crystal structure of the c1 domain of cardiac myosin binding protein-c: implications for...

doi:10.1016/j.jmb.2008.02.044 J. Mol. Biol. (2008) 378, 387–397

Available online at www.sciencedirect.com

Crystal Structure of the C1 domain of CardiacMyosin Binding Protein-C: Implications forHypertrophic Cardiomyopathy

Lata Govada1†, Liz Carpenter2†, Paula C. A. da Fonseca1,John R. Helliwell3,4, Pierre Rizkallah4, Emily Flashman5,Naomi E. Chayen1, Charles Redwood5 and John M. Squire1⁎

1Biomolecular MedicineDepartment, SORA Division,Imperial College London,London SW7 2AZ, UK2Centre for StructuralBiology, Division of MolecularBiosciences, Imperial CollegeLondon, London SW7 2AZ, UK3School of Chemistry,University of Manchester,Oxford Road, Manchester M139PL, UK4STFC Daresbury Laboratory,Warrington, WA4 4AD, UK5Department of CardiovascularMedicine, University of Oxford,Wellcome Trust Centre forHuman Genetics, RooseveltDrive, Oxford OX3 7BN, UK

Received 14 January 2008;received in revised form17 February 2008;accepted 19 February 2008Available online4 March 2008

*Corresponding author. Muscle ConBristol BS8 1TD, UK. E-mail address† L.G. and L.C. contributed equalPresent address: P. C. A. da Fonse

London SW3 6JB, UK.Abbreviations used: MyBP-C, my

subfragment-2; HCM, hypertrophicdiffraction.

0022-2836/$ - see front matter © 2008 E

C-protein is a major component of skeletal and cardiac muscle thick fila-ments. Mutations in the gene encoding cardiac C-protein [cardiac myosinbinding protein-C (cMyBP-C)] are one of the principal causes of hyper-trophic cardiomyopathy. cMyBP-C is a string of globular domains includingeight immunoglobulin-like and three fibronectin-like domains termed C0–C10. It binds to myosin and titin, and probably to actin, and may have botha structural and a regulatory role in muscle function. To help to understandthe pathology of the known mutations, we have solved the structure of theimmunoglobulin-like C1 domain of MyBP-C by X-ray crystallography to aresolution of 1.55 Å. Mutations associated with hypertrophic cardiomyo-pathy are clustered at one end towards the C-terminus, close to the im-portant C1C2 linker, where they alter the structural integrity of this regionand its interactions.

© 2008 Elsevier Ltd. All rights reserved.

Keywords: hypertrophic cardiomyopathy; IgI domain structure; muscleregulation; MyBP-C C1 domain; C-protein
Edited by I. Wilson
traction Group, Department of Physiology and Pharmacology, University of Bristol,: [email protected] to the work.ca, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road,

osin binding protein-C; cMyBP-C, cardiac myosin binding protein-C; S2,cardiomyopathy; ELC, essential light chain; MAD, multiwavelength anomalous

lsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.jmb.2008.02.044

388 Crystal Structure of Cardiac C-protein Domain

Introduction

Myosin binding protein-C (MyBP-C) is a compo-nent of the myosin filaments of skeletal and cardiacmuscles located in the crossbridge-containing C-

Fig. 1 (legend o

zones as seven to nine stripes approximately 43 nmapart.1–3 It is thought to play a role both in theregulation of contractility and in the maintenanceof myosin filament structure.4 The cardiac protein(cMyBP-C), in common with the two skeletal iso-

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389Crystal Structure of Cardiac C-protein Domain

forms, is a modular protein composed of immuno-globulin I (IgI)-like and fibronectin 3-like domains(Fig. 1a).5–7 cMyBP-C also contains certain cardiac-specific features, namely an additional N-terminalIgI-like domain (C0), a nine-residue (LAGGGRRIS)insert within the MyBP-C motif and a 28-amino-acidloop in the C5 domain (Fig. 1a and c).8 The C-terminus of the protein binds to the thick filamentbackbone via interactions with the myosin rodthrough its C7 to C10 domain, primarily throughC10, and also through C7–C10 with titin.9–12 TheC1C2 region has been shown to bind to the sub-fragment-2 (S2) portion of myosin and this interac-tion is abolished by phosphorylation of residueswithin the MyBP-C motif by cAMP-dependentprotein kinase A.5 It has been suggested that thisprovides an additional means of regulation ofcontractility in which interaction between cMyBP-Cand S2 reduces the incorporation of myosin headsinto the contractile cycle.13 This has been supportedby the finding that incubation of skinned myocyteswith exogenous myosin S2 increases contractility.14

Furthermore, skinned cardiac myocytes from a ho-mozygous C-protein knockout mouse model gene-rated greater power output than those from non-transgenic animals.15 In contrast, however, it hasbeen found that treatment of skinned ventricularmyocytes with a C0–C2 fragment was able toactivate force in the absence of calcium.16 Proteinkinase A phosphorylation of cMyBP-C regulates thekinetics of force development17 and hence affects theearly pressure rise rate in systole.18 Recent structuraldata have also suggested that the N-terminal region(possibly the Pro-Ala-rich region on the N-terminalside of C1; Fig. 1a and c)may bind to actin filaments,7

implying a further regulatory role for the protein andalso a possible role in maintaining the thick and thinfilaments in the correct 3-D register.Molecular genetic work has shown that the auto-

somal dominant disease hypertrophic cardiomyo-pathy (HCM) is caused by mutations in at least ninedifferent sarcomeric protein genes.19 MYBPC3,which encodes cMyBP-C, was identified as one ofthese disease genes in 1995,20,21 and since then atleast 60 HCM-causing mutations have been re-ported. Recent studies of large numbers of patients(e.g., Ref. 22) have shown that mutations are mostcommonly found in MYH7, which encodes the α-myosin heavy chain, and in MYBPC3, each account-ing for at least a quarter of affected individuals.

Fig. 1. (a) Schematic representation of the 11 domains [8MyBP-C. (b) Amino acid residues 1–258 of the N-terminal reDNA, including the hexahistidine tag and a thrombin cleavagethe residues seen in crystals of the C1 structure with the sequekey known HCM mutations in the N-terminal domains C0, Clinker with the LAGGGRRIS insertion that can be phosphoryla(d) Multiple sequence alignment of the C1 domain of MyBP-Csites D228N, Y237S, H257P and E258K. Sequence alignmeclustalw] and displayed using the Jalview alignment editor.6 (ELC of human atrial myosin and the C-terminal end of the cMyC0 and C1 that follows. The boxed sequences, rich in lysinesresidues are shown in red.

Numerous mutations inMYBPC3 result in truncatedproteins, many of which lack the C-terminal do-mains necessary for binding to the thick filamentbackbone. There are also, however, numerous mis-sense mutations in cMyBP-C that cause single aminosubstitutions. These mutations are unlikely to act viahaploinsufficiency and the mutant proteins areprobably incorporated relatively normally into thethick filament structure and act via a “poison poly-peptide” mechanism by interfering with a specificfunction of cMyBP-C. For example, the 10 missensemutations within the C1C2 domains and the in-cluded MyBP-C motif (Fig. 1a and c) may alter theinteraction of this region with S2, as HCMmutationsin the corresponding portion of S2 abolish the inter-action with C1C2.23 They may also have an effect onthe binding of the N-terminal region of MyBP-C toactin. Mouse models of cMyBP-C HCM mutationshave shown reduced unloaded shortening velocity24

and reduced power output.25

We have now solved the crystal structure of theimmunoglobulin-like C1 domain of cMyBP-C (Fig.1b and c), the region of the molecule between thePro-Ala-rich C0C1 putative actin-binding linker andthe MyBP-C motif between C1 and C2, which can bephosphorylated and can bind to myosin S2. This hadpreviously been determined by NMR [Protein DataBank (PDB) ID 2avg], but the present X-ray analysisis to higher precision (diffraction resolution 1.55 Å).Solution structures for other MyBP-C domains havealso been determined (PDB IDs 1x5y, 1pd6, 2e7cand 1gxe). Nevertheless, the work presented here isone of the highest-resolution crystal-structure deter-minations of any muscle Ig domain. We have foundthat the various important C1 sites of mutationimplicated in HCM are clustered at one end ofthe C1 domain near to the cMyBP-C motif. Wediscuss details of the local interactions that theseresidues make in the native structure and the likelystructural and functional effects of the knownmutations.

Results

The C1 fold

Initial crystallisation trials of a recombinant frag-ment composed of the human C0C1 domains of the

immunoglobulin (Ig) and 3 fibronectin (Fn)] of cardiacgion C0C1 of cMyBP-C derived from the pET-28a vectorsite (LVPRGS). The sequence highlighted in orange showsnce in the Pro-Ala linker disordered. (c) Illustration of the1 and C2 of human cMyBP-C. Also shown is the C1–C2ted (shown in pink) and is reported to bind to myosin S2.5

from different species. Arrows show key HCM mutationnts were produced using CLUSTALW [www.ebi.ac.uk/e) Sequence alignment of the N-terminal extension on theBP-C C0 domain together with the Pro-Ala linker between(blue), are thought to form an actin binding site.7 Acidic

http://www.ebi.ac.uk/clustalw

http://www.ebi.ac.uk/clustalw

Fig. 3. The X-ray structure of the C1 domain deter-mined here. (a) 3-D structure of the C1 domain of cMyBP-C with the β-strands labelled. (b and c) Superpositions ofcMyBP-C C1 domain [yellow/orange as in left view in (a)]with telokin [(PDB ID 1tlk) in green (b)] and a Z-domain oftitin [PDB ID 1ya5, blue in (c)], indicating the homologybetween the three structures. (d and e) Structure of the C1domain of cMyBP-C in ribbon view; two orientationsshowing the locations of the four main known mutationsassociated with HCM. (f and g) Surface views of the C1domain in the same orientations as (d) and (e) revealingthe surface charge distribution. The C-terminal end wherethe mutations are found is highly acidic (red). All figureswere produced using PyMol [DeLano Scientific LLC,USA, www.delanoscientific.com].

Fig. 2. Silver-stained gel of protein sample and crystalsof cMyBP-C. The commercial molecular mass markerswere run on either side of the gel with lysozyme (mole-cular mass 14 kDa) as a additional marker. A strong bandaround 29 kDa was seen in the original C0C1 protein lane,whereas the redissolved crystals gave a single prominentband at around 13 kDa.


cMyBP-C protein yielded well-diffracting proteincrystals. However, when the protein content of thecrystals was analysed by SDS-PAGE, they werefound to contain only a fragment of the protein. Theexpected C0C1 molecular mass is around 29 kDa,but the band observed by SDS-PAGE indicated thatthe crystals contained mainly a 12-kDa fragment(Fig. 2). Washed crystals were subsequently ana-lysed by mass spectrometry and N-terminal se-quence analysis. The molecular mass of the proteincrystals determined by matrix-assisted laser desorp-tion/ionisation mass spectroscopy was 12,341 Daand the N-terminal sequence was GAPDDPIGLFVM.This indicated that the crystallised fragment beganwith Gly148, which is the C-terminal end of the Pro-Ala linker at the start of the C1 domain (Fig. 1b). Infact, sequences like the 6-kDa Pro-Ala-rich regionhave been implicated in self-proteolysis26,27 and itmay be this that caused the cleavage of the C0 andC1 domains.The human cMyBP-C C1 domain crystallised with

I41 space group symmetry. Structure determinationwas achieved usingmultiwavelength anomalous dif-fraction (MAD)phasing (Materials andMethods) andstructure refinementwas carried out to a resolution of1.55 Å. The X-ray crystallographic structure of the C1domain of cMyBP-C (Fig. 3a; Table 1) consists of allthe residues between 152 and 258, with the exceptionof residues 182 to 185, which were disordered. Thestructure shows a β-sandwich fold typical of mem-bers of the IgI family,23 with two β-sheets containingseven β strands, labelled A to G (Fig. 3a), two veryshort segments of 310 helix, three bends and three

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Table 1. Structure solution by Se-Met MAD and refinement using REFMAC

Protein

Selenomethionine

NativePeak Remote Inflection

Beamline SRS, 10.1 SRS, 10.1 SRS, 10.1 SRS, 14.1Wavelength (Å) 0.9798 0.9756 0.9802 1.488Space group I41 I41 I41 I41Unit cell parameters a, c (Å) 48.79, 95.28 48.80, 95.31 48.58, 94.88 48.85, 95.13Resolution (Å) 25.0–1.9 25.0–2.4 20.0–2.3 20.0–1.45Completeness (%)a 91.0(62.9) 95.3 (88.2) 89.5 (62.8) 82.8 (99.9)Redundancya 9.6 (5.9) 7.0 (4.6) 6.3 (3.7) 5.4 (5.2)⟨I⟩/⟨σI⟩

a 31.8 (7.7) 32.8 (8.8) 24.7 (6.2) 23.4 (4.6)Unique reflections 8004 5432 7784 16,401Rsym (%)a 5.5 (21.2) 4.3 (15.1) 5.3 (19.4) 6.1 (39.5)Refinement resolution range (Å) 20.0–1.9 20.0–1.55No reflections working/test 7444/358 12,035/652Rcryst/Rfree (%) 24.4/29.8 18.4(17.7)b/24.1(22.7)b

rmsdBonds (Å) 0.017 0.011c

Angles (°) 1.87 1.22c

No. of final model bound water molecules 169

Ramachandran plot statisticsResidues in most favoured regions [A,B,L] 86 97.7%Residues in additional allowed regions [a,b,l,p] 2 2.3Residues in generously allowed regions [∼a,∼b,∼ l,∼p] 0 0.0%Residues in disallowed regions [XX] 0 0.0%No. of non-glycine and non-proline residues 88 100%Number of end-residues (excl. Gly and Pro) 3No. of glycine residues 8No. of proline residues 5Total number of residues 104

a Figures in brackets are the respective values for each quantity listed in the outer diffraction resolution shell.b SHELX97: R and Rfree values for F N4σF in brackets. Also highest (0.44 /Å3) and lowest (–0.36 e/Å3) final Fo−Fc electron difference

density.c REFMAC5 values, i.e., prior to split occupancy refinement step.


hydrogen-bonded turns. Strands A, G, F and C formone β-sheet and B, E and D form the other, both withanti-parallel packing of adjacent strands.Despite the fact that the C1 domain proved not to

be amenable to solution by molecular replacementmethods, it is quite similar in structure to a numberof known proteins (details of similarities anddifferences below). These were identified using theDALI server‡ and include adhesion molecules,immunoglobulins, immunoglobulin-like domainsand structural and calmodulin-binding muscle pro-teins. Two of the most structurally homologous pro-teins, telokin, a calmodulin-binding protein (PDB ID1tlk28), and parts of titin (a Z-domain; PDB ID1ya5,29), were superimposed on the C1 domain inFig. 3b and c. In both cases there was a good fit(rmsd, 1.31 and 1.14 Å, respectively) for 89 residues(out of 97 observed in C1), indicating their structuralsimilarity. However, in both of these other casesthere was only a single 310 helix. The similarity isgreatest towards the C-terminal end, possibly indi-cating a conserved common functional role for theC-termini.

‡www.ebi.ac.uk/dali

Location of mutations associated withhypertrophic cardiomyopathy

The four known mutations in the C1 domain ofthe cMyBP-C associated with HCM, as shown in Fig.1c, are D228N, Y237S, H257P and E258K. In order toassess the likely structural consequences of suchmutations, we compared the C1 amino acid se-quences from a number of different sources rangingfrom humans, including the skeletal forms, to axo-lotl (Fig. 1d). Remarkably, the positions of Tyr237 andGlu258 are conserved across all sequences. His257 isconserved in all cardiac types except chicken butabsent in all skeletal forms except human slow.Asp228 shows quite low conservation, being acidicin most cardiac sequences and basic in skeletalsequences. The location of these residues in the C1structure is illustrated in Fig. 3d and e, where it canbe seen that they cluster towards the C-terminalof the C1 domain and are therefore adjacent to theMyBP-C motif that forms the linker to the C2domain. This region of the structure is charac-terised by a highly acidic molecular surface (Fig. 3fand g).The side chain of the highly conserved residue

Tyr237 is located within the hydrophobic core of theC1 domain. In the crystal structure this residue is

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hydrogen-bonded to the backbone carbonyl ofresidue Phe233, which in turn is involved in a webof hydrogen bonds between residues located in theloops towards the C-terminus of the domain,including residues Asp228, Tyr237 and Glu258

Fig. 4 (legend o

(Fig. 3d and e). It is likely, therefore, that the muta-tion T237S would result in the destabilisation of itsinteraction with Phe233 (Fig. 4a and b), due to theloss of their hydrogen bond, and that this wouldpropagate through these C-terminal loops. The

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mutation H257P may well cause similar effects onthese loops, not just due to disruption of the hydro-gen bonds between this residue and both Glu165and Val168 (Fig. 4c and d), but also because of thelocal secondary-structure alterations likely to beimposed by the insertion of a proline residue. On theother hand, the principal effect of the mutationsD228N and E258K, with the side chains of both theseresidues being exposed at the surface of the C1domain structure (Fig. 4c), is most likely to be thealteration of the surface charge of this highly acidicregion of the protein. Overall, the mutations asso-ciated with HCM identified within the C1 domain ofMyBP-C all seem to lead to the disruption of thehighly charged region around the C-terminus of thisdomain.

Discussion

C-protein (MyBP-C) was originally identified byOffer et al.1 as a contaminant of myosin preparationsand it was soon shown to be located in the muscle A-band by Craig and Offer.30 Its binding to the lightmeromyosin part of the myosin rod was shown byMoos et al.31 and the precise location of theinteraction has recently been determined by Flash-man et al.12 The identity of the C-terminal C7 to C10region of MyBP-C as the myosin and titin bindingregion was made mainly by Fischman and hiscollaborators.9,32 That MyBP-C can bind elsewhereon themyosin rod than to light meromyosin, namelythat it can bind to the heavy meromyosin S2 regionof the myosin rod, was shown by Gautel and hiscollaborators,5,23 who have recently solved thestructure of the N-terminal 126-residue per chaincoiled coil of S2 and have shown this region to havethree rings of negative charge, the middle one ofwhich may be associated with C-protein binding.

Binding properties of the C0C1C2C3C4 region ofMyBP-C

For many years it has been thought that MyBP-Ccan interact with actin. This was originally sug-gested by Moos et al.33 and has been supported by anumber of more recent binding studies (see Reviewin Ref. 34). Structural evidence for MyBP-C binding

Fig. 4. (a) Stereo view of the C-terminal region of C1 showimutations, particularly Asp 228, Tyr237, Phe233 and Glu258. (cshowing the surface locations of the Asp228 and Glu258 residradically alter the surface charge. The overviews in (b) anddirections as (a) and (c), respectively. (e and f) Schematic diagextension and (f) the N-terminal end of C0 and the Pro-Ala linkfilaments through the actin-binding motif (AB motif) whose smyosin subfragment 2 (S2) and the neck andmotor of the myosare in orange and the Pro-Ala-rich extension on the ELC (PA lina tropomyosin strand (TM) in green. (f) This has the same myoshown with domains 0, 1, 2, 3, 4 and 5 in yellow, the Pro-Ala-rgreen patch on C1. The MyBP-C motif between C1 and C2envisaged that the binding of C0 and the AB Motif may occLikewise the interaction of C1C2 with myosin S2 will depend

to actin was also provided by Squire et al.7 based onX-ray fibre diffraction studies of whole muscle. Inaddition, these authors noted a sequence similaritybetween the Pro-Ala-rich linker on the N-terminalside of C1 and the extension on the essential lightchain (ELC) of muscle myosin II.35 Two examplesequences are shown in Fig. 1e. In the ELC exten-sion sequence shown here there are 38 residues. Ofthese, the N-terminal residues 1–13, boxed in thesequences in Fig. 1e, were shown by Trayer and hiscolleagues to be actin-binding (e.g., Refs. 36,37). Theother residues are predominantly Pro and Ala, withthe strong suggestion that this region might beextended and rodlike.38–42 A structural model forthis sequence has recently been proposed.43 For afairly extended chain with 3.5 Å per residue, therewould be a total length of around 95 Å for 27residues, whereas for a compact α-helical structurethe length would be about 40 Å. Taking a plausiblestructure to be nearer to the extended end of theseextremes, as in Ref. 43, it is evident that the ex-tension on the ELC might be such that it could crossthe gap between myosin and actin. Indeed there isstructural evidence from electron microscopy fortwo-site binding of myosin heads to actin when theELC extension is present.44 The way in which thismight occur is illustrated schematically in Fig. 4a,based on figures in Refs. 40,43–45.Recent studies of the binding properties of the first

few N-terminal domains of MyBP-C (Fig. 1) byRazumova et al.46 using a variety of constructs,C0C1, C0C1C2, C1C2 and C2C3C4, have shownthat combinations containing the MyBP-C motif,between C1 and C2, affect the sliding velocity ofactin filaments over a skeletal heavy-meromyosin-labelled substrate, whereas constructs lacking thisregion do not. The presence of this motif also re-duces the myosin ATPase rates in solution. Surpris-ingly, they also found that C1C2, hitherto thought tobind only to S2, can in their hands bind to actin aswell. In addition, C2C3C4, which lacked the MyBP-C motif and therefore did not affect actin filamentsliding velocity in unregulated conditions, did re-duce the velocity of regulated filaments at high cal-cium, but not at low calcium, possibly by interactingwith the activated thin-filament regulatory complex.In a separate somewhat contradictory study,

Herron et al.16 showed that constructs like C0C1C2

ng interactions of some of the key residues associated with) Alternative stereo view to (a) of the same region of C1 butues where the mutations D228N and E258K occur, which(d) show the whole C1 domain from the same viewingrams showing the possible binding of (e) the myosin ELC(PA link) between domains C0 and C1 in cMyBP-C to actinequence is given in Fig. 1e. In (e), coloured in red are thein head. The myosin ELC and regulatory light chain (RLC)k) is in blue. A few actin monomers are shown in grey andsin and actin features as in (e), but here part of cMyBP-C isich linker in blue, and the site of mutations in C1 as a lightis shown to have three phosphorylation sites (3P). It isur in resting muscle but will be labile and easily broken.on the state of phosphorylation of the MyBP-C motif.11


and C0C1, but not C0 or C1C2, could activateskinned myocytes in the absence of calcium, pos-sibly providing a myosin-based regulatory systemin addition to the normal troponin–tropomyosinsystem on actin. They also found that this was asarcomere-length-dependent phenomenon, perhapshelping to explain the Frank–Starling length–tensionrelationship in cardiac muscle.47 That the constructsproducing this effect had only the Pro-Ala-richlinker in common suggested to these authors thatthis region can bind to myosin and modify its pro-perties, since, unlike Razumova et al.,46 they couldnot detect binding of either C0 or C0C1 to the I-bandof their preparations. In our own studies, notdetailed here but reported by Govada48 (and alsofound by Redwood et al., unpublished data), weobtained a different result, namely that C0C1 camedown with actin in co-sedimentation assays.In summary, a number of different studies (in-

cluding one on the phosphorylation effects inC-protein knockout mice49 where troponin-I phos-phorylation on its own without MyBP-C phosphor-ylation was ineffective) suggest different propertiesfor the N-terminal region of MyBP-C. However,they all emphasise the vital importance of this re-gion as a regulator of muscle contraction, with boththe Pro-Ala linker and the MyBP-C motif implicatedas major players in this role. The C1 domain onwhich we report here is centrally placed betweenthese linkers, which emphasises its significance; itmay help the S2 binding of MyBP-C under someconditions and its binding to actin under otherconditions.

Implications of known C1 mutations

All of these observations lead to a possible modelfor the behaviour of the N-terminal region of MyBP-C, while acknowledging that its binding partnerswill most probably change according to the condi-tions and state of activity of the muscle. In summary,in some cases the C1C2 region binds to myosin S2and reduces ATPase activity depending on its stateof phosphorylation, whereas in other cases parts ofthe C0 to C4 complex can bind to actin. Figure 4fshows a scheme analogous to that for the ELC ex-tension in Fig. 4e, but this time showing the C1C2region of MyBP-C binding to myosin S2. As in thecase of the extension on the ELC, the Pro-Ala linkerbetween C0 and C1 is shown as an extended rodwith an actin-binding domain, the C-terminal partof C0, on the end (see Fig. 1e, boxed region). It issuggested that in some conditions, such as restingmuscle, the Pro-Ala linker can span the gap across toactin and produce cross-linking between the myosinand actin filaments. It has been observed that theMyBP-Cmeridional X-ray reflection at around 440 Åin patterns from resting muscle,50 which has beenexplained in terms of the N-terminal region ofMyBP-C binding to actin,7 reduces greatly in inten-sity when muscle is activated, suggesting that acti-vation produces considerable disorder in this part ofMyBP-C. It must be presumed from this that the

actin binding by MyBP-C that occurs in restingmuscle is weak and is easily disrupted on activation.With the scenario in Fig. 4f in mind, what is the

role of C1 and how do the observed mutations affectits behaviour? First, the binding of C1C2 to myosinS2 is thought to be largely an electrostatic interac-tion, with the MyBP-C motif between C1 and C2binding to a negatively charged collar in S2.51 Thelarge negative potential on the C-terminal end of C1(Fig. 3f and g) must be presumed to have a majorrole in locating the MyBP-C motif in the right placealong S2 and the damage created by the dramaticchange in charge associated with the E258K andD228N mutations or the disruption caused by theY237S andH257P would therefore not be surprising.Most analyses of the effect of HCM mutations inother sarcomeric proteins such as β-myosin heavychain and the troponin complex have revealed thatthe changes are likely to lead to enhanced contrac-tility,19,52,53 which may give rise to disease viamechanisms involving energetic compromise.54 Wepredict that the HCMmissense mutations within theC1 domain, in common with the mutations withinthe myosin S2 region,23 act to weaken the S2:C1C2attachment in the unphosphorylated state that willproduce a higher rate of crossbridge cycling. Themutations may also have an effect on actin binding.These effects will be brought about as outlinedabove by changes to the side chains at the proposedprotein-binding interfaces.

Materials and Methods

Protein preparation

The C0C1 sequence (residues 1–258) was amplified fromfull-length human cMyBP-C cDNA by PCR and insertedinto the bacterial expression vector pET28a (Novagen,Merck Chemicals Ltd). Transformed BL21(DE3)pLysS cellswere grown and induced with IPTG at 37 °C according tostandard protocols. Cells were lysed in the presence ofprotease inhibitors in 10 mM imidazole, 300 mM sodiumphosphate, 300 mM sodium chloride, pH 8.0, by sonication.Soluble protein was purified from the lysis supernatant byaffinity chromatography using Ni2+-NTA resin [Qiagen(UK) Ltd.] followed by gel filtration using Superdex 75 (GEHealthcare Bio-Sciences AB, Uppsala, Sweden). The pur-ified protein was dialysed into 10 mM imidazole, 50 mMsodium chloride, 0.5 mM dithiothreitol, 50 mM TRIS HCl,pH 7.0, and concentrated to at least 20 mg/ml.The selenomethionine derivative was generated using

B834 cells transformed with the pET28a C0C2 constructgrown in medium containing 0.3 g/l selenomethionine55

and purified in the same manner as the unlabelled form.

Crystallisation methodology and conditions

Crystals of native and selenomethionine-substituted C1domain of cMyBP-C were grown by a modified hanging-drop method in which control of evaporation and arrest ofnucleation were carried out. Two crystal morphologies,both in the native as well as the selenomethionine deriva-tive of the protein (thick rods and pyramids), were ob-


tained by equilibrating at 20 °C a 2-μl drop containing a1:1 mixture of protein solution and crystallising solu-tion consisting of 18% PEG 3350 and 0.1M Hepes pH 7.3over 500 μl of crystallising solution in the reservoir.Crystals of the native formwere obtained 2weeks after theexperiments were set up, whilst the selenomethionine-substituted derivative crystals grew overnight. The rod-shaped crystals grew to a maximum dimension of 400μm×200 μm×50 μm and the pyramids to 300 μm×300μm×100 μm. Whilst the rods diffracted to 1.5 Å thepyramids diffracted only to a resolution of 3 Å.

Data collection and crystallographic parameters

The crystals obtained proved to be difficult to cool at 100K in the presence of cryoprotectants. All data sets collectedwith cryoprotected crystals diffracted to worse than 2.2 Åand had high mosaicities. Crystals cooled directly fromthe drop, in the absence of added cryoprotectants, butwith the 18% polyethylene glycol partially fulfilling such arole, gave diffraction to beyond 1.6 Å, but the diffractionimages were still affected to some extent by ice rings. Thehigh-resolution data used for refinement were measuredon SRS Station 14.156 and processed with a reject fractionof 0.925 in DENZO,57 which effectively removed thereflections that were superimposed on the water rings.This resulted in an overall data completeness of 82.5%.These data proved to be the best for refinement of thestructure; the affected resolution annuli are 4.21 to 3.34,2.32 to 2.20 and 1.95 to 1.89 Å. The data completenessbetween 1.8 and 1.55 Å resolution is 100%.Attempts to solve the structure by molecular replace-

ment using immunoglobulin folds proved unsuccessful,obviously due to the too large rmsd between the searchmodels available and the determined structure. The struc-ture was therefore solved using MAD phasing with threewavelengths collected on selenomethionine protein onbeamline 10.1 at the Synchrotron Radiation Source atDaresbury58 as shown in Table 1. One selenomethioninesite was found using SOLVE,59 which gave an overallfigure of merit of 0.40 (20 to 2.3 Å resolution) and a score of6.16. An initial structure was built using RESOLVE59 andthen rebuilt using O60 and refined with CNS.61 Molecularreplacement with this structure into the 1.55 Å native dataset was followed by density modification and phaseextension to 1.55 Å with DM.62 The selenomethioninestructure and initial refinement of the model was, as usual,just a stepping stone to the main model refinement (to1.55 Å). Indeed the selenomethionine diffraction data areclosely isomorphous to the native protein model (e.g.,SCAELIT R-factor on F 6.4%). Subsequently the structurewas refined with REFMAC563 in the CCP4 suite ofprograms§. The structure showed split occupancies forresidues Lys195, Ser236, Val241, Ser242 and Phe247; thefinal structure was therefore refined in SHELX9764 torefine the split occupancy. The bound water structure wasfirst developed within REFMAC563 then COOT65 andcompleted in SHELX97;64 a total of 169 bound watermolecules were found. Statistics of the Ramachandranplot are given in Table 1.

Protein Data Bank accession codes

Coordinates and structure factors have been depositedin the Protein Data Bank (PDB ID 2v6h).

§www.ccp4.ac.uk

Acknowledgements

We are indebted to Katie King and Paul Robinsonfor help with protein purification. This work wassupported by grants to J.M.S. from the EuropeanMyores Project and to J.M.S., C.R. and E.F. by grantsfrom the British Heart Foundation. NEC acknowl-edges support from the EPSRC (EP/501113/1) andthe OptiCryst Project LSHG-CT-2006-037793. TheSTFC Daresbury Laboratory is thanked for SR beamtime under an award to J.M.S.

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crystal structure of the c1 domain of cardiac myosin binding protein-c: implications for...

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