Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation
Cellular machinery has evolved to prevent or reverse protein misfolding and aggregation , which are damaging to cells and tissues . Bacterial ClpB and its yeast counterpart Hsp104 are members of the Clp/Hsp100 family and function in disaggregation and refolding of protein aggregates together with Hsp70 and its co-chaperones ( Glover and Lindquist , 1998; Goloubinoff et al . , 1999; Motohashi et al . , 1999; Zolkiewski , 1999; Doyle and Wickner , 2009; Haslberger et al . , 2010 ) . ClpB/Hsp104 are conserved in bacteria , fungi , plants and mitochondria and are essential for recovery of cells from heat shock and other proteotoxic stresses ( Parsell et al . , 1994; Mogk et al . , 1999 ) . The oligomeric ring Hsp100 proteins thread substrates through a central channel , via binding to conserved tyrosine residues on flexible loops ( Weber-Ban et al . , 1999; Kim et al . , 2000; Lum et al . , 2004; Weibezahn et al . , 2004; Haslberger et al . , 2008; Tessarz et al . , 2008 ) . They belong to the AAA+ ( ATPases associated with various cellular activities ) superfamily of ATPases , with characteristic α and β subdomains ( Ogura and Wilkinson , 2001; Erzberger and Berger , 2006 ) . ATP binds between the two subdomains and at the subunit interface of adjacent monomers , with a catalytic Arg-finger provided by the neighbouring subunit . While some other members of the Hsp100 family have been crystallised in their oligomeric form ( Bochtler et al . , 2000; Glynn et al . , 2009; Wang et al . , 2011 ) , the atomic structure of ClpB is known only for the monomer ( T . thermophilus ) . ClpB is composed of a N-terminal domain ( ND ) followed by two AAA+ domains ( AAA-1 and AAA-2 ) . An 85 Å long coil-coiled propeller , the middle domain ( MD ) , is inserted into the small subdomain of AAA-1 ( Lee et al . , 2003 ) . It has two blades with mutationally sensitive sites at either end , termed motif 1 and motif 2 . Cryo-EM reconstructions of TthClpB hexamers show a two-tiered molecule accounting for the AAA+ rings , but lack density for the ND . On the other hand , various cryo-EM studies of Hsp104 revealed the presence of an ND ring on top of the AAA+ ones . Although the overall shape and dimensions of the ClpB and Hsp104 hexamers are comparable in the various cryo-EM studies , there are substantial differences in the channel width ( Lee et al . , 2003; Wendler et al . , 2007 , 2009; Lee et al . , 2010 ) . Observation of narrow ( Lee et al . , 2003 , 2010 ) ( 15–30 Å ) vs expanded ( Wendler et al . , 2007 , 2009 ) ( 30–80 Å ) cavities has led to different pseudo-atomic models of the hexamers . In one model , the AAA+ rings are compact , as in the crystal structures of other AAA+ hexamers such as p97 , HslU or SV40 LTag , with the Arg-finger contacting the neighbouring subunit ( Bochtler et al . , 2000; Huyton et al . , 2003; Gai et al . , 2004 ) . In the EM maps used to build this compact model there is little or no density for the MD and it was assumed to extend radially outwards from the ring ( Lee et al . , 2003 , 2010 ) . In the expanded model , the AAA+ domains are more widely separated with the MD intercalated between them , preventing the canonical Arg-finger contacts ( Wendler et al . , 2007 , 2009 ) . An attempt to localize the Hsp104 MD by genetically inserting a lysozyme resulted in a cryo-EM reconstruction of this chimera with visible density for the lysozyme , but not for the MD itself ( Lee et al . , 2010 ) . Since none of the existing cryo-EM structures allows an unambiguous localization of the MD , its position is still a matter of debate ( Desantis and Shorter , 2012 ) . The MD confers unique disaggregase ability to ClpB/Hsp104 ( Kedzierska et al . , 2003; Mogk et al . , 2003 ) and is required for species-specific cooperation with the DnaK-DnaJ ( DnaKJ ) /Hsp70-Hsp40 system ( Sielaff and Tsai , 2010; Miot et al . , 2011; Seyffer et al . , 2012 ) . A direct interaction between the ClpB MD and DnaK has been shown by NMR spectroscopy and site-specific crosslinking and involves the motif 2 tip of the MD and the ATPase domain of DnaK ( Seyffer et al . , 2012; Rosenzweig et al . , 2013 ) . The MD acts to repress ClpB disaggregase activity , and DnaK binding relieves this repression ( Oguchi et al . , 2012; Seyffer et al . , 2012 ) . Point mutations in the MD show that interactions between motif 2 and AAA-1 are critical for regulating ATPase and disaggregase activities ( Haslberger et al . , 2007; Oguchi et al . , 2012; Seyffer et al . , 2012; Lipinska et al . , 2013 ) . Thus , the MD plays an essential role in coupling Hsp70 interaction to ATPase regulation and substrate disaggregation in ClpB/Hsp104 . It is therefore important to understand its structural role in ClpB/Hsp104 hexamers and in the context of the ClpB-DnaKJ bi-chaperone machinery . To address the ambiguities in domain arrangement and to elucidate the working principle of the MD , we performed single particle EM studies of ClpB under conditions where its orientation could be determined more reliably than in previous studies . We used BAP ( ClpB with the ClpA tripeptide for ClpP binding ) , a chimera engineered to bind the ClpP protease via the replacement of a C-terminal ClpB segment with the ClpP binding region of ClpA ( Weibezahn et al . , 2004 ) . This construct has been extensively used to study the ClpB disaggregation mechanism by monitoring substrate proteolysis after delivery to ClpP ( Weibezahn et al . , 2004; Haslberger et al . , 2008; Tessarz et al . , 2008; Mizuno et al . , 2012; Rosenzweig et al . , 2013; Figure 1A ) . Therefore , BAP is suitable for structural studies and has allowed us to obtain maps with visible MD densities that shed light on its regulatory mechanisms . 10 . 7554/eLife . 02481 . 003Figure 1 . 3D structure of the symmetrised BAP-ClpP complex . ( A ) Schematic of the complex showing the threading of a polypeptide . ClpB domains are in yellow , N-terminus; red/orange , AAA-1 domain; green , MD and blue/cyan , AAA-2 domain . ClpP is in grey . ( B ) Negative stain EM image ( left panel ) , class averages of 2:2 BAP-ClpP ( upper right panel ) and cut out 1:1 BAP-ClpP particles ( bottom right panel ) . Scale bars are 150 Å . ( C ) BAP-ClpP structure with a fitted ClpB monomer ( left ) and central slice with fitted atomic coordinates ( right ) . ( D ) AAA-1 and AAA-2 layers . ( E ) Two adjacent AAA-1 domains from the hexameric fit viewed in the plane of the ring . One monomer is in colour and the other in grey . Residues involved in engineered disulphide bonds are shown as spheres . Residues involved in intermolecular cross-links are labeled . The separation between cross-linking Cα pairs ranges from 8 to 14 Å . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 00310 . 7554/eLife . 02481 . 004Figure 1—figure supplement 1 . Complex formation between BAP and ClpP does not change MD protection pattern in HX experiments . ( A ) BAP and ClpP form a stable complex during HX experiments causing stabilization of BAP . Protein samples preincubated with ATPγS ( BAP , BAP-ClpP ) were diluted 20-fold into D2O buffer and incubated for 60 s before quenching the proton-deuteron exchange reaction . The mass of full length BAP after deuteron incorporation was determined . Complex formation with ClpP protects 30 amide protons in BAP from exchange . ( B ) ClpB , BAP and BAP-ClpP were incubated with ATPγS and HX values of peptic peptides were determined after 60 s . Peptide 746–754 ( * , KKIFRPEFI ) includes the ClpP-binding loop of ClpA and is only present in BAP . Relative ( % ) deuterium incorporation levels are given for each peptide . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 00410 . 7554/eLife . 02481 . 005Figure 1—figure supplement 2 . The BAP N-terminus is highly mobile . ( A ) Class averages of BAP-ClpP . The arrows indicate the N-terminal ( yellow ) , AAA-1 ( red ) , AAA-2 ( blue ) and ClpP ( black ) tiers . ( B ) Eigen images showing that high variance is localized in the N-terminal ring . Scale bars are 50 Å . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 00510 . 7554/eLife . 02481 . 006Figure 1—figure supplement 3 . Fourier Shell Correlation plots to estimate EM map resolution . For each sample , FSC curves of C6 and C1 reconstructions are plotted together . The C1 curves show lower resolution in each pair . Example class averages are shown in the left column with corresponding re-projections on the right . Scale bar is 50 Å . FSC plots were deposited in the 3D-EM database EM ( www . emdatabank . org ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 00610 . 7554/eLife . 02481 . 007Figure 1—figure supplement 4 . Crystal structure of E . coli ClpB and EM based model . ( A ) Superimposed chains of E . coli ClpB and T . thermophilus crystal structures . For clarity , the N-terminus is not shown . ( B ) Comparison between the ClpB coordinates fitted into the EM maps ( in colours ) and the ClpB crystal structure ( grey ) . In the EM model the MD is more tilted than in the crystal structures . AAA-2 is rotated by ∼25° , and the N-terminus is rotated by 45° in the plane of the ring . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 007
Using the BAP complex facilitates orientation determination in EM reconstruction , as previously observed for ClpA-ClpP ( Effantin et al . , 2010 ) . Side views of the elongated BAP-ClpP complex are easily recognisable ( Figure 1B ) whereas in 2D projections of ClpB alone , which has a globular shape , it is hard to distinguish side from tilted views . By restricting the dataset to side views the angle assignment is more reliable , and these views are sufficient to generate the full 3D structure ( Figure 1C ) . Using H/D exchange experiments , which report on the solvent accessibility and structural flexibility of amide hydrogens , we found that BAP , either alone or bound to ClpP , displays the same protection pattern as ClpB , implying the same MD conformation ( Figure 1—figure supplement 1 ) . The BAP-ClpP complexes formed by mixing BAP and ClpP at a 1:1 molar ratio of hexamer to heptamer in the presence of ATPγS were stained with uranyl acetate . Complexes containing two BAP hexamers per ClpP double-heptamer were picked for single particle analysis and separated into halves for processing ( Effantin et al . , 2010; Figure 1B ) . Initial analysis revealed the presence of four layers corresponding to the ND , AAA-1 , AAA-2 and ClpP rings . Class averages and eigenimage analysis indicate that the ND is extremely mobile in the BAP hexamer ( Figure 1—figure supplement 2 ) , consistent with crystallographic data on monomeric T . thermophilus ClpB ( Lee et al . , 2003 ) . The ND layer was therefore excluded during later stages of image alignment . Similarly , the region corresponding to ClpP , very useful for the initial analysis , was not included in the refinement because of its symmetry mismatch with ClpB . Using ∼12 , 000 particles , we obtained a 3D map by refining the alignment of the two AAA+ rings only , but then including the whole molecule in the reconstruction , which was at ∼17 Å resolution ( Figure 1C , D , Figure 1—figure supplement 3 ) . In order to simplify the problems of alignment and reconstruction , we initially imposed sixfold symmetry , which blurs the features of the heptameric ClpP ring . Similarly , the mobile ClpB ND is blurred into a solid disc . The BAP hexamer has overall outer dimensions of ∼150 × 100 Å , similar to previous structures of ClpB/Hsp104 ( Parsell et al . , 1994; Lee et al . , 2003; Wendler et al . , 2007 , 2009; Figure 1C ) . It encloses a ∼30 Å wide central channel , comparable in size to that in the crystal structure of ClpC ( Wang et al . , 2011; Figure 1C , D ) . In the reconstruction it is possible to identify regions accounting for all the domains , such as L-shaped densities for the AAA+ domains and a rod-like density for the coiled-coil MD . To interpret the domain interactions , we fitted ClpB atomic coordinates into the EM density . We determined the crystal structure of an ND truncation of E . coli ClpB ( residues 159 to 858; E279A/E432A/E678A mutation; Table 1 ) . The subunit structure is very similar to that of T . thermophilus ClpB ( Figure 1—figure supplement 4 ) . Since none of the available crystal structure conformations fit in the EM map , domains were fitted as separate rigid bodies connected at hinge regions ( Figure 1C ) . For the AAA-1 ring , it was possible to build a hexamer model based on the crystal structure of hexameric ClpC ( Wang et al . , 2011 ) ( PDB code 3PXG ) , a homologue that also displays disaggregation activity in vitro ( Schlothauer et al . , 2003 ) . The resulting ClpB AAA-1 hexamer model was automatically docked into the AAA-1 layer as a rigid body ( Figure 1D ) . This strategy was chosen over the fitting of a single subunit followed by hexamerisation because it is expected to provide a more accurate picture of the subunit interface , which is difficult to determine at the resolution of our EM map . However , this approach did not work for the AAA-2 layer since the ClpB AAA-2 hexamer based on the ClpC crystal structure was not compatible with the density . In this case a ∼40° tilt of the monomers into the plane of the ring was required to obtain the optimal fit , which resembles the pseudo-atomic model of the homologue ClpA AAA-2 ring ( Effantin et al . , 2010 ) . Coordinates of the E . coli ClpB ND ( residues 1 to 148 ) were obtained from the PDB ( 1KHY ) . A single N domain was fitted manually , maintaining the connection to AAA-1 , and then hexamerised by applying symmetry in Chimera ( Pettersen et al . , 2004 ) . 10 . 7554/eLife . 02481 . 008Table 1 . X-ray data collection and refinement statisticsDOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 008ProteinClpB E279A/E432A/E678A ( SeMet ) + ATPWavelength ( Å ) 0 . 9794Space groupP65Unit cell ( Å , ° ) 127 . 34 , 127 . 34 , 119 . 86 , 90 , 90 , 120Molecules1Resolution ( Å ) 81 . 15–3 . 50 ( 3 . 69–3 . 50 ) Reflections measured202965Unique reflections14024Rmerge8 . 8 ( 53 . 2 ) Rpim3 . 0 ( 14 . 9 ) I/σI16 . 8 ( 5 . 4 ) Completeness ( % ) 99 . 9 ( 100 ) Redundancy14 . 5 ( 14 . 8 ) Rwork/Rfree ( % ) 22 . 5/24 . 5Protein residues657 ( 5257 atoms ) ADP2Rmsd bond lengths ( Å ) 0 . 004Rmsd bond angles ( ° ) 0 . 94Average B-Factor Protein99 . 6Average B-Factor ADP87 . 7Ramachandran plot statistics Favored ( % ) 95 . 5 Allowed ( % ) 4 . 1 Generous ( % ) 0 . 4 Disallowed ( % ) 0 PDB entry code4CIU The AAA+ rings have a central opening of ∼30 Å and therefore are not as compact as in one of the previous models ( Lee et al . , 2003 , 2007 , 2010 ) , but not as expanded as in the other ( Wendler et al . , 2007 ) . The Arg-fingers are at the interface between subunits , available to catalyse hydrolysis as expected from mutational studies ( Mogk et al . , 2003; Yamasaki et al . , 2011; Biter et al . , 2012 ) . The ClpB coiled-coil MDs were separately docked in the rod-shaped densities surrounding the AAA-1 ring ( Figure 1D ) , maintaining the connection to the AAA-1 small subdomain . The pseudo-atomic model of one ClpB subunit obtained from this fitting differs from the crystal structure by rotations about the inter-domain hinge points ( Figure 1—figure supplement 4B ) . In this position , the MD contacts the neighbouring AAA-1 via its motif 1 , while motif 2 makes intrasubunit AAA-1 interactions . This is in good agreement with recent biochemical data showing protection of these two motifs upon ClpB oligomerisation and formation of an intermolecular disulphide bond between E344C of AAA-1 and L424C of a neighbouring MD motif 1 ( Oguchi et al . , 2012; Figure 1E ) . Moreover , intramolecular disulphide cross-links engineered between AAA-1 and motif 2 residues in TthClpB , EcoClpB and yeast Hsp104 are also compatible with the fitted structure ( Figure 1E; K476C/E358C , Oguchi et al . , 2012; G175C/R484C , H362C/Q473C E . coli numbering , Lee et al . , 2003; G175C/S499C , Haslberger et al . , 2007; Hsp104 K358C/D484C , Lipinska et al . , 2013 ) . However , this arrangement is not compatible with some of the engineered disulphide bonds observed in yeast Hsp104 ( Desantis et al . , 2014 ) ( D427C/E475C , D427C/E471C and E320C/N467C ) . To investigate possible species-specific structural differences we reconstructed the functional yeast homologue HAP ( Hsp104 with the ClpA tripeptide for ClpP binding ) in complex with ClpP . Using ∼10 , 000 particles and analyzing the data as described above , an independent map of HAP was obtained at ∼21 Å resolution ( Figure 2A , Figure 1—figure supplement 3 ) . The three-layered structure is comparable to BAP and there is density accounting for all domains including the MD , which surrounds the AAA-1 ring . The atomic coordinates of the ND and AAA+ rings obtained from the BAP analysis were fitted as rigid bodies . In order to fit the density , the MD must adopt a more horizontal orientation ( Figure 2A ) . In summary , HAP shows overall the same structural organisation as its bacterial homologue ClpB . 10 . 7554/eLife . 02481 . 009Figure 2 . Independently determined maps and fitted hexameric models of HAP-ClpP , BAPtrap-ClpP and ClpB . ( A ) Negative stain EM map of HAP-ClpP . From left to right: surface side-view , AAA-1 layer and AAA-2 layer . The central channel enclosed by the AAA-2 ring is filled with density . ( B ) Cryo EM map of BAP-ClpP formed with the BAP variant that traps the substrate inside . ( C ) Cryo EM map of wild type ClpB alone . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 009 Since negative stain EM of ClpB ( BAP ) in complex with ClpP gave a clear result different from all previous cryo EM maps of ClpB and Hsp104 , we collected cryo EM data on BAP-ClpP as well as on ClpB alone . The same strategy of using only clearly identifiable side views was applied . Complexes were imaged in the presence of ATPγS and independent maps were obtained by de novo angular reconstitution in each case ( Figure 2B , C ) . For cryo EM of the BAP-ClpP complex , we used a Trap ( E279A/E678A ) variant , which can bind but not hydrolyze ATP due to mutations in both Walker B motifs . We anticipated that the Trap construct would be more stable than the wild type , but data collection was challenging because side views were not abundant . Eventually , ∼4500 particles were collected and the same processing strategy was used as for the negative stain data . This variant is more likely to trap non native substrates in its central channel , and the extra density seen in this complex is likely to arise from denatured protein , possibly ClpP , present in the sample ( Figure 2B ) . In the case of isolated ClpB , side views were sorted on the basis of multivariate statistical analysis ( MSA ) . Briefly , particles representing all views of ClpB were picked , centered and classified by MSA . Only particles belonging to classes representing side-views ( 80°< β <100° ) were extracted and used for further processing . Both maps show overall dimensions and density distributions comparable to the negative stain structure of BAP in complex with ClpP , confirming that the ClpB hexamer structure is not significantly altered either by negative staining or by binding to ClpP . The atomic model derived by fitting the negative stain BAP-ClpP map could be docked as a rigid body into both cryo EM reconstructions , but the MD assumes a more horizontal orientation , similar to that in HAP ( Figure 2 ) . E432A and Y503D are ClpB point mutations at opposite ends of the MD coiled-coil , which result in repressed ( E432A ) and hyperactive ( Y503D ) states ( Oguchi et al . , 2012; Seyffer et al . , 2012 ) . Repressed ClpB-E432A is deficient in DnaK interaction and cannot be activated by its Hsp70 partner . Hyperactive ClpB-Y503D shows high ATPase and substrate unfolding activity even in the absence of Hsp70 ( Oguchi et al . , 2012; Seyffer et al . , 2012 ) . We collected negative stain EM datasets of the BAP-ClpP complexes of these variants and obtained 3D reconstructions of BAP-E432A and BAP-Y503D at 18 Å and 20 Å , respectively ( Figure 3 , Figure 1—figure supplement 3 ) . Starting models were independently generated by angular reconstitution and refined with sixfold symmetry ( Figure 3—figure supplement 1 ) . Both mutants assemble into three layers similar to the wild type and show high variability in the ND ring . Atomic coordinates of the AAA+ rings can be fitted as described for the wild type , using the ClpC hexamer as starting point . Some rearrangement was necessary to fit the ND into density ( Figure 3 ) . The most notable difference between the two maps is a ∼30° difference in orientation of the motif 1 blade of the MD and the loss of the motif 2 density in the hyperactive state ( Figure 4A , B ) . Another difference observed upon alignment of the AAA-1 ring in the repressed , wild-type and hyperactive maps is a ∼15° rotation of the wild-type AAA-2 ring relative to the mutants . 10 . 7554/eLife . 02481 . 010Figure 3 . Structures of repressed and hyperactive BAP-ClpP mutants . ( A ) Side view ( left ) and 35 Å section ( right ) of the BAP-E432A map with the ClpB hexamer fit . ( B ) Fitting of the BAP-E432A AAA+ rings . ( C ) Side view ( left ) and 35 Å section of the fitted BAP-Y503D structure . MD is only partially in density and is more tilted than in the wild type and the BAP-E432A repressed mutant . The ND is smeared out as in all BAP forms due to disorder , but has a more vertical orientation in the hyperactive state . ( D ) Fitting of the BAP-Y503D AAA+ rings . The motif 2 region protrudes from the density . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01010 . 7554/eLife . 02481 . 011Figure 3—figure supplement 1 . Test of EM map refinements after interchange of starting models . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01110 . 7554/eLife . 02481 . 012Figure 4 . Analysis of MD movements . ( A ) Repressed ( red ) , wild-type ( yellow ) and hyperactive ( green ) forms of BAP-ClpP with a fitted subunit . ( B ) Distances ( D ) are shown between Cα atoms of Q427 ( motif 1 ) and Q502 ( motif 2 ) of neighbouring subunits based on the symmetrised EM reconstructions of wild-type ClpB and MD mutants . ( C ) Fluorescence energy transfer between motif 1 and motif 2 of adjacent ClpB subunits . Emission spectra of ClpB-Q427W-Q502C-IAEDANS in the monomeric ( high salt ) , oligomeric ( low salt ) and ATP state ( 2 mM ATP ) are shown . Effects of MD mutations on FRET efficiency were determined . The ratio of acceptor to donor fluorescence ( derived from the areas under the curves ) was calculated as readout for FRET efficiency . ( D ) Intermolecular disulfide crosslinking between MD motif 1 and motif 2 of adjacent subunits . Reduced and oxidized ClpB-K431C-S499C and repressed ( E432A ) and hyperactive ( Y503D ) variants were analyzed by non-reducing SDS-PAGE . Positions of monomers ( 1 ) and crosslinked dimers ( 2 ) , trimers ( 3 ) , tetramers ( 4 ) , pentamers ( 5 ) and hexamers ( 6 ) are indicated . A protein standard is given . CoPhe at 25 µM was used as oxidizing agent . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01210 . 7554/eLife . 02481 . 013Figure 4—figure supplement 1 . Contacts between adjacent MDs in the EM model compared to those found in the ClpB crystal structures . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01310 . 7554/eLife . 02481 . 014Figure 4—figure supplement 2 . Tryptophan emission spectra of unlabeled ClpB-Q427W-Q502C and of corresponding MD mutants in the monomeric ( HS , high salt ) , oligomeric ( LS , low salt ) and ATP-loaded state ( LS + 2 mM ATP ) are given . High salt ( 400 mM KCl ) prevents ClpB oligomerisation . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 014 In the BAP-E432A repressed mutant the MD is clearly visible around the AAA-1 ring . It has a horizontal orientation so that motif 2 forms a contact with motif 1 of the neighbouring MD ( Figure 3B , Figure 4B ) . This packing of the MD is supported by biochemical data showing overprotection of motif 2 in this mutant ( Oguchi et al . , 2012 ) . Moreover , a very similar intersubunit motif 1-motif 2 contact is seen in all the ClpB crystal structures , in which ClpB monomers are arranged in a spiral assembly ( TthClpB , 1QVR , Lee et al . , 2003; EcoClpB current study ) ( Figure 4—figure supplement 1 ) . In the Y503D hyperactive form there is density for motif 1 in a more tilted orientation , but there is no density visible for motif 2 , suggesting that this region becomes either disordered or highly mobile ( Figure 3C , D ) . This finding is in accordance with H/D exchange data showing deprotection of this region in the BAP-Y503D mutant ( Oguchi et al . , 2012 ) . We conclude that , in the Y503D hyperactive form , the motif 1 arm of the MD is tilted downwards so that it can no longer contact motif 2 of the adjacent MD , and binds to a lower region of the neighbouring AAA-1 domain . Motif 2 is mobile and solvent exposed ( Figure 3C , D , Figure 4A , B ) . Alignment of AAA-1 domains of wild type , repressed and hyperactive forms of ClpB/BAP confirms that the MD rotates ∼30° from a tilted orientation in the hyperactive state , to a horizontal one in the repressed state , with the wild type occupying an intermediate orientation ( Figure 4B ) . In this movement motif 1 switches from being protected against AAA-1 to engaging motif 2 in trans . These different orientations of the MD have opposite consequences for DnaK binding . The horizontal MD position , with motif 1 contacting motif 2 of the neighbouring subunit , is incompatible with DnaK binding at the tip of motif 2 ( Seyffer et al . , 2012; Rosenzweig et al . , 2013 ) . In contrast , tilting of the MD exposes motif 2 for DnaK interaction . To confirm the close proximity of motif 1 and motif 2 of neighbouring MDs we performed fluorescence resonance energy transfer ( FRET ) experiments using Q427W ( motif 1 ) as FRET donor and IAEDANS labeled Q502C ( motif 2 ) as FRET acceptor . The FRET pair ( Förster radius of 22 Å ) has a distance of 19 . 1 Å in the wild type model and was introduced into the tryptophan-free ClpB-W462Y/W543L variant . In addition we coupled the FRET pair to E432A and Y503D mutations , to monitor the consequences of repressed and hyperactive states on FRET efficiency . The distance between the FRET partners is either reduced ( 11 . 3 Å ) or increased ( 35 . 9 Å ) in models of the repressed and hyperactive variants , respectively ( Figure 4B ) . All ClpB variants were IAEDANS labeled with similar efficiencies ( 70–80%; Figure 4—figure supplement 2 ) . High IAEDANS fluorescence and thus FRET efficiency was observed for wild type ClpB and E432A upon ClpB oligomerization . Increase in acceptor fluorescence in general correlated with reduced tryptophan emission except for ClpB wild type upon oligomerization . In contrast , IAEDANS fluorescence remained low under all conditions tested when the FRET pair was linked to Y503D ( Figure 4C ) . Furthermore , to test for direct interaction between motif 1 and motif 2 , we introduced cysteine residues into motif 1 ( K431C ) and motif 2 ( S499C ) and analyzed whether intermolecular crosslinks form under oxidizing conditions . Low Cu-Phenanthrolin concentrations ( 25 μM ) yielded a ladder of crosslink products from ClpB dimers to hexamers . Although non-specific dimer formation was observed in single cysteine variants , higher oligomers were only found in double cysteine variants ( Figure 4D ) . Introducing the activating ( Y503D ) mutation to ClpB-K431C/S499C decreased but did not abolish crosslink formation , suggesting rapid fluctuation of MDs between different conformations even in the hyperactive state ( Figure 4D ) . These findings support the interaction of MD motifs 1 and 2 from adjacent subunits as observed in the EM reconstructions and strengthening or loosening of the contact in repressed and hyperactive states , respectively . Mutating MD motif 1 can impair Hsp70 interaction and consequently protein disaggregation ( Oguchi et al . , 2012; Seyffer et al . , 2012 ) . On the other hand a ClpB motif 1 deletion variant retains substantial disaggregation activity ( Seyffer et al . , 2012; del Castillo et al . , 2011 ) , providing no clarification of its role in protein disaggregation . Our EM structural data indicate that motif 1 acts as a crucial component of ClpB regulation by stabilizing MDs in a horizontal position through interaction with an adjacent motif 2 . Indeed , ClpB-ΔM1 ( ΔE410-Y455 ) shows hyperstimulation of ATPase activity in the presence of the substrate casein compared to wild-type ClpB , resembling the deregulated ATPase activity of hyperactive ClpB variants lacking motif 2 or the entire MD ( Oguchi et al . , 2012; Figure 5A ) . The basal ATPase activity of ClpB-ΔM1 was reduced in comparison to wild type due to partial oligomerization defects , in agreement with earlier reports ( Oguchi et al . , 2012; del Castillo et al . , 2011 ) . ClpB-ΔM1 oligomerization defects were more pronounced than in ClpB-ΔM1/M2 , suggesting that motif 2 in absence of motif 1 might impede hexamer formation . 10 . 7554/eLife . 02481 . 015Figure 5 . Deletion of MD motif 1 causes ClpB activation . ( A ) Basal and substrate-stimulated ATPase activities of ClpB wild type and indicated MD deletions were determined in the absence and presence of casein . Relative ATPase activations by casein were calculated ( inset ) . ( B ) Unfolding of Casein-YFP by BAP wild type and indicated MD deletions in the presence of ClpP was monitored by YFP fluorescence . Initial YFP fluorescence was set to 100% . ΔM1: ΔE410-Y455 , ΔM2: ΔS456-E520 , ΔM1/M2: ΔE410-E520 . ( C ) E . coli ΔclpB cells expressing the indicated plasmid-encoded clpB alleles under control of an IPTG-regulatable promoter were grown overnight at 30°C . Various dilutions ( 10−1–10−7 ) were spotted on LB plates containing the indicated IPTG concentration and incubated at 37°C for 24 hr . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 015 Next we tested whether hyperstimulation of ATPase activity is linked to high unfolding power . We employed BAP variants of the respective deletion constructs and tested for unfolding and degradation of casein-YFP in presence of ClpP . BAP-ΔM1 unfolds the YFP moiety of casein-YFP , an activity only observed for hyperactive BAP variants but not wild type BAP ( Oguchi et al . , 2012; Figure 5B ) . Loss of ClpB regulation in ClpB-ΔM1 was also linked to severe toxicity upon expression in E . coli ΔclpB mutant cells ( Figure 5C ) . However , higher expression levels of ClpB-ΔM1 than of ClpB-Y503D are required to observe the same degree of toxicity probably because deletion of motif 1 results in oligomerisation defects ( Oguchi et al . , 2012; Figure 5C ) . In conclusion ClpB-ΔM1 exhibits the three major characteristics of hyperactive ClpB variants ( high ATPase and unfolding activities linked to cellular toxicity ) , demonstrating the crucial regulatory role for motif 1 in interacting with an adjacent motif 2 to ensure tight activity control of ClpB . As mentioned above , we imposed 6-fold symmetry as a first approximation , to simplify the alignment and reconstruction problem . Nevertheless , crystal structures of the AAA+ protein ClpX show that the homo-hexameric assembly can be markedly asymmetric ( Glynn et al . , 2009; Kon et al . , 2012; Stinson et al . , 2013 ) . We therefore reanalysed our negative stain EM data without imposing symmetry , in order to study the conformational variability within the hexamer . Using ∼15 , 000 , ∼10 , 000 and ∼9000 particles for wild-type BAP , BAP-E432A and BAP-Y503D we obtained asymmetric maps at 21 Å , 21 Å and 25 Å resolution , respectively ( Figure 6A , Figure 1—figure supplement 3 , Figure 6—figure supplement 1 ) . Although the numbers of particles were similar to those used for sixfold analysis , the resolution is only slightly worse and the maps are comparable in quality to the symmetrised ones , consistent with the structure being asymmetric . The structure of the hyperactive mutant is less defined and has lower resolution than the other two , particularly for the MDs , probably owing to their higher mobility . Therefore , only wild-type BAP and BAP-E432A were used for further analysis . 10 . 7554/eLife . 02481 . 016Figure 6 . Variations in MD orientations around the ring . ( A ) Asymmetric reconstructions of repressed BAP-E432A , wild type BAP-ClpP and hyperactive BAP-Y503 . ( B ) Side views of the BAP-ClpP wild type structure rotated successively 60° . Red MDs are oriented horizontally and make motif 1 to motif 2 contact as in the repressed state . Yellow MDs have slightly tilted orientations similar to the wild type . Green MDs are tilted as in the hyperactive state . One of the MDs contacts AAA-2 of the same subunit ( last right panel ) . ( C ) Equivalent views of the BAP E432A-ClpP repressed mutant structure . Color code for the MD orientations is the same as above . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01610 . 7554/eLife . 02481 . 017Figure 6—figure supplement 1 . Plots showing the angular distribution of single particles ( red dots ) around the Euler sphere for each asymmetric reconstruction . Angle search was done with 2° sampling . The equatorial band reflects the Euler angle distribution around the BAP-ClpP central axis . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01710 . 7554/eLife . 02481 . 018Figure 6—figure supplement 2 . Open and closed conformations of AAA+ domains can be fitted into the asymmetric ClpB map . ( A ) Superimposition of an open ( blue ) and a closed ( grey ) nucleotide pocket ( ClpX 3HWS ) . ADP is depicted in green spheres . ( B ) EcoClpB AAA-2 dimers built with open , intermediate and closed conformations based on ClpX crystallographic dimers . ( C ) AAA-1 and AAA-2 layers of asymmetric wild-type ClpB reconstruction plus indication of nucleotide occupancy . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 01810 . 7554/eLife . 02481 . 019Figure 6—figure supplement 3 . Asymmetric nucleotide binding to ClpB . ( A–D ) Instrumental responses of successive injections of ADP ( 8 . 39 mM , 1 . 92 mM , 5 . 82 mM and 4 . 05 mM ADP for ClpB-WT , ClpB-K212A , ClpB-E432A , ClpB-Y503D , respectively ) into a solution of ClpB-WT ( 678 μM ) , ClpB-K212A ( 239 μM ) , ClpB-E432A ( 467 μM ) and ClpB-Y503D ( 396 μM ) . The molar ratio of ADP to ClpB hexamer is indicated . Raw isothermal titration calorimetry data are shown . Integrated data after base-line-correction and fitting to the respective binding isotherms to a single-site binding model are given ( lower panel ) . The number of ADP binding sites was calculated . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 019 The asymmetric structures show similar density distributions in the AAA+ rings , even though not all six subunits in a ring can be aligned simultaneously owing to 5°–15° variations in rotational orientation . This rotational variability may explain the apparent ∼15° rotation of the AAA-2 wild-type ring relative to the mutants observed in the symmetrised reconstructions . As expected from the eigenimage analysis , the ND ring is not very well defined and the density does not account for all six NDs . Therefore , we did not attempt any atomic structure fitting into this region . Docking of the atomic structures of AAA-1 , AAA-2 and MD was performed as follows . Structures of AAA+ proteins crystallised in their hexameric assemblies show that the large and small subdomains of the AAA fold assume a range of conformations that can be clustered into open or closed forms ( Cho and Vale , 2012; Stinson et al . , 2013; Figure 6—figure supplement 2 ) . The closed conformation is ATP-binding competent and there are intermediate forms that might also represent weak binding states . Using a gallery of available AAA+ crystal structures we modelled open , closed and intermediate conformations of ClpB AAA-1 and AAA-2 . We also created AAA+ dimers of adjacent ClpB subunits based on ClpX pseudo-hexameric crystal structures ( Glynn et al . , 2009; Stinson et al . , 2013; Figure 6—figure supplement 2 ) that were fitted as rigid bodies into the asymmetric reconstructions . Crystallographic dimers are likely to provide more realistic models of subunit interfaces than can be deduced by fitting individual subunits into low-resolution maps . The AAA-2 ring density could be almost entirely interpreted using this approach and the fit suggests that 3 to 5 subunits are sufficiently closed to bind ATP ( Figure 6—figure supplement 2 ) . We analyzed nucleotide ( ADP ) binding to wild type ClpB by isothermal calorimetry ( ITC ) revealing a binding stoichiometry of 7 . 5 ± 0 . 1 ADP per hexamer ( Figure 6—figure supplement 3 ) . ITC experiments using ClpB-K212A , which is deficient in nucleotide binding in AAA-1 , allowed us to determine a binding stoichiometry of 3 . 7 ± 0 . 3 ADP in AAA-2 of the mutant hexamer ( Figure 6—figure supplement 3 ) . The same ADP binding stoichiometry was also found for the repressed and hyperactive variants ( Figure 6—figure supplement 3 ) . The deduced stoichiometries are in good agreement with the distinct AAA-2 conformations observed in the asymmetric EM reconstructions . Similar calculations have been reported for the AAA+ proteins MCM , ClpX and HslU ( Moreau et al . , 2007; Yakamavich et al . , 2008 ) . The AAA-1 ring is more asymmetric than AAA-2 and it is not easily interpretable by fitting crystallographic dimer models . There is sufficient density to guide rigid body fitting of all AAA-1 domains , but this fitting does not allow deductions of nucleotide occupancy ( Figure 6—figure supplement 2 ) . Asymmetric reconstructions of both wild-type BAP and repressed mutant display clear densities accounting for the MDs that lie outside the AAA-1 ring and assume different orientations similar to those observed in the symmetrised maps of wild type and mutants . In the wild-type asymmetric reconstruction , the MD orientation ranges from horizontal as in the repressed state to highly tilted , similar to the hyperactive state , passing through the intermediate wild type-like state ( Figure 6B ) . In one subunit , motif 1 contacts AAA-2 of the same subunit , suggesting a route of allosteric communication between AAA-1 and AAA-2 . Additionally , this contact is compatible with recently reported engineered disulphide bonds between motif 1 residues and AAA-2 in Hsp104 and ClpB ( Desantis et al . , 2014 ) . The tilted MDs are clustered together , consistent with the release of motif 1-motif 2 contacts freeing adjacent subunits . In the asymmetric reconstruction of the repressed state , five MDs are found in the horizontal orientation , followed by a sixth for which there is no clear density ( Figure 6C ) . In the wild type two MDs can make the motif 1 to motif 2 contact , while in the repressed state up to five MDs are compatible with this contact ( Figure 6B , C ) . Conversely , in the wild type at least two MDs exist in a clearly activated state but in the repressed state none of the MDs are visible in the tilted , hyperactive conformation . As a consequence , the MD conformations in BAP-E432A do not support Hsp70 binding and ATPase activation , in agreement with previous findings ( Oguchi et al . , 2012; Seyffer et al . , 2012 ) . However , ClpB wild type hexamers harbour two MDs that favour Hsp70 recruitment , potentially priming the ClpB ring for further activation .
The BAP construct of ClpB complexed with ClpP , combined with a conservative approach of using only clearly identifiable side views and basing the analysis mainly on negative stain images ( with independent confirmation from cryo EM ) enabled us to unambiguously locate all subunit domains in the oligomer , including the coiled-coil MD propeller . Although the quoted resolutions for some previous structures of ClpB and Hsp104 were better , the globular shape , flexibility and asymmetry of these hexamers reduce the reliability of orientation assignment . Moreover , previous published structures of ClpB/Hsp104 were obtained by cryo EM only , which provides a lower signal-to-noise ratio ( SNR ) than negative stain data . Rigid body fitting of E . coli ClpB atomic coordinates ( PDB code 4CIU ) into our new maps reveals that the MD is not projecting outwards from the hexamer ( Lee et al . , 2003 , 2007 , 2010 ) nor intercalated between subunits ( Wendler et al . , 2007 , 2009 ) , but is instead lying on the surface of the ClpB hexameric ring with a variable degree of tilt ( Figure 4A , Figure 6B , C ) . A similar position of the MD is seen for Hsp104 , underlining the conserved activity and mechanism of these disaggregases . This new MD arrangement is in excellent agreement with recent , extensive biochemical analysis of the MD ( Oguchi et al . , 2012 ) . It was proposed that the MD works as a molecular toggle switching from a repressed state in which both motif 1 and 2 ends of the propeller are protected , to an active state where motif 2 is deprotected , exposing the binding site for DnaK/Hsp70 ( Oguchi et al . , 2012; Seyffer et al . , 2012; Rosenzweig et al . , 2013 ) . Our EM reconstructions of repressed , hyperactive and wild type ClpB reveal a lever-like movement of the MD that switches from the repressed state with head-to-tail motif 1-motif 2 binding between adjacent subunits , to a mobile , activated state with motif 2 free and available for binding to DnaK ( Figure 7A ) . The wild type conformation is intermediate between these states and thus poised for switching . 10 . 7554/eLife . 02481 . 020Figure 7 . Interaction between the ClpB hexamer and DnaK . ( A ) Binding of DnaK is only possible when the MD is tilted ( green ) . DnaK cannot bind when two adjacent MDs occupy a more horizontal position ( red ) as it would clash with motif 1 of the neighbouring ClpB subunit . ( B ) Cartoon of an opened-out ClpB ring with two DnaK molecules bound . The MDs involved in DnaK binding are shown in green , and those that cannot bind to DnaK are shown in red . Release of the ends of the MD favours activation of adjacent subunits , and the active cluster can move around the ring in a wave-like manner . The model of DnaK binding to the MD is based on the NMR study in Seyffer et al . ( 2012 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 02481 . 020 Our data thus explain the critical role of motif 1 , which regulates the accessibility of motif 2 and consequently ClpB activity . Confirming its important regulatory role , deletion of motif 1 results in hyperactive ATPase and unfolding activity of ClpB-ΔM1 as well as cell toxicity ( Figure 5 ) , representing key characteristics of the hyperactive state ( Oguchi et al . , 2012 ) . Moreover , our maps suggest that motif 1 , through its contacts with either the adjacent motif 2 or AAA-1 , plays a key role in direct communication between neighbouring subunits , which must therefore act in a coordinated manner . It has become clear that AAA+ proteins are highly dynamic molecular motors unlikely to exist in a homogeneous structural state . Therefore we generated asymmetric reconstructions of ClpB . Although the resolution of the asymmetric structures is not sufficient to support a detailed mechanistic model , these reconstructions provide the first visualization of the MD conformational flexibility that was inferred from biochemical analysis ( Lee et al . , 2003; Haslberger et al . , 2007; Oguchi et al . , 2012 ) . The asymmetric structures show that the MD orientation varies around the ring occupying the repressed , wild type-like and hyperactive positions described by the symmetrised averages . The variable tilts of MDs observed around the ring suggest that 2 to 4 adjacent subunits are available for DnaK binding in the wild-type vs only 1 in the repressed mutant ( Figure 6B , C ) . This is consistent with the estimated stoichiometry of 2–5 molecules of DnaK per ClpB hexamer required for activation ( Seyffer et al . , 2012; Desantis et al . , 2014 ) . It also suggests that at least four subunits must have detached MDs to allow activity , perhaps through movements of the AAA+ domains , in agreement with the number of ClpX subunits that hydrolyze ATP in a coordinated manner to unfold GFP in single molecule experiments ( Sen et al . , 2013 ) . Moreover , we calculated an ADP binding stoichiometry of 4 for both wild type and mutants ( Figure 6—figure supplement 3 ) , which indicates that although ATP hydrolysis is strongly affected , detachment of the MD does not change the nucleotide binding . The cryo EM reconstruction of wild-type ClpB suggests that the various MD conformations exist only transiently , poised between hyperactive and repressed states , with the balance shifted slightly towards repressive motif 1-motif 2 contacts ( Figure 2 ) . DnaK binding to an accessible motif 2 stabilizes the MD in a tilted conformation , thereby in turn breaking the repressive contacts with MDs in neighbouring subunits . Thus , an initial encounter of DnaK will facilitate DnaK binding in the neighbouring MDs . In this model , breakage or formation of motif 1-motif 2 contacts provides a mechanistic basis for signalling DnaK binding or dissociation in a wavelike manner around the ClpB ring . ( Figure 7B ) . The model predicts spatial proximity of multiple DnaK molecules , which is the case for aggregated but not soluble DnaK substrates , directing ClpB activity specifically to protein aggregates . Moreover , the activation of ClpB by DnaK binding , combined with movements of the highly mobile N domain , might act to deliver the substrate to the channel entrance , where it would be engaged for threading , unfolding and consequent extraction from the aggregate .
E . coli strains used were derivatives of MC4100 . Mutant derivatives of ClpB/BAP were generated by PCR mutagenesis and standard cloning techniques in pDS56 and were verified by sequencing . Wild type and mutant ClpB were purified using Ni-IDA ( Macherey–Nagel ) and size exclusion chromatography ( Superdex S200 , Amersham ) following standard protocols . Purifications of DnaK , DnaJ , GrpE , ClpP , Luciferase and Casein-YFP were performed as described previously ( Oguchi et al . , 2012 ) . Pyruvate kinase and α–casein were purchased from Sigma . Protein concentrations were determined with the Bio-Rad Bradford assay . BAP ( HAP ) -ClpP complexes were formed in 20 mM Tris–HCl , pH 7 . 5 , 20 mM KCl , 15 mM MgCl2 , 1 mM DTT and 2 mM ATPγS . Proteins were applied to glow-discharged carbon coated grids ( EM sciences ) , previously coated with 5 kDa poly-lysine ( Sigma-Aldrich , UK ) to positively charge the surface . Samples were stained with 2% uranyl acetate . For cryo-EM imaging , BAPtrap-ClpP was applied to holey carbon grids coated with a thin carbon film and pretreated with poly-lysine while ClpB specimens were loaded onto lacey carbon grids . Cryo-EM specimens were vitrified in a Vitrobot ( FEI , UK ) . Images were recorded on a 4k Gatan ( UK ) CCD camera at a magnification of 50 , 000Χ for negatively stained specimens ( pixel size 2 . 2 Ε; underfocus range: 0 . 5–1 . 2 μm ) and of 80 , 000 × for cryo specimens ( pixel size 1 . 4 Å; underfocus range: 1 . 5 µM–4 μm ) . All data were collected on a Tecnai F20 FEG operated at 200 kV under low dose conditions . The contrast transfer function ( CTF ) for each CCD frame was determined with CTFFIND3 ( Mindell and Grigorieff , 2003 ) and corrected by phase flipping using Bshow1 . 6 ( Heymann and Belnap , 2007 ) . Side views of BAP-ClpP 2:2 complexes were manually picked using Boxer ( Ludtke et al . , 1999 ) and extracted into 256 × 256 boxes . The boxed particles were band-pass filtered between 300 and 10 Å for the negative stain dataset and between 300 and 5 Å for the cryo dataset . They were then normalized to the same mean and standard deviation . Particles were initially aligned to the total sum of 10–20 vertically oriented particles using SPIDER ( Frank et al . , 1996 ) . Individual 1:1 BAP-ClpP complexes were extracted with circular masks and classified by MSA in IMAGIC-5 ( van Heel et al . , 1996 ) to remove images that did not represent BAP-ClpP , yielding 17 , 470 particles of wild type BAP-ClpP , 12 , 588 of BAP-E432A-ClpP , 9436 of BAP-Y503D-ClpP and 12 , 568 of HAP-ClpP . The BAPtrap-ClpP cryo dataset included 4592 particles . At this stage , particles were high-pass filtered to 160 Å and initial class averages of 20–30 images each were obtained by MSA . All alignments of 1:1 BAP-ClpP complexes were done limiting the in-plane rotation to ±20° . Upon further classification , 5–10 good classes were used to generate a starting model by angular reconstitution ( van Heel et al . , 2000 ) . Alternative starting models were also created by applying sixfold symmetry to single classes and then using the resultant 3D map , generally composed of three discs corresponding to the three layers of the molecule , to generate an anchor set for Euler angle assignment . A low-resolution density map was independently created for each dataset by angular reconstitution with sixfold symmetry . Particle orientations were refined in multiple cycles of AP SHC alignment in SPIDER , MSA and angular reconstitution in IMAGIC and the resulting 3D reconstruction , filtered to 30 Å , was used as an initial model for projection matching in SPIDER . By applying a rectangular mask , only the AAA+ layers were refined . After 8–10 cycles of projection matching , using progressively smaller angular sampling steps ( 4°–1° ) and filtering the 3D to the estimated resolution at each cycle , final structures were generated of the whole complex and their resolution was estimated by Fourier shell correlation with a 0 . 5 correlation cutoff . Based on cross correlation coefficient , around 70–80% of each dataset was included in the final reconstruction . The differences between the mutant structures were tested by refining with interchanged starting models . In both cases , the original mutant structure was recovered despite the use of the other map as a starting model for projection matching ( Figure 3—figure supplement 1 ) . For cryo EM of ClpB alone , the views were randomly oriented and the initial strategy was to extract clearly identifiable side views by MSA and classification . 7606 side views were used to generate a starting model by angular reconstitution , which was refined to 29 Å resolution ( Figure 1—figure supplement 3 ) by projection matching . For the reconstructions without imposed symmetry , particle orientations were determined using either the sixfold symmetrised map filtered to 50 Å or a sphere obtained from the average of all particles as a starting model . Subsequently , particle orientations were refined with ∼10 cycles of projection matching without imposed symmetry . The particle orientations were well distributed around the BAP-ClpP central axis ( Figure 6—figure supplement 1 ) . The resolution was estimated by Fourier shell correlation ( FSC ) at 0 . 5 ( Figure 1—figure supplement 3 ) . Docking was done using the crystal structures of E . coli ClpB-E432A ( current study , PDB code: 4CIU ) and E . coli ClpB ND ( PDB code 1KHY ) . A homology model of Hsp104 , obtained using Phyre2 ( Kelley and Sternberg , 2009 ) was used for fitting into the HAP reconstruction . A hexameric ClpB/Hsp104 AAA-1 ring was modelled on the ClpC hexamer crystal structure ( PDB code 3PXG ) and was automatically fitted as a rigid body into the symmetrised maps using the UCSF Chimera package ( Pettersen et al . , 2004 ) . The N-terminal , the middle and the AAA-2 domains were first fitted manually and then local fitting was optimized in Chimera , followed by symmetrisation . In the asymmetric reconstructions , dimers of adjacent ClpB subunits were modelled on crystal structures of ClpX hexamers and fitted as rigid bodies in Chimera . Steady-state ATP hydrolysis rates were determined in buffer A ( 50 mM Tris pH 7 . 5 , 5 mM MgCl2 , 20 mM KCl , 2 mM DTT ) as described ( Haslberger et al . , 2007 ) . ClpB disaggregation activities were determined by following the refolding of aggregated firefly Luciferase according to published protocols ( Haslberger et al . , 2007 ) . Chaperones were used at the following concentrations: 1 μM ClpB ( wild type or derivatives ) , 1 μM DnaK , 0 . 2 μM DnaJ , 0 . 1 μM GrpE . Oligomerisation of ClpB variants was tested as described previously ( Mogk et al . , 2003; Haslberger et al . , 2007 ) . Site-specific labelling of ClpB using 1 , 5-IAEDANS , 5- ( [ ( 2-iodoacetyl ) amino]ethylamino ) naphthalene-1-sulfonic acid ( Invitrogen , Germany ) was performed according to the manufacturers' instructions . The intrinsic tryptophan fluorescence of ClpB variant harbouring a single tryptophan ( ClpB*-Q427CW , 1 μM each ) was measured on a Perkin-Elmer ( Germany ) LS50B spectrofluorimeter at 25°C in low salt buffer A ( 50 mM Tris pH 7 . 5 , 5 mM MgCl2 , 20 mM KCl , 2 mM DTT ) . Tryptophan and IAEDANS emission spectra of labeled ClpB variants were recorded in high and low salt buffer A ( supplemented with 400 mM KCl or 20 mM KCl , respectively ) in the absence or presence of 2 mM nucleotide between 300 and 550 nm at a fixed excitation wavelength of 290 nm . The Förster radius of the Trp-IAEDANS FRET pair was calculated as 22 Å ( Jeganathan et al . , 2006 ) . For formation of disulfide bridges ClpB cysteine variants were first dialyzed to remove DTT . Cysteine oxidation was achieved by adding 25 μM Cu-Phenanthroline to 4 μM ClpB and incubating the mixture for 1 min at room temperature . Oxidation and disulfide bond formation was stopped by addition of 50 mM iodoacetamide and SDS-sample buffer containing 5 mM EDTA . Crosslink products were analyzed by a non-reducing SDS gradient gel ( 3–8% ) . Casein-YFP unfolding and degradation assays were carried out using 6 μM BAP ( wild type or variants ) , 9 μM ClpP and 0 . 5 μM Casein-YFP . Degradation of Casein-YFP was determined by monitoring YFP fluorescence at 535 nm ( excitation wavelength 515 nm ) at a Perkin–Elmer LS50B spectrofluorimeter . E . coli cells harbouring plasmid-encoded clpB alleles were grown in the absence of IPTG overnight at 30°C . Serial dilutions were prepared , spotted on LB-plates containing different IPTG concentrations and incubated for 24 hr at 37°C . ClpB wild type and variants were extensively dialyzed against low salt buffer A ( 50 mM Tris [pH 7 . 5] , 25 mM KCl , 20 mM MgCl2 , 5% glycerol ) . Isothermal titration calorimetry ( ITC ) was performed using an ITC calorimeter ( iTC200Microcalorimeter , MicroCal , Germany ) . Consecutive injections of nucleotide into a 300 μl cell containing ClpB were performed after sample equilibration at 30°C . Integration and fitting of ITC data were performed using ORIGIN software ( GE , Germany ) . ClpB and ADP concentrations were determined by UV absorbance at 280 nm . HX-MS experiments were performed similar to those described earlier ( Oguchi et al . , 2012 ) . Briefly , ClpB ( 100 pmol ) , BAP ( 100 pmol ) or BAP-ClpP complex ( 100 pmol and 200 pmol respectively ) were incubated for 3 min at 30°C in low salt buffer A ( 50 mM Tris , pH 7 . 5 , 25 mM KCl , 20 mM MgCl2 , 2 mM DTT ) in presence of ATP or ATPγS and diluted 20-fold into D2O-based MDH buffer to initiate amide proton-deuteron exchange . The exchange reaction was stopped after 1 min by the addition of 1 volume of ice-cold quench buffer ( 0 . 4 M K-phosphate buffer , pH 2 . 2 ) . Quenched samples were immediately injected into the HPLC setup , with ( peptide analysis ) or without ( full length protein analysis ) online peptic digest , and analyzed on an electrospray ionization quadrupole time-of-flight mass spectrometer ( QSTAR Pulsar , Applied Biosystems ) as described in Rist et al . ( 2003 ) . Analysis of deuteron incorporation into peptide was performed by using AnalystQS software ( Applied Biosystems/MDS SCIEX , Germany ) . The EM maps have been deposited in the 3D-EM database ( www . emdatabank . org ) with accession codes EMD-2555 ( BAP-E432A C6 ) , EMD-2556 ( BAP-E432A C1 ) , EMD-2557 ( BAP wild type C6 ) , and EMD-2558 ( BAP wild type C1 ) , EMD-2559 ( BAP-Y503D C6 ) , EMD-2560 ( BAP-Y503D C1 ) , EMD-2561 ( HAP ) , EMD-2562 ( BAPtrap cryo ) and EMD-2563 ( ClpB wild type cryo ) . The corresponding FSC curves have also been deposited . The crystal structure atomic coordinates of E . coli ClpB E279A/E432A/E678A + ATP have been deposited in the PDB database ( http://www . ebi . ac . uk/pdbe/ ) with entry code 4CIU . PDB models based on the EM maps have also been deposited with codes 4D2Q ( BAP-E432A C6 ) ; 4D2U ( BAP wild type C6 ) and 4D2X ( BAP-Y503D C6 ) .
The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system . Essential for disaggregation , the ClpB middle domain ( MD ) is a coiled-coil propeller that binds Hsp70 . Although the ClpB subunit structure is known , positioning of the MD in the hexamer and its mechanism of action are unclear . We obtained electron microscopy ( EM ) structures of the BAP variant of ClpB that binds the protease ClpP , clearly revealing MD density on the surface of the ClpB ring . Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer . Adjacent , horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction . Tilting of the MD breaks this contact , allowing Hsp70 binding , and releasing the contact in adjacent subunits . Our data suggest a wavelike activation of ClpB subunits around the ring .
Proteins are long chain-like molecules that twist and fold into complex three-dimensional shapes in order to carry out their functions . High temperatures or other types of stress can cause proteins to fold incorrectly , and misfolded proteins can form clumps ( or aggregates ) that are harmful to cells . Additional proteins called chaperones are therefore used by cells to help proteins to fold correctly , or to refold poorly folded proteins . ClpB proteins ( and related proteins ) are chaperones found in bacteria , fungi and plants; these proteins co-operate with other chaperones to rescue misfolded proteins that have aggregated—an activity that helps cells to survive heat shock and other stresses . Six ClpB proteins work together to form a ring-shaped complex , and the misfolded protein is unfolded by threading it through the centre of this ring . Each ClpB protein also has a middle domain that acts to switch the complex on and off as needed . The middle domains are known to form coiled-coils , with protein helices coiled together like the strands of a rope . However , previous efforts to work out the structure of the ClpB complex did not clearly establish where these coiled-coils were positioned relative to the rest of the ring . Now Carroni et al . have used image processing to overcome these problems and reveal that the middle domains are wrapped around the outer edge of the ring complex . Analysis of ClpB mutants that lock the complex in either an off or on state revealed that the middle domains are linked head-to tail to encircle the ring when the complex is off . However , when the complex switches on , the middle domains let go of each other and tilt , allowing the ring to change shape . Carroni et al . suggest that the exposed ends of the middle domains are free to bind to other chaperones ( those that work to refold the unfolded proteins ) , thereby activating the complex . Although Carroni et al . have revealed how the ClpB ring complex is activated , further work is needed to understand exactly how the unlocked ring works to rescue misfolded proteins from aggregates within cells .