Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin
Class III myosins ( Myo3 ) , together with class IX myosins , are two special groups of the myosin superfamily as these two sub-families of actin motors contain enzymatically active domains and thus are regarded as motorized signaling molecules ( Bähler , 2000 ) . The first member of Myo3 was identified in Drosophila photoreceptors and named as NinaC ( neither inactivation nor afterpotential C ) ( Montell and Rubin , 1988 ) . There are two paralogs of Myo3 in vertebrate , Myo3a and Myo3b , both of which are known to express in vertebrate retina and cochlea ( Dose and Burnside , 2000 , 2002; Shin et al . , 2013 ) . It is believed that they may play partially redundant roles as transporters that are crucial for vertebrate photoreceptor and stereocilia ultrastructure maintenance ( Manor et al . , 2012; Mecklenburg et al . , 2015; Merritt et al . , 2012 ) . Myo3 across different species all contain an N-terminal S/T kinase domain before their motor head . The kinase domain has been reported to regulate the motor’s ATPase activity ( Komaba et al . , 2010; Quintero et al . , 2010 ) . The tail regions of Myo3 from different species are less conserved . Drosophila NinaC contains a PDZ binding motif at its very C-terminus capable of binding to a master scaffold protein called INAD ( Inactivation no afterpotential D ) ( Wes et al . , 1999 ) . Vertebrate Myo3 tails share a conserved vertebrate specific domain referred to as tail homology I motif ( THDI ) ( Figure 1A ) ( Dose et al . , 2003 ) . The THDI mediates binding of Myo3 to its cargo protein Espin1 ( Ectoplasmic specialization protein 1 ) and allows Myo3 to transport Espin1 to the tips of actin bundle-based structures such as filopodia and stereocilia . Once tip localized , Espin1 WH2 domain promotes the elongation of actin protrusions ( Merritt et al . , 2012; Salles et al . , 2009 ) . However , the detailed molecular basis governing the Myo3 and Espin1 interaction is not clear . 10 . 7554/eLife . 12856 . 003Figure 1 . Biochemical characterizations of the Myo3/Espin1 interaction . ( A ) Domain organizations of Espin1 , Myo3a and Myo3b . ( B ) Sequence alignment of THDI of Myo3a and Myo3b showing that there are a pair of repeating sequences within THDI , which we term as ARB1 and ARB2 . Hs , human; Mm , mouse; Gg , chicken; Xt , Xenopus tropicalis; Dr , Danio rerio . ( C ) ITC results showing that Myo3b-ARB12 ( C1 ) as well as each individual site ( C2 for ARB1 and C3 for ARB2 ) can bind to Espin1-AR with strong affinities . ( D ) FPLC-MALS showing that ARB12 and Espin1-AR form a 1:2 complex . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 00310 . 7554/eLife . 12856 . 004Figure 1—figure supplement 1 . ITC results of Myo3a-ARBs binding to Espin1-AR . ( A–C ) ITC results showing that Myo3a-ARB12 ( A ) as well as each individual ARB ( B for ARB1 and C for ARB2 ) can bind to Espin1-AR with strong affinities . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 00410 . 7554/eLife . 12856 . 005Figure 1—figure supplement 2 . Analytical gel filtration chromatography analysis of the Espin1-AR and Myo3b-ARB12 interaction . ( A–C ) The profiles of mixtures of Trx-Espin1-AR and Myo3b-ARB12 with different molar-ratios . The black and red curves in each panel represent the elution profiles of Trx-Espin1-AR alone and Myo3b-ARB12 alone , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 005 Espin1 was first identified in Sertoli cell-spermatid junctions ( Bartles et al . , 1996 ) , encoded by the gene Espin . Later , shorter spliced isoforms of Espin gene products ( Espin2B , Espin3A and Espin4 ) were shown to be expressed in other F-actin rich structures such as brush border microvilli and Purkinje cell dendritic spines ( Bartles et al . , 1998; Sekerkova et al . , 2003 ) . They share a common 14 kDa C-terminal actin binding domain ( ABD; Figure 1A ) , which was reported to be necessary and sufficient for F-actin bundling activity ( Bartles , 2000; Bartles et al . , 1998 ) . Besides the ABD , all Espin isoforms contain a WH2 motif which can bind to actin monomer and a proline rich ( PR ) region which can interact with profilins ( Sekerkova et al . , 2006 ) . Espin2B contains one more PR region and an extra actin binding site ( xAB ) at the N-terminus ( Chen et al . , 1999 ) . Espin1 is the longest isoform in the family and contains a stretch of ankyrin repeats ( AR ) in its N-terminus ( Figure 1A ) . The AR of Espin1 is responsible for directly interacting with Myo3 tail THDI ( Merritt et al . , 2012; Salles et al . , 2009 ) . Recently , it was reported that Espin1 contains an AR binding sequence immediately C-terminal to xAB , and binding of AR to this sequence prevents xAB from binding to actin ( Zheng et al . , 2014 ) . As such , the actin binding activity of the N-terminal part of Espin1 is auto-inhibited , and this AR-binding region is named as the auto-inhibitory region ( AI , Figure 1A ) . The authors also proposed that Myo3 binding can release the auto-inhibition and increase the diameter of Espin1-promoted actin bundles ( Zheng et al . , 2014 ) . Myo3a/Myo3b are known to co-localize with Espin1 at the tips of stereocilia of hair cells ( Merritt et al . , 2012; Salles et al . , 2009; Schneider et al . , 2006 ) . Importantly , co-expression of Myo3a and Espin1 can further stimulate elongation of stereocilia in cultured organ of Corti hair cells ( Salles et al . , 2009 ) . When expressed in heterologous cells , Myo3a gets enriched at the tip of filopodia ( Les Erickson et al . , 2003; Salles et al . , 2009; Schneider et al . , 2006 ) . Mutations of human Myo3a gene is known to cause progressive non-syndromic hearing loss , DFNB30 ( Walsh et al . , 2002 ) . Also , transgenic mice with DFNB30 mutation undergo age-dependent outer hair cell degeneration ( Walsh et al . , 2011 ) . The jerker mouse carrying espin frame-shift mutation suffers from hair cells degeneration , deafness and vestibular dysfunction ( Sekerkova et al . , 2011; Zheng et al . , 2000 ) . Mutations in espin were also reported to be associated with non-syndromic autosomal recessive deafness DFNB36 and non-syndromic autosomal dominant deafness ( Boulouiz et al . , 2008; Donaudy et al . , 2006; Naz et al . , 2004 ) . A prominent phenotype of the jerker mice is that their hair cell stereocilia are uniformly thinner and shorter , degenerate faster than those of the wild type littermates ( Sekerkova et al . , 2011; Zheng et al . , 2000 ) . These genetic findings convincingly point to critical roles of espin in stereocilia development and maintenance , likely by promoting the assembly and stabilization of parallel actin filament bundles in stereocilia ( Bartles , 2000; Sekerkova et al . , 2006 ) . Here , we discover that both Myo3a and Myo3b contain two highly similar repeat sequences in their THDI region , each capable of independently binding to Espin1-AR with high affinity . The high resolution crystal structures of each of the two binding sequences from Myo3b in complex with Espin1-AR not only reveal the molecular basis governing the specific interaction between Myo3 and Espin1 , but also allow us to discern the Myo3-mediated release of the auto-inhibition mechanism of Espin1 . Based on these structural findings , we predict that binding of Myo3 to Espin1 can cluster Espin1 and thus enable Espin1 to further cross-link actin filaments into higher order fibers . Consistent with this prediction , we demonstrate by electron and fluorescence microscopic studies that binding of Myo3 to Espin1 can further promote formation of Espin1-mediated thicker actin bundles .
First we analyzed the sequences of the reported Espin1-binding THDI regions of both Myo3a and Myo3b , and found that the region contains a pair of repeating sequences in its N- and C-terminal halves ( Figure 1A and B , denoted as ARB1 and ARB2 for Espin1 ankyrin repeats binding region 1 and 2 as detailed below ) . Using isothermal titration calorimetry ( ITC ) -based quantitative binding assay , we found that purified THDI from both Myo3a and Myo3b can bind to Espin1-AR with high affinities ( Kd in the range of tens of nanomolars; Figure 1C1 and Figure 1—figure supplement 1A ) . Inspection of the ITC-based titration curves indicated that the binding stoichiometry of the Myo3 THDI and Espin1-AR clearly deviates from the value of 1:1 ( Figure 1C1 and Figure 1—figure supplement 1A ) . We thus hypothesized that each of the two repeat sequences in Myo3 THDI may independently bind to Espin1-AR , forming a 2:1 stoichiometric complex . We verified this prediction by gel filtration chromatography and static light scattering experiments ( Figure 1—figure supplement 2 and Figure 1D ) . In the gel filtration analysis , addition of an equivalent molar amount of Espin1-AR to Myo3b-THDI resulted in a complex peak with a smaller elution volume ( Figure 1—figure supplement 2A ) . Addition of another molar equivalent of Espin1-AR further shifted the complex peak to a smaller elution volume ( Figure 1—figure supplement 2B ) . However , further addition of Espin1-AR did not change the elution volume of the complex any more , indicating that Myo3b THDI is saturated by the binding of two molar ratios Espin1-AR ( Figure 1—figure supplement 2C ) . To accurately determine the stoichiometry , we used fast protein liquid chromatography ( FPLC ) coupled with multi-angle light scattering ( FPLC-MALS ) to calculate the molecular mass of the Myo3b THDI and Espin1-AR complex . When mixed Myo3b THDI with saturated amount of Trx-tagged Espin1-AR , the fitted molecular weight of the complex peak ( ~109 . 5 kDa ) matches well with the theoretical molecular weight of 117 kDa for the ( Trx-Espin1-AR ) 2/Myo3b-THDI complex ( Figure 1D ) , confirming that Myo3 THDI contains two Espin1-AR binding sites . Next , we divided Myo3b-THDI into two fragments , each corresponding to ARB1 and ARB2 as shown in Figure 1B . Both Myo3 ARB1 and ARB2 bind to Espin1-AR with affinities also in the range of tens of nanomolars and each with a 1:1 stoichiometry ( Figure 1C2 and 1C3 , and Figure 1—figure supplement 1B and C ) , indicating that the two repeating sequences in Myo3-THDI can independently bind to Espin1-AR with comparable affinities . The 1:2 stoichiometry between Myo3 and Espin1 is consistent with a previous finding that human Myo3a THDI lacking exon 33 ( exon 33 mainly encodes ARB2 ) can still interact with Espin1 ( Salles et al . , 2009 ) . To understand the molecular basis of the Espin1/Myo3 interaction , we solved the crystal structure of the Espin1-AR/Myo3b-ARB1 complex at 1 . 65 Å resolution ( Table 1 ) . The structure revealed that Espin1-AR contains 10 ANK repeats ( Figure 2 ) , instead of 8 as predicted earlier ( Bartles et al . , 1996 ) . The repeats 2–9 each contains the signature ‘TPLH’ sequence at the N-terminus of the αA helix , so can be viewed as the canonical ANK repeats . Like shown in the recently determined structures of the 24 ANK repeats scaffold protein ankyrin-B ( Wang et al . , 2014 ) and the 9 ANK repeats RNase L ( Han et al . , 2012 ) , the two ANK repeats capping the two termini of Espin1-AR do not contain the ‘TPLH’ sequence ( Figure 2—figure supplement 1 ) . We believe that these two non-canonical ANK repeats capping the two termini of Espin1-AR mainly play a structural stabilization role of the entire AR fold . The 10 ANK repeats form a left-handed superhelix with the αA helices forming the inner groove and the αB helices forming the outer surface ( Figure 2—figure supplement 2 ) . Clear additional electron densities lining the inner groove of the ANK repeats allowed us to build the bound Myo3b-ARB1 peptide model with high confidence ( Figure 2A ) . The ARB1 binds to Espin1-AR in an antiparallel manner , similar to the binding mode between ANK repeats from Ankyrin R/B/G and their targets ( Wang et al . , 2014 ) as well as between ANKRA2/RFXANK and their targets ( Xu et al . , 2012 ) , suggesting that elongated inner grooves are common target binding sites of ANK repeats in general . The ARB1 spans nearly the entire inner groove , covering ~1230 Å2 of solvent accessible area . The N-terminal of ARB1 adopts an extended structure and binds to the C-terminal of Espin1-AR . The C-terminal of ARB1 forms an α-helix and binds to the N-terminal half of Espin1-AR ( Figure 2B ) . The amino acid sequences of Espin1-AR from different vertebrate species as well as of the mammalian paralogs Espin-like proteins ( Shin et al . , 2013 ) are highly conserved ( Figure 2—figure supplement 1 ) . We mapped the sequence conservation profile to the structure of Espin1-AR and found that the residues in the inner groove of AR are highly conserved . In particular the residues in the ARB1 binding surface are essentially totally conserved ( Figure 2C ) . 10 . 7554/eLife . 12856 . 006Table 1 . Statistics of X-ray Crystallographic Data Collection and Model refinement Numbers in parentheses represent the value for the highest resolution shell . a . Rmerge=Σ Ii- <I> / ΣIi , where Ii is the intensity of measured reflection and <I> is the mean intensity of all symmetry-related reflections . b . Rcryst=Σ Fcalc – Fobs /ΣFobs , where Fobs and Fcalc are observed and calculated structure factors . c . Rfree= ΣTFcalc – Fobs /ΣFobs , where T is a test data set of about 5% of the total unique reflections randomly chosen and set aside prior to refinement . d . B factors and Ramachandran plot statistics are calculated using MOLPROBITY ( Chen et al . , 2010 ) . e . CC* and CC1/2 were defined by Karplus and Diederichs ( Karplus and Diederichs , 2012 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 006Data setsEspin1-AR/Myo3b-ARB1 5ET1 Espin1-AR/Myo3b-ARB2 5ET0 Space group P21 P2 Wavelength ( Å ) 0 . 9791 0 . 9795 Unit Cell Parameters ( Å ) a=72 . 74 , b=71 . 14 , c=76 . 88 α=γ=90° , β=96 . 88° a=39 . 74 , b=68 . 78 , c=173 . 45 α=γ=90° , β=90 . 04° Resolution range ( Å ) 50-1 . 65 ( 1 . 68–1 . 65 ) 50-2 . 30 ( 2 . 42–2 . 30 ) No . of unique reflections 93433 ( 4625 ) 39636 ( 5866 ) Redundancy 3 . 7 ( 3 . 7 ) 3 . 7 ( 3 . 8 ) I/σ 18 . 5 ( 1 . 7 ) 7 . 7 ( 1 . 9 ) Completeness ( % ) 99 . 8 ( 99 . 9 ) 94 . 9 ( 96 . 6 ) Rmergea ( % ) 8 . 9 ( 91 . 6 ) 10 . 3 ( 79 . 9 ) CC* for the highest resolution shell e 0 . 866 0 . 878 CCi/2 for the highest resolution shell e 0 . 599 0 . 627 Structure refinement Resolution ( Å ) 50-1 . 65 ( 1 . 71–1 . 65 ) 10-2 . 3 ( 2 . 38–2 . 30 ) Rcryst b/Rfree c ( % ) 16 . 94/19 . 11 ( 25 . 77/28 . 64 ) 22 . 32/25 . 34 ( 26 . 74/30 . 90 ) rmsd bonds ( Å ) / angles ( ° ) 0 . 006 / 0 . 795 0 . 010 / 1 . 113 Average B factor ( Å2 ) d 23 . 2 60 . 5 No . of atoms Protein atoms 5374 4985 Water 378 23 Ligands 30 0 No . of reflections Working set 89061 37660 Test set 4345 1925 Ramachandran plot regions d Favored ( % ) 98 . 9 98 . 4 Allowed ( % ) 1 . 1 1 . 6 Outliers ( % ) 0 0 10 . 7554/eLife . 12856 . 007Figure 2 . The overall structure of the Myo3-ARB/Espin1-AR complex . ( A ) An omit map showing the binding of Myo3b-ARB1 to Espin1-AR . The Fo-Fc density map was generated by deleting the Myo3b-ARB1 part from the final model and contoured at 3 . 0σ . The Myo3b-ARB1 fitting the electron density is displayed in the stick model . ( B ) The overall structure of the Myo3b-ARB1/Espin1-AR complex . The Espin1-AR is shown in cylinders , Myo3b-ARB1 is shown with the ribbon diagram and colored in magenta . ( C ) The amino acid conservation map of Espin1-AR . The conservation map was calculated based on the sequence alignment of vertebrate Espin1 and mammalian Espin-like proteins shown in Figure 2—figure supplement 1 . The identical residues are colored in dark blue; the strongly similar residues are colored in blue; the weakly similar residues are colored in light blue; the variable residues are colored in white . ( D ) The overall structure of Myo3b-ARB2/Espin1-AR complex . The Espin1-AR is shown in cylinders , Myo3b-ARB2 is shown in ribbon diagram and colored in dark purple . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 00710 . 7554/eLife . 12856 . 008Figure 2—figure supplement 1 . Structural-based sequence alignments of AR of Espin1 from different vertebrate species and Espin-like proteins from mammals . Residues that are identical and highly similar are shown in red and yellow boxes , respectively . Residues that are directly involved in ARBs binding are highlighted with solid magenta stars . Negatively charged residues in region 3 that extend the negatively charged pockets ( also refer to Figure 3 ) are highlighted with unfilled magenta stars . The alignment was created using ESPript 3 . 0 ( http://espript . ibcp . fr/ESPript/ESPript/ ) . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 00810 . 7554/eLife . 12856 . 009Figure 2—figure supplement 2 . Superhelical model of Espin1-AR . The superhelical model of Espin1-AR viewed from the top ( left ) and side ( right ) . To illustrate the curvature nature of the ANK repeats , we artificially connected several Espin1-AR units in a head-to-tail manner , and showing one Espin1-AR unit using the color mode as used in Figure 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 009 We have also determined the Espin1-AR/Myo3b-ARB2 complex at a resolution of 2 . 3 Å ( Table 1 ) . The structure of the complex and the binding mode of Myo3b-ARB2 to Espin1-AR are highly similar to what are observed in the Espin1-AR/Myo3b-ARB1 complex ( Figure 2D ) , directly confirming our earlier conclusion that the two repeat sequences in Myo3-THDI bind to Espin1-AR with similar binding mode and affinity . We tried very hard to crystallize the Espin1-AR/Myo3-THDI complexes without success , presumably due to flexibilities of the connection sequences between ARB1 and ARB2 of Myo3 . Since the two complex structures are essentially the same , here we only describe the detailed interactions observed in the Espin1-AR/Myo3b-ARB1 structure , which was resolved at a higher resolution . The Espin1-AR/Myo3b-ARB1 interface can be arbitrarily divided into three regions ( Figure 3A ) . The first binding site is formed by the repeats 2–4 of Espin1-AR and binds to the C-terminal α-helix of ARB1 ( Figure 3A1 ) . Two absolutely conserved tyrosine residues ( Tyr1267ARB1 and Tyr1268ARB1 , the double tyrosine ( ‘YY’ ) motif; Figure 3B ) and Leu1271ARB1 in the next turn insert into the hydrophobic pocket in the N-terminal of Espin1-AR ( Figure 3A1 ) . In addition , the hydroxyl groups of the ‘YY’ motif also make hydrogen bonds . Mutations of these two tyrosine residues to alanine greatly decreased Myo3b-ARB1’s binding to Espin1-AR ( Figure 3C , Figure 3—figure supplement 1B ) . Similarly , mutation of Leu110 in the Espin1-AR hydrophobic pocket to a polar residue aspartic acid decreased the affinity by ~10-fold ( Figure 3C , Figure 3—figure supplement 1C ) . Furthermore , the carboxyl group of Asp1264ARB1 makes hydrogen bonds with Asn69 and Ser103 . Mutation of this residue together with Glu1263ARB1 to alanines decreased the affinity by ~10-fold ( Figure 3C , Figure 3—figure supplement 1D ) . The second region is composed of Espin1 repeats 5–8 and binds to the middle-stretch of ARB1 with an extended conformation ( Figure 3A2 ) . Both the side chains and backbone carbonyl of Leu1259ARB1 are involved in the interaction . Asp205 located in the finger loop between repeat 6 and 7 forms a salt bridge with Lys1257ARB1 and a hydrogen bond with Gln1254ARB1 . Mutation of Lys1257ARB1 into a reversed charged residue glutamic acid , together with Leu1259ARB1 to alanine substitution also decreased the affinity by about ~10-fold ( Figure 3C , Figure 3—figure supplement 1E ) . The third region involves inner groove of the Espin1-AR repeats 8–10 , which is highly enriched with negatively charged residues ( Figure 3A3 ) . The two highly conserved Arg residues at the beginning of Myo3b-ARB1 ( Arg1251ARB1 and Arg1252ARB1 ) insert into the negatively charged pocket ( Figure 3A3 ) . Mutating these two arginine residues to reverse charged residues glutamic acid decreased Myo3B-ARB1 binding to Espin1-AR by ~15-fold ( Figure 3C , Figure 3—figure supplement 1F ) . By analyzing the sequence of the ARBs from Myo3 , we found that there exist more positively charged residues in addition to the highly conserved Arg residues at the further N-terminal end of ARB2 from both Myo3a and Myo3b ( Figure 3B ) . We anticipate that these additional positively charged residues might also be involved in the binding , as there remain unoccupied , negatively charged surfaces in the third region of the Espin1-AR/Myo3b-ARB1 structure ( Figure 3A3 ) . Indeed , substitutions of the more N-terminal positively charged residues of Myo3b-ARB2 ( Arg1282ARB2 and Lys1283ARB2 ) with alanines decreased its binding to Espin1-AR by ~10-fold ( Figure 3C , Figure 3—figure supplement 1I ) . 10 . 7554/eLife . 12856 . 010Figure 3 . The detailed Myo3/Espin1-AR interaction . ( A ) The Myo3b-ARB1/Espin1-AR interface is divided into three regions corresponding to the ‘YY’ motif ( A1 ) , the ‘KxL’ motif ( A2 ) and the N-terminal positively charged residues ( A3 ) of Myo3b-ARB1 . The residues tested with the mutagenesis experiments are highlighted with boxes . The side chains or main chains of the residues involved in the interactions are highlighted in the stick model . Charge-charge and hydrogen bonding interaction are highlighted by dashed lines . The electrostatic surface potentials were calculated using PyMol . ( B ) Sequence alignment of Myo3-ARBs showing the conservation of ARBs . The conserved residues involved in the binding are highlighted with solid green triangles . The variable residues involved in the binding are highlighted with solid blue triangles . The two positively charged residues in ARB2 that are not resolved in the structure are highlighted with unfilled triangles . The sequence logo beneath the alignment was generated using WebLogo ( Crooks et al . , 2004 ) . ( C ) ITC derived dissociation constants showing that mutations of the critical residues in the interface invariably weakened the binding . The original ITC data are shown in Figure 3—figure supplement 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 01010 . 7554/eLife . 12856 . 011Figure 3—figure supplement 1 . The ITC titration curves for calculating the dissociation constants shown in Figure 3C . Note that Panels A & G are the same as Figure 1C2 and C3 . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 01110 . 7554/eLife . 12856 . 012Figure 3—figure supplement 2 . Comparison of Myo3b-ARB2/Espin1-AR and Myo3b-ARB1/Espin1-AR structures . ( A ) An omit map showing the binding of Myo3b-ARB2 to Espin1-AR . The Fo-Fc density map was generated by deleting the Myo3b-ARB2 part from the final model and contoured at 2 . 5σ . The Myo3b-ARB2 fitting the electron density is displayed in the stick model . ( B and C ) The Myo3b-ARB2/Espin1-AR interface corresponding to the ‘YY’ motif ( B ) and the ‘KxL’ motif ( C ) . The side chains or main chains of the residues involved in the interactions are highlighted in the stick model . Charge-charge and hydrogen bonding interaction are highlighted by dashed lines . As a comparison , the structure of Myo3b-ARB1/Espin1-AR is transparent . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 012 Comparing the structures of Espin1-AR in complex with Myo3b-ARB1 and Myo3b-ARB2 , the ‘YY’ motif and the ‘KxL’ motif are essentially in the same places ( Figure 3—figure supplement 2B and C ) . Despite the high similarity , there are still a few minor differences . First of all , the C-terminal α-helix of ARB2 is shorter . The interaction is mediated by a hydrogen bond between Asp1294 and Tyr144 , instead of the more extensive interaction observed in Myo3b-ARB1 complex ( Figure 3—figure supplement 2B ) . Moreover , the positively charged residues in the N-terminus of ARB2 cannot be reliably built , probably due to the high salt concentration in the crystallization buffer ( 1 . 6 M ammonium sulfate ) . Nonetheless , clear electron density can be observed near the negatively charged surface ( Figure 3—figure supplement 2A ) . Indeed , substitutions of these positively charged residues with Ala weakened the binding ( Figure 3C and Figure 3—figure supplement 1G–I ) . Furthermore , the involvement of more positively charged residues of ARB2 may compensate for the less extensive interaction in its shorter C-terminal helix , thus resulting in a similar binding affinity to Espin1-AR as ARB1 does ( 107 nM for ARB1 vs 53 nM for ARB2 , Figure 1C2 and C3 ) . It was reported that a conserved region following the xAB segment of Espin1 can interact with the N-terminal AR ( Figure 4A ) and inhibit the actin binding activity of xAB ( Zheng et al . , 2014 ) . By comparing the sequence of AI ( aa 496–529 ) with the consensus sequence of Myo3-ARBs , we find that Espin1-AI bears high sequence homology with Myo3-ARBs ( e . g . the completely conserved ‘YY’ motif , the central ‘KxL’ motif , and the N-terminal positively charged residues; Figure 4B ) . Thus , we predict that AI may bind to Espin1-AR with a similar binding mode as Myo3-ARBs do . We used ITC-based binding assay to test this prediction , and found that Espin1-AI can indeed bind to Espin1-AR , albeit with a more moderate affinity than Myo3-ARBs ( Kd of 1 . 32 μM vs 0 . 05~0 . 1 μM ) ( Figure 4C1 ) . Fully consistent with our structure-based sequence alignment analysis , substitutions of the two tyrosines in Espin1-AI to alanines greatly weakened its binding to Espin1-AR ( Figure 4C2 ) . Given that the AI segment ( aa 496–529 ) is immediately C-terminal to xAB ( aa 462–487 ) of Espin1 ( Figure 4A ) , one might envision that the interaction between Espin1 AR and AI can conformationally mask the xAB’s actin binding activity and thus renders Espin1 in an auto-inhibited conformation . 10 . 7554/eLife . 12856 . 013Figure 4 . Biochemical characterization of the Espin1 auto-inhibition . ( A ) Domain organization of Espin1 showing that the Espin1-AI in the middle may bind to Espin1-AR at the N-terminus . ( B ) Sequence alignment of Espin1-AI from different vertebrate species , and comparison of Espin1-AI with the consensus sequence of Myo3-ARBs as shown in Figure 3D . ( C ) ITC result showing that Espin1-AI binds to Espin1-AR with a moderate affinity ( C1 ) . Mutation of the ‘YY’ motif to alanine greatly decrease the binding ( C2 ) . ( D ) ITC results showing that Myo3a-ARB1 can still bind to Espin1-1-529 ( D1 ) and Espin1-FL ( D2 ) with a sub-micromolar affinity . In contrast , Myo3a-ARB1 binds to Espin1-AR ( D3 ) and Espin1-1-494 with comparable strong affinities ( D4 ) . Panel D3 is the same as Figure 1—figure supplement 1B . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 01310 . 7554/eLife . 12856 . 014Figure 4—figure supplement 1 . FPLC-MALS results of Espin1-1-529 and Espin1-FL . The FPLC-SLS results showing that Espin1-1-529 ( left ) and Espin1-FL ( right ) each exists as a monomer in solution . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 014 It was shown that a synthetic peptide encompassing the Myo3a-ARB1 sequence identified here can stimulate the actin binding activity of xAB ( Zheng et al . , 2014 ) . Based on our analysis , the most likely mechanism for Myo3a/b-ARB1-mediated stimulation of xAB’s actin binding may be due to the release of AI binding from Espin1-AR by direct competition of Myo3a/b-ARB1 binding . We designed biochemistry experiments to support the above model . If Myo3-ARB1 can indeed compete with AI for binding to Espin1-AR , then Myo3-ARB1 must still be able to bind to the auto-inhibited Espin1 but with an affinity weaker than binding to the isolated Espin-1-AR . We obtained highly purified N-terminal auto-inhibitory fragment of Espin1 spanning from AR to AI ( denoted as Espin1-1-529 , Figure 4A ) and the full length Espin1 ( denoted as Espin1-FL ) , and found that both proteins exist as monomer in solution ( Figure 4—figure supplement 1 ) , indicating that Espin1 auto-inhibition is intra-molecular in nature . ITC-based assay further showed that Myo3a-ARB1 can indeed bind to both Espin1-1-529 and Espin1-FL and with a weaker binding affinity than binding to Espin1-AR ( Figure 4D1–3 ) , consistent with a partially blocked Espin1-AR binding groove by AI . We also noticed that the ITC titration reactions of Myo3a-ARB1 to Espin1-1-529 and Espin1-FL are endothermic ( Figure 4D1 and 4D2 ) instead of the exothermic reactions between Myo3a-ARB1 titrating to Espin1-AR ( Figure 4D3 ) , further indicating that the binding of Myo3a-ARB1 to the auto-inhibited Espin1 is not a simple direct association process between ARB1 and AR . To provide further proof , we truncated Espin1 from the C-terminus just before the AI ( i . e . , aa 1–494 , denoted as Espin1-1-494 ) and found that Espin1-1-494 binds to Myo3a-ARB1 with an affinity similar to that between Espin1-AR and Myo3a-ARB1 ( Figure 4D3 ) , indicating that AI is indeed responsible for the decreased binding of Espin1 to Myo3a-ARB1 . Myo3a is known to localize at the tip of filopodia when transfected in heterologous cells like HeLa or COS7 cells , whereas Myo3b alone cannot tip-localize as it lacks ABM ( Les Erickson et al . , 2003; Salles et al . , 2009 ) . However , when co-expressed with Espin1 , Myo3b can bind to Espin1 and localize to the tip of filopodia ( Manor et al . , 2012; Merritt et al . , 2012 ) . Similarly , Myo3a lacking ABM can only tip-localize when co-expressed with Espin1 . Deletion of the Myo3 kinase domain is known to render the motor in a constant active state in promoting the length of filopodia , thus we used Myo3 constructs lacking the kinase domain in the subsequent experiments ( Les Erickson et al . , 2003; Quintero et al . , 2010; 2013 ) . We first tested the role of Myo3/Espin1 binding on Myo3a’s ability to tip-localize . To test our biochemical findings and to determine the impact of Myo3-ARB ‘YY’ motifs on Myo3-Espin1 interaction , Espin1 transportation ( i . e . , tip localization ) and filopodia elongation , we co-expressed various Myo3aΔKΔABM ( lacking the kinase domain and the ABM ) and Myo3bΔK constructs with Espin1 in COS7 cells . Since both Myo3aΔKΔABM and Myo3bΔK cannot tip-localize by its own , we reasoned that when co-expressed with Espin1 , the Myo3 and Espin1 tip localization levels will determine the intactness of their mutual binding . As expected , both GFP-tagged wild type Myo3a ( lacking the kinase domain and the ABM , denoted as ΔKΔABM ) and RFP-tagged Espin1 localized to the tip of filopodia when co-transfected in COS7 cells ( Figure 5A1 ) . Mutation of the ‘YY’ motif of either of the ARBs ( denoted as mARB1 and mARB2 ) only had a moderate or even unobservable effect on filopodia tip localization for both Myo3a and Espin1 ( Figure 5B ) , indicating that ARB1 and ARB2 may play some redundant functions in this overexpression system . Mutations of both ‘YY’ motifs ( mARB1+mARB2 , denoted as mARB ) significantly reduced the filopodia tip localization of Myo3a ( Figure 5A2 and Figure 5B ) . Similarly , deletion of one ARB had a moderate effect and deletion of both ARBs had a much more severe effect on filopodia tip localization of Myo3a ( Figure 5A3 and Figure 5B ) . We have also tested the effect of the corresponding set of mutations or deletions of ARBs on Myo3b , and observed similar results ( Figure 5C and Figure 5—figure supplement 1 ) as those of Myo3a . It is worth noting that the ratio of tip to cell body protein level of Myo3b is significantly less than that of Myo3a ( Figure 5B and C ) , which is also consistent with previously reported findings ( Manor et al . , 2012; Merritt et al . , 2012 ) , but the detailed mechanism for this difference is unknown . The above results demonstrate that both ARBs in Myo3a or Myo3b are important for filopodia tip localizations of Myo3 and Espin1 . 10 . 7554/eLife . 12856 . 015Figure 5 . Myo3-ARBs/Espin1 interaction is critical for the filopodia tip localizations of Espin1 and Myo3 . ( A ) Representative fluorescence images of COS7 cells co-expressing RFP-Espin1 and various GFP-Myo3a experimental constructs . A1 , Myo3aΔKΔABM WT; A2 , Myo3aΔKΔABM mARB; A3 , Myo3aΔKΔABM dARB . Scale bar: 5 µm . ( B ) Quantifications of the tip to cell body ratios of GFP-Myo3a ( or its mutants ) and RFP-Espin1 based on the experiments shown in panel A . ( C ) Quantifications of the tip to cell body ratios of GFP-Myo3b ( or its mutants ) and RFP-Espin1 when expressed in COS7 cells . The representative images for this group of experiments are shown in Figure 5—figure supplement 1 . Values are means ± SEM and analyzed with Two-tailed Student’s t test; *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 01510 . 7554/eLife . 12856 . 016Figure 5—figure supplement 1 . ARBs are required for both Myo3b and Espin1 filopodia tip localization . Representative COS7 cells images of RFP-Espin1 co-transfected with different constructs of GFP-Myo3b . Row 1 , Myo3bΔKΔABM WT; Row 2 , Myo3bΔKΔABM mARB; Row 3 , Myo3bΔKΔABM dARB . Scale bar: 5 µm . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 016 Our above structural and biochemical characterizations of the Myo3 and Espin1 interactions point to a likely regulatory role of Myo3 on Espin1’s actin binding and bundling activity . It can be deduced that binding of Myo3-ARBs can first release the auto-inhibited conformation of Espin1 . Perhaps more importantly , formation of Myo3/Espin1 complex leads to two Espin1 molcules to be juxtaposed to each other , forming a Myo3 cross-linked Espin1 dimer . Due to the large space between the N-terminal AR and C-terminal ABD in Espin1 ( Figure 1A ) , this Myo3-mediated cross-linking positions the two copies of Espin1 ABD at a distance considerably larger than those allowed by other known actin cross-linking proteins . Therefore , we predicted that binding of Myo3 may stimulate higher order actin bundling activity of Espin1 . We used both fluorescent microscopy ( FM ) and transmission electron microscopy ( TEM ) techniques to exam the Espin1-mediated F-actin bundles with and without the binding of Myo3 ARBs ( Figure 6 ) . Under FM , the F-actin alone showed only background signal when probed by fluorescence-labeled phalloidin since individual F-actin is too small to be resolved ( Figure 6B , left ) . This is consistent with a previous report ( Zheng et al . , 2014 ) , and also directly revealed by our TEM study showing the nm sized F-actin filaments ( Figure 6B , right ) . When Espin1 was added into the F-actin solution , uniform needle like bundles could be observed under FM . TEM showed that the bundle size is ~190 ± 8 nm in width ( mean ± SEM ) ( Figure 6A1 and C , Figure 6—figure supplement 1A ) . According to the auto-inhibited model , these actin bundles are probably induced by the C-terminal ABD of Espin1 . By adding ARB1 or ARB2 to Espin1 and F-actin containing solution , some of the actin bundles were cross-linked and formed clusters under FM and TEM ( Figure 6A2 and 3 ) . Similar phenomena have also been observed earlier using Myo3/Espin1/actin co-polymerization bundling assay ( Zheng et al . , 2014 ) , instead of the post-polymerization bundling assay employed in this study . From the TEM images , the diameter of the cluster is slightly larger than Espin1 only ( Figure 6A2 and A3 , Figure 6—figure supplement 1B and C ) . Quantification of the F-actin bundle width from the TEM images revealed that the addition of ARBs slightly increased the width of the actin bundles , with 216 ± 9 nm for the ARB1 group and 218 ± 9 nm for the ARB2 group ( Figure 6C ) . The above data suggest that the cluster is formed by the Espin1-ABD mediated actin bundles and the ARB1/ARB2 binding releases the xAB , providing an additional actin binding site for forming slightly wider and more branched actin bundles . Interestingly , when ARB12 were added to Espin1 and F-actin containing solution , significantly thicker , elongated , and less branched bundles were observed under FM ( Figure 6A4 , left ) , a morphology clearly distinct from that with the addition of either ARB1 alone or ARB2 alone . When examined under TEM , the thicker bundles appear to be composed of several thinner actin bundles as observed in the Espin1-ABD-promoted actin bundles ( Figure 6A4 , right and Figure 6—figure supplement 1D ) , indicating that Myo3-ARB12-mediated binding of Espin1 can promote/stabilize parallel actin fiber formation presumably due to Myo3 mediated cross-linking of Espin1 . Quantification of the width of actin bundles formed in the presence the Myo3-ARB12 and Espin1 showed that the average width of the actin bundles are significantly larger ( 330 ± 20; with the thickest bundles reaching ~600 nm in width ) than the other three groups ( Figure 6C ) . As a control , Myo3a-ARB12 alone could not cause bundling of F-actin ( Figure 6—figure supplement 1E ) . Based on these results , we propose a model that , first the Espin1-ABD is able to bundle F-actins , forming the thin F-actin bundles; then the two Espin1 binding sites located at the tail of Myo3 not only release the auto-inhibition of but also cross-link Espin1 , further assembling the thin actin-bundles into thicker parallel actin bundle fibers ( Figure 6D ) . Therefore , one can envision that a key function of the Myo3/Espin1 interaction is to promote formation of thicker parallel actin bundle fibers in cellular structures such as stereocilia . 10 . 7554/eLife . 12856 . 017Figure 6 . Myo3 binding promotes Espin1’s higher order actin bundling activity . ( A ) Representative images of actin bundles induced by Espin1 with and without the presence of various forms of Myo3-ARBs under fluorescent microscopy ( left ) or transmission electron microscopy ( right ) . A1: F-actin+Espin1 with a 4:1 molar ratio mixing; A2: F-actin+Espin1+ARB1 with a 4:1:1 molar ratio mixing; A3: F-actin+Espin1 +ARB2 with a 4:1:1 molar ratio mixing; A4: F-actin+Espin1+ARB12 with a 4:1:0 . 5 molar ratio mixing . The scale of each column is the same and is indicated at the top panel . ( B ) Representative images of F-actin only under fluorescent microscopy ( left ) and transmission electron microscopy ( right ) . ( C ) Distribution of the width of actin bundles from the different groups of experiments . Black: F-actin+Espin1; red: F-actin+Espin1+ARB1; green: F-actin+Espin1+ARB2; blue: F-actin+Espin1+ARB12 . Statistics are performed by box plot as well as Two-tailed Student’s t test . *p<0 . 05 , **p<0 . 01 , ***p<0 . 001 . ( D ) Cartoon diagram showing Myo3 mediated higher order actin bundling by Espin1 . Without Myo3 , the thin actin bundles were induced by Espin1-ABD ( left ) . With Myo3 , the two ARBs can bring two Espin1 together , facilitating the formation of higher order actin bundles ( right ) . The xAB region of this Myo3-activated Espin1 and the ABM of Myo3a may further stabilize the higher order actin bundles . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 01710 . 7554/eLife . 12856 . 018Figure 6—figure supplement 1 . Representative TEM images of actin bundles . ( A–D ) Representative images of actin bundles induced by Espin1 with and without the presence of various forms of Myo3-ARBs under transmission electron microscopy . A: F-actin+Espin1 with a 4:1 molar ratio mixing; B: F-actin+Espin1+ARB1 with a 4:1:1 molar ratio mixing; A3: F-actin+Espin1+ARB2 with a 4:1:1 molar ratio mixing; C: F-actin+Espin1+ARB12 with a 4:1:0 . 5 molar ratio mixing . The scale of each column is the same and is indicated at the left panel . ( E ) Representative images of actin bundles induced by ARB12 only with a 4:0 . 5 molar ratio mixing under transmission electron microscopy at different magnifications . Scale bar: 0 . 5 µm for the top panel and 100 nm for the bottom panel . DOI: http://dx . doi . org/10 . 7554/eLife . 12856 . 018
The most important findings of this study are the structure-based discovery of two strong and independent Espin1 binding sites in the tail region of each class III myosins , and Myo3 binding-induced auto-inhibition release of Espin1 . These discoveries , together with our cell-based filopodia formation and localization assay and microscopic-based actin bundling assay , provide compelling evidences showing that Myo3 and Espin1 work together to assemble and promote higher order parallel actin bundle formation in cellular processes such as stereocilia . We believe that our model reveals the probable underlying molecular mechanism of hearing loss development ( stereocilia degeneration ) in humans with mutations in Myo3a ( DFNB30 ) ( Walsh et al . , 2002 ) . It is believed that the delayed hearing loss phenotype in DFNB30 subjects could be due to Myo3b compensatory mechanism ( Manor et al . , 2012 ) , a hypothesis supported by our results as well . With the help of the motor domain-mediated high affinity binding of Myo3 to actin filaments ( Dose et al . , 2007; Kambara et al . , 2006 ) , the full-length Myo3/Espin1 complex may have even higher actin fiber bundling activity than the Myo3-ABR12/Espin1 complex studied in this work . As such , our study reveals a previously unrecognized direct myosin binding-induced actin bundling activity regulation mechanism of an actin filament cross-linker protein . It also demonstrates an intimate synergistic action mechanism between two classes of actin binding proteins , namely actin filament-based myosin motors and actin filament cross-linking protein , in controlling actin fiber size and stability . It has been reported that another unconventional myosin in stereocilia , Myosin XVa , can form a complex with an actin capping protein Eps8 to regulate stereocilia elongations ( Manor et al . , 2011 ) , suggesting that working together with actin binding proteins to regulate actin skeletal dynamics might be a new mode of function for unconventional myosins . Fitting with this model , stereocilia in jerker mice are thinner in their diameters likely due to loss of Espin1-mediated higher order actin fiber assembly , easier to form tapered structures and easier to degenerate presumably due to stability decrease of less bundled actin fibers in stereocilia ( Sekerkova et al . , 2011; Zheng et al . , 2000 ) . As one of the three known actin cross-linkers in stereocilia , the abundance of Espin is relatively low compared to fascin and plastin ( Shin et al . , 2013 ) . It has been proposed that Espin may play a regulating role in elongation and widening of actin bundle by concentrating at the tip of stereocilia instead of major structural cross-linking roles ( Avenarius et al . , 2014; Loomis et al . , 2003 ) . Interestingly , the abundance of Myo3 ( both Myo3a and Myo3b together ) is similar to that of Espin ( a few hundred copies per stereocilium ) ( Shin et al . , 2013 ) , indicating that Myo3 and Espin1 may work together to regulate higher order actin bundle structure formation and stability in stereocilia . Both Myo3 and Espin1 are known to concentrate at the tips of stereocilia in hair cells and at the tips of filopodia when expressed in heterologous cells ( Merritt et al . , 2012; Salles et al . , 2009; Schneider et al . , 2006 ) . It is possible that stabilization of the growing end of the actin filaments at the plus ends by both Myo3 and Espin1 is a critical driving force for actin filament elongations ( Avenarius et al . , 2014; Loomis et al . , 2003; Shin et al . , 2013 ) . The Myo3 binding-mediated cross-linking of Espin1 cargo is in sharp contrast to the cargo binding-mediated motor dimerization and processivity induction known in a number of other unconventional myosins ( myosin V , VI and VII ) ( Lu et al . , 2014; Sakai et al . , 2011; Shi et al . , 2014; Yu et al . , 2009 ) . Such large differences in their cargo bindings probably match with the distinct functional properties of these myosins . For a fast-moving cargo transporting motor such as myosin VI , it is important that the motor assumes as a dimer and gets activated for moving in one direction upon binding to a cognate cargo . In such a case , the binding of a cargo protein to the motor tail exerts large impact on the motor activity regulations . As a high duty ratio motor , processive and rapid movement along the actin filaments is not likely to be the main function of Myo3 , although the motor can still move towards the plus end of actin filaments ( Les Erickson et al . , 2003; Merritt et al . , 2012; Salles et al . , 2009; Schneider et al . , 2006 ) . Instead , one of Myo3’s key functions appears to coordinate with its cargo protein Espin1 to regulate Espin1-mediated actin filaments assembly and stability . Whether such myosin binding-mediated cargo activity regulation is also adopted by other unconventional myosins , particularly for those not known to play transporting roles , is an interesting research topic in the future . We provide detailed structural information regarding Myo3b/Espin1 interaction . We demonstrate that the Espin1 binding sites in Myo3a and Myo3b are essentially identical and therefore are predicted to be functionally interchangeable . These findings provide a molecular explanation for the partially redundant functions of Myo3a and Myo3b in hair cells . This analysis also predicts that Espin-like proteins can also bind to Myo3 as Espin1 does , suggesting that the functions of Espin1 and Espin-like protein may also be partially redundant in tissues like stereocilia ( Shin et al . , 2013 ) . Such redundancies suggest that mutation of single myo3a or myo3b or defects in espin1 or espin-like may not always develop severe phenotypes in vertebrates . Moreover , we note that forked , the Drosophila ortholog of Espin , is expressed in bristle cells , a cell type that is enriched in bundled actin filaments ( Petersen et al . , 1994 ) . Drosophila Forked protein is predicted to contain five ANK repeats , corresponding to Espin1 repeats 5–9 . The structures solved here of mammalian Espin1-AR should help for identifying potential binding partners of Forked-AR . Although Myo3a and Myo3b are highly similar , there are clear differences in terms of filopodia tip localizations ( Manor et al . , 2012; Merritt et al . , 2012 ) . Myo3a localize to the very tip of filopodia , while Myo3b distributes in a wider tip-base gradient ( Merritt et al . , 2012 ) . The different tip localization patterns of Myo3a and Myo3b does not originate from the unique ABM of Myo3a ( Manor et al . , 2012 ) . Our study here suggests that their Espin1 binding THDI regions are not responsible for different localizations of Myo3a and Myo3b along stereocilia/filopodia either . It is possible that the kinetics and the F-actin binding affinities of their motor domains or other variable regions in their tails may contribute to such differences . Most of the unconventional myosins use their globular cargo binding domains to recognize their cargoes ( Lu et al . , 2014 ) . However , Myo3 use their unstructured , yet highly conserved tails to recognize globular domains from their cargoes . Myo3 and another unconventional myosins , Myosin XIX , are not predicted to contain folded globular domains in their tail regions ( Lu et al . , 2014 ) . Based on the results in this study , we anticipate that these two myosins might use different unstructured fragments in their tails to specifically recognize various cargoes . A very recent report showed that another region in Myo3a’s tail , which is located immediately N-terminal to the Espin1 binding ARB12 region , can bind to MORN4 ( Mecklenburg et al . , 2015 ) , the mammalian ortholog of Drosophila Retinophilin . It is noted that Retinophilin can also bind to Drosophila NinaC ( Venkatachalam et al . , 2010 ) . Future studies are required to elucidate the molecular basis governing the Myo3/MORN4 and NinaC/Retinophilin interactions for better understanding of the interactions .
The coding sequences of Myo3b-ARB12 ( Accession Number: NP_796350 . 2 , aa 1234–1333 ) and Espin1-AR ( Accession Number: NP_997570 . 1 , aa 1–352 ) were PCR amplified from mouse cDNA library . The full-length human Myo3a and Espin1 plasmids have been described earlier ( Merritt et al . , 2012; Salles et al . , 2009 ) . The mouse Myo3b-ARB1 ( aa 1234–1279 ) , Myo3b-ARB2 ( aa 1280–1333 ) , Espin1-AR ( aa 1–352 ) , human Myo3a-ARB1 ( aa 1488–1520 ) , Myo3a-ARB2 ( aa 1521–1553 ) , Espin1-AR ( aa 1–352 ) , Espin1-AI ( aa 496–529 ) , Espin1-1-494 , Espin1-1-529 and Espin1-FL were cloned into an in-house modified pET32a vector ( Liu et al . , 2011 ) . The mouse Myo3b-ARB12 and human Myo3a-ARB12 ( aa 1488–1553 ) were cloned into a pETM . 3C vector . All truncations and point mutations of Myo3 and Espin1 used in the current study were created with the standard PCR-based mutagenesis method and confirmed by DNA sequencing . For heterologous cell expressions , the full-length human Myo3a and deletions or mutations were cloned into a modified EGFP vector and the full-length human Espin1 was cloned into a modified RFP vector . All proteins were expressed in Escherichia coli BL21 ( DE3 ) except for Myo3a-ARB12 and Myo3a-ARB2 which were expressed in Escherichia coli Rosetta ( DE3 ) . The N-terminal thioredoxin-His6-tagged or His6-tagged proteins were purified with a Ni Sepharose 6 Fast Flow column and subsequent Superdex-200 prep grade size-exclusion chromatography . Protein samples ( typically 100 µl at a concentration of 50 µM pre-equilibrated with column buffer ) was injected into an AKTA FPLC system with a Superose-12 10/300 GL column ( GE Healthcare ) using the column buffer of 50 mM Tris-HCl ( pH 7 . 8 ) , 1 mM DTT , 1 mM EDTA , and 100 mM NaCl . The chromatography system was coupled to a multi-angle light scattering system equipped with a 18 angles static light scattering detector ( Dawn , Wyatt ) and a differential refractive index detector ( Optilab , Wyatt ) . The elution profiles were analyzed using the ASTRA 6 software ( Wyatt ) . Crystals of the Espin1-AR/Myo3b-ARB1 complex and Espin1-AR/Myo3b-ARB2 complex ( both in 50 mM Tris-HCl , pH 7 . 8 , 100 mM NaCl , 1 mM EDTA , 1 mM DTT buffer ) were obtained by sitting drop vapor diffusion methods at 16°C . The crystals of the Espin1-AR/Myo3b-ARB1 complex were grown in buffer containing 0 . 2 M lithium acetate and 20% w/v PEG3350 and soaked in crystallization solution containing additional 25% glycerol for cryoprotection . The crystals of Espin1-AR/Myo3b-ARB2 complex were grown in buffer containing 1 . 6 M ammonium sulfate , 0 . 1 M Tris pH 8 . 0 and soaked in crystallization solution containing higher concentration of ammonium sulfate for cryoprotection . Diffraction data were collected at the Shanghai Synchrotron Radiation Facility BL17U at 100 K . Data were processed and scaled using HKL2000 ( Otwinowski and Minor , 1997 ) . Structure of the Espin1-AR/Myo3b-ARB1 complex was solved by molecular replacement with the model of short ANK repeats ( 1N0R ) using PHASER ( Mccoy et al . , 2007 ) . Structure of the Espin1-AR/Myo3b-ARB2 complex was also solved by molecular replacement with the 10 ANK repeats of Espin1 in the previous structure as the search model . Phases were greatly improved after auto-building by Buccaneer ( Cowtan , 2006 ) . Further manual model building and refinement were completed iteratively using COOT ( Emsley et al . , 2010 ) and PHENIX ( Adams et al . , 2010 ) . The final model was validated by MolProbity ( Chen et al . , 2010 ) . The final refinement statistics are summarized in Table 1 . All structure figures were prepared by PyMOL ( http://www . pymol . org ) . The coordinates of the structures reported in this work have been deposited to PDB under the access codes of 5ET1 and 5ET0 for the Espin1-AR/Myo3b-ARB1 and Espin1-AR/Myo3b-ARB2 structures , respectively . Isothermal titration calorimetry ( ITC ) measurements were carried out on a MicroCal iTC200 at 25°C , except for the two endothermic titrations which were performed at 16°C . Titration buffer contained 50 mM Tris-HCl pH 7 . 8 , 1 mM DTT , 1 mM EDTA and 200 mM NaCl . Each titration point was performed by injecting a 2 μL aliquot of a protein sample from a syringe into a protein sample in the cell at a time interval of 120 s to ensure that the titration peak returned to the baseline . The titration data were analyzed by Origin7 . 0 ( Microcal ) . COS7 cells were cultured in Dulbecco's Modified Eagle Medium ( Corning ) supplemented with 1 mM Sodium Pyruvate , 4 mM L-glutamine , 4 . 5 g/L D-Glucose , 10% fetal bovine serum ( FBS ) ( Gemini ) , and 100 units of penicillin-streptomycin ( Corning ) . Cultured COS7 cells were maintained at 37°C with 5% CO2 in air . For transfections , 30–40 × 103 cells were plated on acid washed 22 mm square 1 . 5# glass coverslips and allowed to adhere over-night . 24 hr later , cells were transiently transfected using FUGENE HD transfection reagent ( Promega ) as per manufacturer’s protocol and imaged after ~20–30 hr . For live cell imaging , the coverslips with transfected cells were placed in rose chambers filled with Opti-MEM media without phenol red ( Life Technologies ) and supplemented with 5% FBS ( Gemini ) and 100 units of Penicillin-streptomycin . Images were acquired by using a TE2000-PFS fluorescence microscope ( Nikon Instruments ) with a 60x/1 . 4 N . A . phase objective . Image acquisition was managed by NIS-Elements AR ( Nikon Instruments ) and the tip to call body measurements were done using ImageJ ( ND2 plugin ) , as described previously ( Quintero et al . , 2010 ) . Data are expressed as mean ± SEM . The mutant groups were compared with the wild type groups by two-tailed student’s t test . Rabbit skeletal muscle actin ( Cytoskeleton ) were hydrated in 5 mM Tris-HCl , 0 . 2 mM CaCl2 , 0 . 2 mM ATP , 0 . 5 mM DTT , pH 8 . 0 , on ice for 1 hr and centrifuged at 150 , 000 g for 10 min at 4°C . Actin concentration in supernatant was determined by NanoDrop . Actin was polymerized at room temperature for 1 hr after adding one-tenth volume of 10×polymerizing buffer ( 500 mM KCl , 20 mM MgCl2 , 10 mM ATP ) . Bundles were prepared by mixing 5 µM F-actin with 1 . 25 µM Espin1 and incubating at room temperature for 1 hr , with or without 1 . 25 µM Myo3a-ARB1/2 or 0 . 625 µM Myo3a-ARB12 added in the mixtures . For fluorescence microscopy , F-actin were labeled with Alexa Fluor 555 Phalloidin . Aliquots ( 5 µl ) were delivered onto microscope slides . The cover slips were then placed over the drop of samples gently . All the samples were imaged using a Fixed Stage Upright Microscope ( Olympus ) . Samples for TEM ( FEI Tecnai 20 ) were adsorbed to glow-discharged , carbon-coated formvar films on copper grids for 1 min and negatively stained with 0 . 75% ( m/v ) uranium formate for 30 s .
Class III myosins ( Myo3 ) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells , and their defects cause hereditary hearing impairments . Myo3 interacts with Espin1 through its tail homology I motif ( THDI ) , however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization . Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1 , revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1 . The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding , but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism . We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1 .
A mammal’s sense of hearing and balance are helped by so-called hair cells within the inner ear . These cells are named after their long , hair-like protrusions called stereocilia , and mutations in the genes involved in stereocilia development and maintenance can lead to hearing loss in humans . Damage to the stereocilia caused by excessive exposure to loud noises can also have the same effect . Stereocilia are full of filaments made of a protein called actin . Other proteins called class III myosins and Espin are also both required for normal development of stereocilia . Mutations in the genes that encode these proteins can cause hereditary deafness in humans . However , it remains unclear exactly how these two proteins ( myosins and Espin ) interact with each other in stereocilia . Using biochemical and structural studies , Liu , Li et al . have now discovered that the so-called ‘tail’ part of the myosins contains a pair of repeated sequences that can each interact with an Espin protein called Espin1 . This interaction allows each myosin to cross-link two Espin1 proteins . Espins assemble actin filaments into bundles , and further experiments showed that this cross-linking interaction between myosins and Espins helped this process , which is linked to stereocilia development and maintenance . Mammals actually have two related copies of class III myosins that play overlapping but slightly different roles . The next challenge will be to try to understand the differences between these related proteins , as well as to try to uncover the roles of other forms of Espin in stereocilia .