
Let's take a look at some of the best options you can book with points and miles, as well as how much it'll cost you to live like a high roller without having to shell out tens — or hundreds — of thousands of dollars on your trip.
One of Sir Richard Branson's private island escapes in the British Virgin Islands, Necker Island is a special place. The island itself is a portrait of Caribbean paradise, with lush vegetation, abundant wildlife and, of course, pristine beaches fronting vibrant turquoise waters.
The island's getaway package has a capacity of just 40 people housed across 15 rooms, which are a combination of guest rooms in a single "Great House" along with several private villas scattered across the island. For larger groups, the island can accommodate up to 54 people.
This is the Caribbean, so you can expect a complete raft of water activities at your disposal including snorkeling, diving, sailing, paddleboarding, kayaking, swimming and so much more. Back on land, you'll have access to just about all the amenities you'd expect at a luxury resort — multiple pools, a sumptuous spa, daily fitness classes, tennis courts and more.
The island places a large emphasis on dining and drinking, so you'll have plenty of options to choose from. There's the Great House, which has a rooftop deck perfectly suited for sunset meals, a beach pavilion that seats up to 40 guests at one table, Turtle Beach where you can enjoy a more casual beach picnic or cocktails, and even a floating sushi bar that's placed in the main beach pool from which you can enjoy fresh sushi and sashimi.
How to book Necker Island
While Necker Island typically needs to be reserved in its entirety, it offers certain weeks — known as Celebration Weeks — during which guests can book a single room on the island. Even better, these rooms can be booked with points, though you'll need a lot of them. If you're a Virgin Atlantic Flying Club Silver or Gold elite status member, you can book a room for two during a Celebration Week for the small sum of 1.5 million Virgin Points.
Located in Seychelles, one of the world's most sought-after holiday destinations, North Island has just 11 villas on an island that belongs completely to the resort, which means you'll be sharing it with hardly any other guests at all.
Each villa is massive, with two bedrooms and more than 5,000 square feet of living space, along with private plunge pools. They're surrounded by the island's dramatic landscape dotted with jagged peaks and vibrant green jungles and culminating in beautiful white sand beaches and warm crystal-clear waters.
While at North Island, you'll never get bored thanks to the wide variety of activities on offer like fishing, diving, snorkeling, kayaking, paddleboarding, sunset cruises and island explorations on foot or by buggy.
How to book North Island
North Island joined the Marriott Bonvoy portfolio several years ago, and you can now use Bonvoy points to book a stay. Note that you must stay a minimum of four nights, and each night will set you back in the ballpark of 385,000 Bonvoy points. Plus, you'll be on the hook for a helicopter transfer that runs about $920 per person, per direction. Considering, though, that rooms routinely sell for well over $6,000 per night, this could represent a phenomenal use of Marriott points, provided you have a whole lot of points that are waiting to be used.
If you're looking to get started on building that Bonvoy account balance, consider signing up for the Marriott Bonvoy Brilliant™ American Express® Card. The card currently comes with an intro bonus of 75,000 Bonvoy points, which you'll get after spending $3,000 in the first three months of holding the card.
Situated on a private island just off the coast of the Thai tourist hotspot of Phuket, The Naka Island provides a private island experience at a great price. Once you arrive at the island via a speedboat transfer, you can take advantage of all it has to offer including pristine beaches, oh-so-Thailand limestone cliffs and even coconut groves.
The resort has just 67 villas and 23 deluxe rooms and suites. Each offers stunning views of Phang Nga Bay, which is dotted with dramatic limestone formations. Beachfront villas feature private plunge pools and direct access to the stunning beaches of the island.
Guests can choose between a number of dining options including a beachfront signature restaurant, an all-day casual restaurant, private dining and a bar right on the beach.
How to book Naka Island
The Naka Island is a Marriott Bonvoy Category 6 property meaning off-peak nights cost 40,000 points, standard nights cost 50,000 and peak nights cost 60,000 points per night. And, this means it's eligible for the free night certificates that come with certain premium Marriott co-branded cards like the Marriott Bonvoy Brilliant™ American Express® Card.
Note, however, that Marriott is doing away with its award charts in March 2022. So if you're thinking about a stay, lock it in now before the chain implements a dynamic pricing model for award nights, meaning the cost per night will vary.
Another entry in the British Virgin Islands, the Scrub Island resort is located on its very own island adjacent to Tortola.
The resort offers accommodations from "regular" guest rooms to a massive six-bedroom villa complete with its own swimming pool.
Elsewhere on the resort, you can enjoy three private beaches, a wide array of watersports and activities, multiple options for eating and drinking and more.
How to book Scrub Island
Scrub Island is a Marriott Bonvoy Category 8 property, meaning free nights cost 70,000 points on off-peak nights, 85,000 points on standard nights and 100,000 points on peak nights.
Note again, though, that Marriott is doing away with its award charts in March 2022, so if you're thinking about a stay, lock it in now before the chain implements a dynamic pricing model for award nights.
The Caribbean may be the first place that comes to mind when thinking about private islands, but you don't want to miss this private island experience in Venice, Italy. Situated on an island called Isola delle Rose, this five-star JW Marriott property offers a serene escape from crowded Venetian streets.
The hotel provides a private water shuttle to ferry guests between the hotel and the city at regular intervals, but you won't be bored if you decide to spend a day — or two — at the resort.
The island spans 40 acres filled with verdant gardens and olive groves. The resort itself offers numerous luxury amenities including a full-service spa, restaurants and bars, a gorgeous infinity pool with panoramic views of the Venetian skyline and more.
The JW Marriott Venice is a Category 7 Marriott Bonvoy property, meaning free nights will cost you 50,000 points for off-peak nights, 60,000 points for standard nights and 70,000 points for peak nights. If you're able to find off-peak availability, you can use a free-night certificate that comes with certain premium Marriott co-branded cards like the Marriott Bonvoy Brilliant™ American Express® Card.
Once again, remember that Marriott is eliminating award charts in March 2022, so if you're thinking about a stay, lock it in now before the chain implements a dynamic pricing model for award nights.
Part of the Small Luxury Hotels of the World collection, Calala Island is a very intimate resort in the Caribbean Sea off the coast of Nicaragua, a location the resort refers to as the "NiCaribbean."
The entire resort has only four — yes, four — suites available to book. The resort embraces a more "rustic" approach to luxury and exclusivity. Don't expect to be wowed with high-end technology or state-of-the-art rooms here. But what you can count on is a glorious stay on what's essentially your own private island on a beautiful stretch of beach.
The resort also employs an "ultra-all-inclusive" model, which means everything you do — from airport transfers to eating to drinking — is included in the price of entry, which means you can get an impressive value staying here, especially if you use points.
How to book Calala Island
Thanks to Hyatt's somewhat new partnership with Small Luxury Hotels of the World, you can use World of Hyatt points to book a stay at Calala Island. The property is a Category 8 World of Hyatt property, meaning free nights will cost you 35,000 points on off-peak nights, 40,000 points on standard nights and 45,000 points on peak nights.
World of Hyatt points are easy to earn thanks to the transfer partnership the program has with Chase Ultimate Rewards. Points earned with cards like the Chase Sapphire Preferred® Card and Chase Sapphire Reserve® can be transferred at a 1:1 rate to World of Hyatt, making a stay very much within reach.
For example, the Chase Sapphire Preferred comes with a sign-up bonus of 60,000 points after spending $4,000 in the first three months from account opening. That bonus can put you well on your way toward a point redemption at Calala Island.
Another private island hotel in Thailand, Cape Fahn is located just off the coast of Ko Samui, one of the country's most popular vacation islands.
There are just 22 villas — all of which come with a private pool — on the resort. Guests can enjoy two on-site restaurants where private dinners on the beach can be arranged, a full-service spa, a shared resort pool and a fitness center all while taking in the stunning views of the Gulf of Thailand from the island's gorgeous beach.
Cape Fahn is a Category 5 World of Hyatt hotel, meaning free nights will cost you 17,000 points on off-peak nights, 20,000 points on standard nights and 23,000 points on peak nights.
Remember, if you have Chase Ultimate Rewards points, you can transfer them to World of Hyatt at a 1:1 rate, making it relatively easy to book a private island escape. In fact, the Chase Sapphire Preferred's sign-up bonus alone can get you three free nights if you travel on off-peak dates — with points to spare.
Situated on its very own island in Australia's Whitsundays archipelago in the heart of the Great Barrier Reef, the InterContinental Hayman Island offers the amenities of a large resort in a secluded and exclusive private island setting.
Simply arriving at Hayman Island is dramatic. After you book your stay, you'll be asked to choose your preference for your transfer between Hamilton and Hayman Islands. You can choose between a private charter, helicopter or boat — any of which will surely set the tone for what's sure to be a luxurious and memorable vacation.
The resort itself offers several distinct wings of rooms including residential-style villas with multiple bedrooms, full kitchens, private pools and more. There are five restaurants and bars to choose from, ranging from a signature fine-dining experience to a relaxed poolside bar and grill serving up fresh light bites and refreshing cocktails.
This resort's best amenity, though, is its unparalleled location among Australia's Great Barrier Reef. As expected, the resort places a strong emphasis on educating guests about and protecting its natural environment. Of course, you'll be able to participate in typical water activities like snorkeling, diving, fishing, sailing and so much more.
How to book InterContinental Hayman Island
The InterContinental Hayman Island is a member of IHG's Rewards Club program, meaning you can use IHG points to book free nights. However, IHG doesn't maintain an award chart, meaning the rate you pay will depend on the specific nights you'd like to travel. You can expect award nights to regularly cost in excess of 100,000 points per night.
To earn IHG Rewards points quickly, consider the IHG® Rewards Club Premier Credit Card, which currently has a sign-up bonus of 125,000 points after spending $3,000 within the first three months.
underscored explore
underscored travel
Previous articleDonations pour in for Bob Saget's favorite charity
Next articleExclusive! Here's the real reason why Jacqueline chose not to do Nagarjuna Akkineni's 'The Ghost'; Details inside! – Times of India
The best sales to shop today: Anker, Aurate, Dick's Sporting Goods and more | CNN Underscored
Good news for travellers! Thailand to resume quarantine-free entry for vaccinated visitors from Feb
20 Chewy pet items our readers can't get enough of | CNN Underscored
Russia will have to pay 'heavy price' if it invades Ukraine, warns US president Joe Biden
<!-- -->"I will unveil the hologram statue on 23rd January, Netaji's birth anniversary," PM Modi tweeted.New Delhi: A statue of freedom icon Subhas Chandra...


Description: UDO § 8.B.2.B and UDO § Appendix C – Variance of Use to permit a Child Care Home as the primary use in the R1 (Low Density Single-Family Residential) zoning district.
a) UDO § 9.I.1.C.2.b – Variance of Use to permit a Wireless Telecommunications Service Facility in the R2 (Low to Moderate Density Single Family Residential) zoning district that does not meet the minimum 500 foot setback from the property line adjoining to a residential zoning district.
b) UDO § 9.I.3.A.2 – Variance of Development Standards to permit the construction of an antenna support structure that does not meet the minimum setback from adjoining property zoned or used for a residential use.
a) UDO § 9.A.2 – Variance of Development Standards to permit construction on a lot with no frontage.
b) UDO § Table 8.B – Variance of Development Standards to permit the creation of lots in the R2 (Low to Moderate Density Single Family Residential) zoning district that do not meet the minimum lot width requirement (110 feet minimum required; 84.99 feet requested).
Description: UDO § Table 8.C – Variance of Development Standards to permit the installation of a chiller in the required rear yard setback for the DT (Downtown) zoning district.
a) UDO § 8.D.1.D and UDO § Appendix C – Conditional Use to permit a church, temple, place of worship use in the I-1 (Light Industrial) zoning district.
b) UDO § 10.0.3 – Variance of Development Standards to allow the reduction of the number of required parking spaces onsite.
a) UDO § Table 12.0.7.E – Variance of Development Standards to permit the use of existing canopy trees instead of the installation of evergreen trees in the landscape buffer between a PB zoning district and a residential use.
b) UDO § 12.0.5.C – Variance of Development Standards to allow the elimination of interior parking lot landscaping within a proposed fenced area.
c) UDO § Table 12.0.6 – Variance of Development Standards to allow the elimination of building base landscaping within the proposed fenced area and the reduction of the building base area to a 5 foot strip around the building along Sheridan Road.
d) UDO § Table 12.0.5.D.2 – Variance of Development Standards application to permit the reduction of perimeter parking lot plantings within the proposed fenced area.
e) UDO § 10.0.4.D.1 – Variance of Development Standards application to permit the elimination of curbing within the proposed fenced area.
f) UDO § 4.B.6 – Variance of Development Standards application to permit a reduction of architectural requirements for non-residential buildings.
g) UDO § 12.0.8.B.2 – Variance of Development Standards application to permit the screening of a portion of a service yard with a vinyl coated chain link fence instead of an opaque fence or wall.
Description: UDO § 4.D.3.H, UDO § 8.B.2.B and UDO § Appendix C – Variance of Use application to request amendment of conditions related to application 13J-13-6046.
Description: UDO § 8.B.2.B and UDO § Appendix C – Variance of Use application to permit Mineral/Sand/Gravel extraction on urban property in an R1 (Low Density Single Family Residential) zoning district.
Description: UDO § 10.0.3.B – Variance of Development Standards to permit a reduction of required off-street parking for a retail store. (38 required, 30 permitted by 2017 variance, 28 requested).
Description: UDO § 8.B.2.B and UDO § Appendix C – Variance of Use to permit a commercial office in the R1 (Low Density Single-Family Residential) zoning district.


text:
The History of Worcester Animal Rescue League
Worcester Animal Rescue League, also known as WARL, was founded in 1912 by a group of Worcester women to help over-worked horses, stray cats and dogs. They were dedicated women who remained with WARL for the remainder of their lives.
We've been around for a while
From 1916 to 1929 WARL was located at 447 Grove Street in Worcester. Even then WARL was taking in animals from beyond the city of Worcester and expanding its scope to needy animals other than dogs, cats and horses. The shelter purchased its first 'automobile truck' to rescue animals. In just one year, staff traveled 5,395 miles on 233 trips.
The 1925 census showed 3,885 animals, which was an increase of 262 animals from the previous year. Not only did WARL need larger quarters, it was operating at a deficit. There was an appeal in the annual report: "Will not the humane Worcester public help a society which cares yearly for nearly 4,000 animals?"
The public stepped forward with donations and bequeathments that led to the purchase of the 5-acre Deacon Fisher estate in 1930. That is Worcester Animal Rescue League's present location at 139 Holden Street in Worcester.
September 17, 1951 was the grand opening of the current shelter building, although it was increased to 1700 square feet in 1975, and there have been other minor renovations.
Celebrating 110 years of care and compassion to the neediest of animals in Central Massachusetts.


text:
SAM Lab
Pratt School of Engineering
Duke ECE
ECE 196 Syllabus
Jeffrey Krolik
CIEMAS 3465
ECE Box 90291
Email: jk@ee.duke.edu
As a signal processing group, our research applies to a vast range of applications from cellphones to autonomous vehicles. However, information extraction techniques are enhanced by incorporating knowledge of the particular problem. Much our work involves radar and sonar where the physical phenomenology is well understood but still remains problematic for target detection and tracking. However, the skills and tools used to solve these problems are broadly applicable.
Interested in these topics?
We are actively looking for students interested in the following:
Multiple sensors working together offer much more information than a single sensor. Cellphones and headphones use additional microphones to cancel noise from the environment. We design algorithms that exploit data captured by sensors at multiple locations.
Uncertainty or randomness of data is quantified through statistics. This allows automatic ways to find signals hidden underneath noise like tracking people through walls using our indoor radar or separating the speech of several people talking in a room.
Electromagnetic and acoustic waves propagate in predictable ways. This phenomenology can be exploited to extract information about the sources, such as location, or about the environment, such as reflecting structures.
Relevant applications of our work
Multipath Radar
Radar systems in urban environments have complex multipath due to buildings and other structures. Indoor radar systems also experience this on a smaller scale where the goal is through-the-wall sensing.
Multipath Sonar
Passive and active sonar systems are used for situational awareness in the ocean. Applications include underwater localization, maneverable underwater unmanned vehicles, and source waveform development.
Cellphones are often used in noisy environments such as airports and malls. Noise cancellation techniques exploit the differences between the talker and noise in order to improve voice quality. Radios in cellphones also must deal with multipath.
Unmanned sensing platforms have to be aware of the surrounding environment. Data from multiple sensors are combined to provide better understanding of their relative location to other objects.
If you're interested in joining us, please apply here to the ECE Ph.D. graduate program and reference our projects in your application. Summer internship opportunities for graduate students in our group also exist at partnering laboratories such as the Naval Research Laboratory, MIT Lincoln Laboratory, and the NATO Centre for Maritime Research & Experimentation. Applicants with U.S. citizenship or permanent	residency status are preferred.


Natural Resources Wales (NRW) is asking people not to start bonfires or leave rubbish behind when visiting nature reserves, as it can threaten the wildlife that lives there.
Marker posts and public safety signs from the nature reserve had been used to fuel a bonfire, and there was broken glass and other debris found around their vehicles.
Large amounts of plastic bottles, cans, food wrappers and general rubbish were also collected over the bank holiday weekend at the site, which is run by NRW.
"Nature reserves are fantastic places for people to enjoy and get closer to nature, but they also provide a home to the rich, diverse and valuable wildlife we have in Wales.
"We do ask all visitors to be considerate when they visit, and to be aware of the harmful effect that fires, fireworks, and leaving rubbish can have on the reserve.
Bonfires could be devastating to the site and its wildlife, and a risk to human lives, because the long dry grass on could easily catch fire from a wayward spark or unextinguished log.
Any rubbish left on the reserve can also contain chemicals which, if eaten by animals, can cause distress or fatalities.
"Wildlife is an important part of our environment, our heritage and our culture here in Wales.
Some nature reserves across Wales are Special Sites of Scientific interest (SSSI) or Special Areas of Conservation (SAC), this means they are protected by law and any damage could be considered a criminal act.
If you see people lighting fires or setting off fireworks on a National Nature Reserve, please contact the Natural Resources Wales 24 hour incident hotline on 0800 807060.


pvc outdoor chaise lounge chairs outdoor patio furniture patio best of patio furniture ideas modern patio outdoor patio furniture outdoor christmas decorations wholesale.
modern chaise lounge chairs outdoor outdoor chair cushions modern chaise lounge chairs of outdoor chair cushions outdoor wicker chair outdoor christmas decorations diy.
outdoor lounge chair cushions outdoor lounge ir cushion cushions furniture with patio covers cover where to buy ab outdoor lounge chair cushions sunbrella.
cheap outdoor lounge chairs lounge chairs for inside the pool brilliant chaise lounge chair white intended for plastic chaise lounge lounge chairs affordable outdoor chaise lounge chairs.
gray indoor outdoor rug outdoor rug grey indoor outdoor rug grey ivory indoor outdoor rug grey bone indoor outdoor kulpmont gray indoor outdoor rug.
outdoor lounge chair cushions clearance patio furniture patio lounge chair cushions clearance furniture mall clearance patio furniture with fire outdoor christmas decorations canada.
target outdoor lounge chairs target pool chairs chaise lounge chairs outdoor target wicker chaise lounges pool chaise chaise lounge chair target outdoor chaise lounge chairs.


text:
Q: TypeError: new(): invalid data type 'str' from ( Training a Deep learning model using import torch ) The actual code
import torch
from torch import nn
import re

# Define the vocabulary of the input and output sequences
vocab = ["The", "quick", "brown", "fox", "jumps", "over", "the", "lazy", "dog", ".",
         "old", "man", "and", "sea", "A", "story", "as", "time", "cat", "in", "hat",
         "To", "be", "or", "not", "to", "be"]

# Define the input and output sizes
input_size = len(vocab)
output_size = len(vocab)

# Define the other parameters of the  model
hidden_size = 256
n_layers = 2
dropout = 0.2
batch_size = 64
learning_rate = 0.01

class MyLSTMModel(nn.Module):
    def __init__(self, input_size, hidden_size, num_layers, dropout, batch_size):
        super().__init__()
        self.input_size = input_size
        self.hidden_size = hidden_size
        self.num_layers = num_layers
        self.dropout = dropout
        self.batch_size = batch_size

        # Define the LSTM cell to use in the  layer
        self.lstm_cell = nn.LSTM(input_size=input_size, hidden_size=hidden_size, num_layers=num_layers, dropout=dropout)

        # Define the architecture of the  using the LSTM cell
        self.model = nn.LSTM(input_size=input_size, hidden_size=hidden_size, num_layers=num_layers, dropout=dropout)

    def forward(self, input_seq, hidden):
        # Preprocess the input string to lowercase and remove punctuation marks
        input_seq = str(input_seq).lower()
        input_seq = re.sub(r'[^\w\s]', '', input_seq)

        # Convert the input string to a list of integers, where each integer represents the index of a word in the vocab list
        input_seq = [vocab.index(word) for word in input_seq.split() if word in vocab]

        # Convert the list of integers to a tensor
        input_seq = torch.tensor(input_seq).unsqueeze(0)

        # Reshape the input tensor to have three dimensions
        input_seq = input_seq.unsqueeze(1)

        # Apply the LSTM cell to the input sequence
        hidden = self.init_hidden()
        output, hidden = self.lstm_cell(input_seq, hidden)
        return output, hidden

    def loss(self, output, target_seq):
        # Convert the target string to a list of integers, where each integer represents the index of a word in the vocab list
        target_seq = [vocab.index(word) for word in target_seq.split() if word in vocab]

        # Convert the list of integers to a tensor
        target_seq = torch.tensor(target_seq)

       # Calculate the cross-entropy loss between the output and the target sequence, using a sequence loss function
        output = output.view(-1, output_size)
        target_seq = target_seq.view(-1)
        loss = nn.CrossEntropyLoss()(output, target_seq)
        return loss

    def init_hidden(self):
        # Initialize the hidden state with zeros
        return torch.zeros(self.num_layers, self.batch_size, self.hidden_size).unsqueeze(0)

    def train(self, train_data):
        # Loop through each training example
        for input_seq, target_seq in train_data:
            # Convert input and target sequences to tensors
            input_seq = [vocab.index(word) for word in input_seq.split()]
            input_seq = torch.tensor(input_seq)

            target_seq = torch.Tensor(target_seq)

            # Set initial hidden state to all zeros
            hidden = torch.zeros(1, self.hidden_size)

            # Forward pass to get the output sequence
            output, hidden = self.forward(input_seq, hidden)

            # Calculate the loss between the output sequence and the target sequence
            loss = self.loss(output, target_seq)

            # Backward pass to compute the gradients of the loss with respect to the model's parameters
            loss.backward()

            # Update the model's parameters using the optimizer object
            optimizer = torch.optim.SGD(self.parameters(), lr=learning_rate)
            optimizer.step()

# Create an instance of the MyLSTMModel class
model = MyLSTMModel(input_size, hidden_size, n_layers, dropout, batch_size)

# Define some example training data
train_data = [("The quick brown fox jumps over the lazy dog", "A story as old as time"),
              ("To be or not to be", "That is the question")]

# Train the model on the training data
model.train(train_data)

i run the code as a normal python script
my code goal

The code defines a MyLSTMModel class which extends the nn.Module class from PyTorch.
This class contains methods for defining the model architecture, initializing the hidden state of the model,
calculating the loss, training the model, and generating text using the trained model.
The MyLSTMModel class uses an LSTM (Long Short-Term Memory) layer to process the input sequence
and generate an output sequence. LSTM is a type of recurrent neural network that is particularly
well-suited for processing sequences of data. It can remember important information from earlier in
the sequence and use that information to make predictions about the future. In this case, the LSTM layer
is used to generate text based on the input sequence.

*but I get this error when I run the code:*

Traceback (most recent call last):
File "D:\path", line 121, in
model.train(train_data)
File "D:\path"", line 95, in train
target_seq = torch.Tensor(target_seq)
TypeError: new(): invalid data type 'str'


Last night a small group of IPs from Oracle, including myself and Jess, went out for a few pints in Reading. It was a lovely night which started off in a pub with a remarkable amount of character, right in the middle of Reading in the main pedestrian zone: the Hobgoblin. A lovely pub that is, with great beers, lots of guest ales, and a very cosy atmosphere. This is the sort of place that has a no mobile phones sticker on the door. Lovely! We then moved on to another pub, The Outback (I think), which was perfectly reasonable but nothing too special. All in all a nice night. I really need to get used to going out more often: I had a hangover after just 4 pints!
Tomorrow, Sam, Ben and I are off to IBM in Hursley (near Southampton) to take part in the first heat of the British Computer Society Programming Competition 2005. I've spent most of the evening brushing up on things like parsers, compilers, and various data structures and algorithms that I really should know but have forgotten about. We're also allowed to bring in all sorts of printed matter so I'm printing out whatever I think could come in handy!
So, wish us luck! I wonder if we'll even get through the first heat, but we can but try.
Good luck Chris! I'm sure you'll do well.


This is my first showreel. It features my work until early 2013. Some projects are from my time at SAE Cologne and some from my studies at MD.h Berlin.
Dominik Burbaum showreel 2013 from Dominik Burbaum on Vimeo.