mutations in β-myosin s2 that cause familial hypertrophic cardiomyopathy (fhc) abolish the...

Article No. jmbi.1999.2522 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 286, 933±949

Mutations in bbb-Myosin S2 that cause FamilialHypertrophic Cardiomyopathy (FHC) Abolish theInteraction with the Regulatory Domain ofMyosin-binding Protein-C

Mathias Gruen1 and Mathias Gautel1,2*

1Max-Planck-Institut fuÈ rMolekulare PhysiologieRheinlanddamm 20144139, Dortmund, Germany2European Molecular BiologyLaboratory, Postfach 10 22 09
69012, Heidelberg, Germany
*Corresponding author

Huxley & Niedergerke, 1954

E-mail address of the [email protected]

Abbreviations used: FHC, familiacardiomyopathy; MyBP, myosin-bincAPK, cAMP-dependent protein kinmeromyosin; HMM, heavy meromytitration calorimetry; CD, circular dmyosin heavy chain.

0022-2836/99/080933±17 $30.00/0

The myosin ®laments of striated muscle contain a family of enigmaticmyosin-binding proteins (MyBP), MyBP-C and MyBP-H. These modularproteins of the intracellular immunoglobulin superfamily contain uniquedomains near their N termini. The N-terminal domain of cardiac MyBP-C,the MyBP-C motif, contains additional phosphorylation sites and mayregulate contraction in a phosphorylation dependent way. In contrast tothe C terminus, which binds to the light meromyosin portion of the myo-sin rod, the interactions of this domain are unknown. We demonstratethat fragments of MyBP-C containing the MyBP-C motif localise to thesarcomeric A-band in cardiomyocytes and isolated myo®brils, withoutaffecting sarcomere structure. The binding site for the MyBP-C motifresides in the N-terminal 126 residues of the S2 segment of the myosinrod. In this region, several mutations in b-myosin are associated withFHC; however, their molecular implications remained unclear. We showthat two representative FHC mutations in b-myosin S2, R870H andE924K, drastically reduce MyBP-C binding (Kd � 60 mM for R870H com-pared with a Kd of � 5 mM for the wild-type) down to undetectable levels(E924K). These mutations do not affect the coiled-coil structure of myo-sin. We suggest that the regulatory function of MyBP-C is mediated bythe interaction with S2, and that mutations in b-myosin S2 may act byaltering the interactions with MyBP-C.

# 1999 Academic Press

Keywords: myosin; myosin-binding protein-C; coiled-coil; hypertrophic
cardiomyopathy
Introduction

The contraction of striated muscle is the functionof a complex macromolecular machine in whichthe sliding of thin ®laments (composed of F-actin)and thick ®laments (composed of myosin;Figure 1(a)) past each other is responsible formuscle shortening and force generation (H.E.

; A.F. Huxley &

ing author:

l hypertrophicding protein(s);ase; LMM, lightosin; ITC, isothermal

ichroism; MHC,

Hanson, 1954). A number of other proteins areassociated with thick and thin ®laments and aregenerally assumed to play a regulatory role inmuscle contraction. In the case of the thin-®lamentassociated proteins, tropomyosin and the troponincomplex, the cooperative/allosteric nature of thisregulatory mechanism is beginning to emerge(Lehrer & Geeves, 1998).

The thick ®laments contain a family of associ-ated proteins discovered in 1972 (Offer, 1972), themyosin-binding proteins C and H (MyBP-C orMyBP-H). MyBP-C is arranged regularly along thethick ®lament in seven to nine positions, whichcoincide with every third level of myosin heads(Offer, 1972; Dennis et al., 1984; Bennett et al.,1986). The exact function of the myosin-bindingproteins is still unresolved. At least four isoforms
of MyBP-C are expressed in ®bre-type speci®c
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Figure 1. (a) The myosin molecule is a polar 470 kDa protein composed of the coiled-coil rod domain and the twomotor domains (subfragment S1) on the myosin heavy chain (MHC). The myosin rod is composed of the light mero-myosin subfragment, which is responsible for the ®lament-forming properties, and subfragment S2, which forms a¯exible link from the heads to the ®lament. The S2 segment contains a proteolytically susceptible region in theC-terminal half, which is believed to act as a ¯exible hinge. In the helical portion of the neck of S1, one essential andone regulatory light chain are bound close to the head-tail junction (see also review by Goldman, 1998). For simpli-city, myosin is drawn as a linearly extended molecule. (b) The domain structure of the cardiac myosin-binding pro-tein C (c-MyBP-C). The polar molecule bears a signal transduction module at the N terminus, the MyBP-C motif(Gautel et al., 1995). Several cardiac-speci®c features are present apart from the isoform-speci®c phosphorylation sitesand are marked by asterisks. The C-terminal region contains a high-af®nity LMM-binding site in C10 (Okagaki et al.,1993) and a low-af®nity titin-binding region (Freiburg & Gautel, 1996). (c) Multiple sequence alignment of the humanand chicken, slow and fast skeletal and cardiac isoforms of MyBP-C. The phosphorylation sites for cAMP-dependentprotein kinase (cAPK) are shaded in grey; the cardiac-speci®c insertion LAGGGRRIS contains an additional cAPKsite (asterisk). Note the conservation between isoforms and species, which argues for a crucial control function of thismodule. Identical residues are boxed. An isoform-speci®c region rich in charged residues is underlined. Ggc, chickencardiac MBP-C (EMBL U38949); Hsc, human cardiac (EMBL X84075); Ggf, chicken fast (EMBL UM31209); Hsf,human fast (EMBL X73113); Hss, human slow (EMBL X73114).

934 Myosin S2 Interactions with MyBP-C

ways (Yamamoto & Moos, 1983; Gautel et al.,1998). Their early appearance in the assembly ofthick ®laments was taken as a suggestion that theirmain function was in the regulation of myosin ®la-ment assembly (Obinata et al., 1984; BaÈhler et al.,
1985; Lin et al., 1994; Koshida et al., 1995). How-
ever, the discovery that the cardiac isoform can bephosphorylated in a dynamic way by cAMP-dependent protein kinase (cAPK) as well as by anassociated Ca2�/calmodulin-dependent kinasesuggested a role in the regulation of muscle con-
traction (Jeacocke & England, 1980; Hartzell &

Myosin S2 Interactions with MyBP-C 935

Titus 1982; Hartzell & Glass, 1984; Garvey et al.,1988; Schlender & Bean, 1991; Gautel et al., 1995).

MyBP-C is a modular protein built from immu-noglobulin and ®bronectin-like domains (Einheber& Fischman, 1990; FuÈ rst et al., 1992; Weber et al.,1993; Gautel et al., 1995; Yasuda et al., 1995)(Figure 1(b)). The C-terminal region interacts withthe light meromyosin portion (LMM) of myosin(Moos et al. 1975., Okagaki et al., 1993;Alyonycheva et al., 1997) as well as with titin(Labeit et al., 1992; Soteriou et al., 1993; Freiburg &Gautel, 1996), thus anchoring the protein to thethick ®lament shaft and specifying its sarcomericlocalisation (Gilbert et al., 1995).

Phosphorylation occurs at isoform-speci®c sitesin a domain speci®c for MyBP-C in the N-terminalregion of the protein (Gautel et al., 1995). This 100-residue domain, the MyBP-C motif, is highly con-served between all isoforms of MyBP-C andbetween avians and mammals (Figure 1(c)). Thissuggests that it interacts with a common andequally conserved binding partner, which, how-ever, has not been identi®ed to date. The extent ofsequence identity between various MyBP-C iso-forms and their cross-species conservation(Figure l(c)) suggest further that the function ofthis module is speci®ed by the conserved regions,and that the phosphorylation sites found in the car-diac isoform represent an additional feature.

The MyBP-C molecule has a length of about40 nm (Offer et al., 1973; Hartzell & Sale, 1985;FuÈ rst et al., 1992). This length is suf®cient to bridgebetween thick and thin ®laments, and indeed,in vitro interaction with F-actin has been reported(Moos et al., 1978). On the other hand, it wasreported that MyBP-C phosphorylation candirectly affect the conformation of the myosinheads on isolated, cardiac thick ®laments(Weinberg & Winegrad, 1996). This suggests ratherthat MyBP-C interacts with a ligand on the thick®lament, most plausibly myosin.

The complex and multiple protein-protein inter-actions in the sarcomere are highly interdependent.Mutations in a number of sarcomeric proteins havebeen identi®ed that cause familial hypertrophiccardiomyopathy (Vikstrom & Leinwand, 1996;Towbin, 1998). MyBP-C was identi®ed as one ofthe genes affected (Bonne et al., 1995; Gautel et al.,1995; Watkin et al., 1995), and a plethora of MyBP-C mutations have been identi®ed (Carrie et al.,1997; Rottbauer et al., 1997; Yu et al., 1997; Niimuraet al., 1998). Interestingly, most of these mutationsare predicted to encode mutant proteins that areC-terminally deleted due to splice donor or accep-tor-site mutations (Carrier et al., 1997; Towbin,1998). These N-terminal fragments lack their myo-sin and titin-binding regions. The deleted mutantprotein could not be detected in myocardial biop-sies, because it is either rapidly degraded or noteven translated, and it was therefore proposed thatalterations in protein stoichiometry during myo®-brillogenesis could account for the chronic deterio-
ration of myocardial function (Rottbauer et al.,
1997). On the other hand, very low levels of thetruncated fragments of MyBP-C could act as poi-son peptides that might exert an adverse effectonly over the long period of time that MyBP-C-associated FHC seems to take to manifest itselfclinically (Watkins et al., 1995). Similarly unclearare the molecular implications of a number ofmutations in b-myosin that are associated withFHC (Vosberg, 1994; Rayment et al., 1995;Vikstrom & Leinwand, 1996). Whereas mostmutations can be speci®cally ascribed to impairedfunctions of the motor domain, the implications ofthe mutations in the S2 segment of the myosin rodare obscure.

The understanding of the function of MyBP-Ctherefore requires the identi®cation of the proteininteractions of the N-terminal MyBP-C motif. Thiswill be a signi®cant advance on our way towardsan understanding of the biological function of thisenigmatic protein and of the molecular pathologyof FHC. We describe here the interactions of theregulatory domain of MyBP-C and an unexpectedlink to FHC mutations in b-myosin.

Results

MyBP-C fragments containing the regulatorydomain are incorporated into the sarcomere

Can the N-terminal region of MyBP-C interactwith other sarcomeric components in intact myo®-brils? To try and answer this question, we trans-fected neonatal rat cardiomyocytes (NRC) with theepitope-tagged fragment C1C2 of cardiac MyBP-C(the regulatory MyBP-C motif ¯anked by the twoadjacent Ig-like domains C1 and C2, which arerequired for stability; Figure 1(a)) as well as a con-trol construct, c-C2C5 (see Figure 1(a)). In the cellstransfected with c-C1C2, the protein is detectedassociated with myo®brils and in a striated pat-tern. Immuno¯uorescence of these cells with anantibody against the Z-disk portion of titin (Younget al., 1998) shows regular cross-striated myo®brils,demonstrating that the presence of c-C1C2 doesnot impair myo®brillogenesis or myo®bril integrity(Figure 2(a) to (c)). At higher magni®cation, thetransfected MyBP-C fragment is visible in broadbands of about 1.6 mm, which alternate with theZ-disks, labelled by the titin antibody (Figure 2(d)to (g)). In some cases, clear doublet bands can beseen (arrowheads in Figure 2(d)). This patternis consistent with an association with theA-bands. The c-C2C5 label was found distributeddiffusely (not shown). To gain a more biochemi-cally de®ned assay for this myo®bril association,we developed a ¯uorescence-based binding assaywith native myo®brils, using the labelled fragmentc-C1C2 as well as the control construct c-C2C5.Cardiac or skeletal myo®brils were incubated with¯uorescent c-C1C2 (c-C1C2*) and the myo®brilswere examined by ¯uorescence microscopy for sar-comeric localisation of the labelled protein. The
c-C1C2* label was found in a cross-striated pattern,

Figure 2. The N-terminal MyBP-C fragment c-C1C2 localises to the A-band in cardiomyocytes. In transfected, neo-natal rat cardiomyocytes, the c-C1C2 fragment (stained with anti-T7 in (a), (c), (d) and (f) is detected in associationwith myo®brils and in a striated pattern (arrowhead in (a) and (d)). The formation of myo®brils, visualised by stain-ing for the titin Z-disk portion (stained with anti-Z1Z2-ra in (b) and (c)) is unimpaired as compared to an untrans-fected control (asterisk) cell. Superposition of both labels shows an alternating pattern for both labels (c: T7, green;titin, red). Higher magni®cation reveals that the c-C1C2 (d) label is found in a 1.6 mm broad stripe (arrowheads in(d)) between the Z-disks labelled against titin (e). Both labels form an alternating pattern with no superposition (f: T7,green; titin, red). Scale bars represent 10 mm in (a) to (c); 5 mm in (d) to (f).


again suggesting its association with myo®brillarproteins (Figure 3). The phase-contrast images ofthe labelled myo®brils show that the labelled frag-ment localises to the A-band (Figure 3(b)). Theclear formation of doublets, excluding the barezone at the thick ®lament centre, suggests associ-ation with the head region of myosin rather thanthe tail. The control fragment c-C2C5* which doesnot contain the MyBP-C motif, shows no appreci-able sarcomeric binding (Figure 3(e)). These resultssuggest that, in addition to the previously charac-terised sorting-motif in the C-terminal region(Okagaki et al., 1993; Gilbert et al., 1995), theN-terminal region of MyBP-C contains bindingsites that can autonomously interact with a proteinof the thick ®lament. The labelled s-C1C2* frag-ment of the slow isoform of MyBP-C shows similarmyo®brillar binding (Figure 3(g)), suggesting thatthe A-band association of this fragment is a con-served property of the different isoforms.

The regulatory MyBP-C motif is a myosin-binding domain

Thick ®laments contain three major protein com-ponents: myosin, titin and MyBP-C. Since previousexperiments had shown that the N-terminal regionof MyBP-C does not interact with titin (Freiburg &Gautel, 1996), we suspected an interaction of C1C2with myosin and used a cosedimentation assay toinvestigate this interaction. Myosin can be sedi-
mented in the ultracentrifuge due to its ®lament
forming properties, which are mediated by thelight meromyosin (LMM) portion (see Figure 1(a)).In these assays, non-®lamentous or non-interactingproteins remain in the supernatant. In order to pro-vide optimal steric access to the sub-moleculardomains of the myosin molecule, myosin mini®la-ments were used that consisted of only 16-18 myo-sin molecules (Reisler et al., 1986). When myosinmini®laments were mixed with c-C1C2 fragment,the protein was consistently found in the pellet(Figure 4(a)). The C1C2 fragments alone showedno precipitation, signifying an interaction betweenthe two proteins. In addition, the skeletal slow iso-form of MyBP-C, s-C1C2, was assayed for its myo-sin-binding properties. Both MyBP-C isoforms(cardiac and slow) bind to mini®laments(Figure 4(a)). The observation that C1C2 binds tomyosin irrespective of the MyBP-C isoformsuggests further that this interaction is a generalfeature of the MyBP-C molecule.

To specify this interaction within the C1C2 frag-ment, the overlapping fragments c-C0C1 andc-C2C5 were examined in the same manner. Con-trols contained bovine serum albumin (BSA; notshown), which shows no detectable cosedimenta-tion. Figure 4(b) shows that cosedimentation ofc-C0C1 and c-C2C5 with myosin is not mediatedby the Ig-like domains, as both ¯anking constructsc-C0C1 and c-C2C5 show no interaction (compar-able to BSA). This localises the myosin-binding siteof C1C2 to the �100 residue MyBP-C motif, which
was identi®ed as the phosphorylation domain of

Figure 3. A myo®bril-bindingassay reveals that the C1C2 frag-ment of MyBP-C associates withthe sarcomeric A-band. Cardiac((a) and (b)) or skeletal ((c) to (h))myo®brils were incubated with¯uorescence-labelled c-C1C2* orc-C2C5*. The c-C1C2* fragment isfound in a striated pattern in doub-let bands (A and C) and compari-son with the phase-contrast images(b), (d), (f) and (h) shows the labelto be localised to the A-band((b) and (d)). The c-C2C5* fragmentcould not be found in associationwith sarcomeric structures ((e) and(f)), indicating a very weak orabsent interaction. The slow MyBP-C s-C1C2* fragment is similarlybound to the A-band ((g) and (h)).The arrowheads mark the centre ofthe same A-band in each micro-graph. Scale bar represents 10 mm.


cardiac MyBP-C (Gautel et al., 1995). Furthermore,these results con®rm those of the ¯uorescence-based myo®bril-binding assay and identify myosinas the thick-®lament ligand of the MyBP-C motif.

The af®nity of the myosin interaction was deter-mined by titration of myosin mini®laments withc-C1C2. The mixtures were sedimented and pro-teins in supernatants and pellets quanti®ed asdescribed in Materials and Methods. The bindingwas found to be saturable at a 1:1 stoichiometry ofthe complex (one mole c-C1C2 per mole of myo-sin), and a binding constant of 6.8 mM was deter-mined using a quadratic ®t (Figure 4(c)).

The proximal 126 residues of myosinsubfragment S2 contain the binding site forthe MyBP-C motif

Which region of myosin, if not LMM, wouldinteract with the MyBP-C motif? In order to mapthe interaction on myosin, we investigated thecompetitive effect of soluble, proteolytic myosinsubfragments on the binding of c-C1C2 in cosedi-mentation assays. Any soluble myosin subfrag-ment containing the MyBP-C binding site isexpected to reduce the presence of c-C1C2 in thepellet, due to its competition with the mini®la-ments for MyBP-C binding. The subfragment
heavy meromyosin (HMM) reduces the presence of
c-C1C2 in the pellet (Figure 5(b)), thus clearlyexcluding subfragment LMM as the binding site.Subfragment S1, which represents only the myosinheads, shows no competition (Figure 5(c)),indirectly implying that binding occurs in the S2portion of the myosin molecule. To gain a morevariable tool for the suspected interaction in thisregion, we used recombinant S2, where mutationscan be introduced in a controlled way. Expressionof recombinant S2 (human b-myosin) in Escherichiacoli yielded a poorly separable mixture of predomi-nantly C-terminally truncated S2 of 34 kDa (corre-sponding to native short S2, the N-terminal portionof long S2; Lu, 1980) and traces of the desired full-length S2 of 59 kDa. The mini-S2 competes forC1C2 binding to full-length myosin in a mannersimilar to that of HMM (Figure 5(d)), indicatingthat it contains all the features necessary for bind-ing. For further sub-mapping, we expressed theN-terminal portion of mini-S2, S2-�, in E. coli.S2-� contains the 126 residues 848 to 963 of thehuman cardiac b-MHC and spans the 18 N-term-inal heptad repeats of the S2 coiled-coil. This frag-ment competes for c-C1C2 binding (Figure 5(e)) inthe same manner as HMM and mini-S2. Theseresults demonstrate that myosin contains a bindingsite for the regulatory domain of MyBP-C in the
proximal 126 residues of S2.

Figure 4 (legend opposite)


Figure 5. The binding site for theMyBP-C motif is localised withinthe proximal 126 residues of myo-sin S2. Protein compositions ofsupernatants (S) and pellets (P)after ultracentrifuge sedimentationof a mixture of 2.5 mM myosinmini®laments and 10 mM c-C1C2containing the following proteo-lytic myosin subfragments: nocompetitor (a), 6 mM HMM(b), 5 mM S1 (c), 9 mM mini S2 (d)and 1.5,3 and 8 mM S2-� (e-1, e-2and e-3). The amount of pelletloaded from these independentexperiments varies by a factor of 2

for (d) and (e) compared to the control. The myosin fragments HMM and the subfragments mini-S2 and S2-�reduce the amount of C1C2 in the myosin pellet. The arrow marks the position of the C1C2 fragment, a smallarrow marks the S2-� fragment in (e).


We veri®ed the interaction of c-C1C2 and itsslow ®bre equivalent, s-C1C2, with S2-A by anindependent method, isothermal titration calorime-try (ITC). This method allows us to measure pro-tein interactions in solution and hence does notrequire surface immobilisation. ITC provides adirect route to the complete thermodynamiccharacterisation of bimolecular equilibrium inter-actions. One component is titrated against theother and the molar reaction enthalpy �RH

O as wellas the association constant KB(�1/Kd) can be deter-mined by direct measurement of the heat of thereaction. Furthermore, the stoichiometry n of theinteraction can be obtained from the molar ratio atthe equivalence point (Ladbury & Chowdhry,1996, and references therein). The pro®le of thetitration curve is dictated by the total concentrationof the macromolecule in the cell (Mtot) and by theassociation constant KB. The product of these quan-tities de®nes the experimental window in whichcon®dence of data ®tting is highest. It is only therange of Mtot � KB from �1 to 1000 that allowsaccurate determination of KB (Wiseman et al.,1989); outside this window, the titration curves aretoo shallow or too steep, respectively. Figure 6shows titrations of c-C1C2 and s-C1C2 with S2-�carried out at 15 �C. The results are summarised inTable 1.

The Kd values obtained are internally consistent
and in good agreement with the binding constant
Figure 4. (a) The C1C2 fragment contains a myosin-bindinnatants (S) and pellets (P) of the cosedimentation assay of aution of c-C1C2 (2) (blind controls); and a mixture of 2.5 mMThe c-C1C2 fragment is found in the pellet only in the pre(s-C1C2 7.5 mM, mini®laments, l mM) is found in the myosinlised in the MyBP-C motif. In the cosedimentation assay withC1C2 fragment is found in the pellet. c-COC1 (1), c-C1C2 (ments, 10 mM. Arrowheads in (a) and (b) indicate the positmyosin mini®laments was titrated with various concentrationtrifugation and the complex that was formed was quanti®edthe concentration of c-C1C2 (�bound) in the complex versuthe data yields saturation at 2.6 (�0.2) mM and a bindingmyosin form a complex of one C1C2 fragment per myosin.

determined in the cosedimentation assay (6.8 mM).However, for the higher concentration of c-C1C2, astoichiometry for the interaction of 0.85 was deter-mined as well as a reaction enthalpy 15 % belowthe value for the lower concentration of c-C1C2.Although this is a tolerably small variation, it canbe explained by the fact that C1C2 tends to aggre-gate in low salt buffers at higher concentrations asdescribed earlier for the native protein (Starr &Offer, 1978). For this reason, titrations at higherconcentrations of C1C2 were not carried out(which would place the experiment in a morefavourable range of Mtot � KB). Additionally, thetitration of c-C1C2 (20 mM) with S2-� (350 mM)was repeated at 25 �C and the parameters n (1.11),�RH

O (ÿ6730 cal/mol) and Kd (11 mM) were deter-mined (data not shown). However, this decreasingaf®nity at 25 �C places the experiment(Mtot � KB � 2) close to the lower edge of the acces-sible window.

Structurally silent mutations in the myosin S2coiled-coil that cause FHC abolish theinteraction with the MyBP-C motif

The myosin molecule gene is the target for var-ious mutations that cause FHC (Rayment et al.,1995; Vikstrom & Leinwand, 1996). Whereas mostFHC mutations in b-MHC can be structurally and
functionally interpreted (Rayment et al., 1995), the
g site as detected in cosedimentation assays. The super-2.5 mM myosin mini®lament solution (1), a 10 mM sol-

myosin mini®laments and 10 mM c-C1C2 (3) are shown.sence of myosin. Similarly, the slow MyBP-C fragment

pellet (4). (b) The myosin-binding site in C1C2 is loca-overlapping N-terminal fragments of MyBP-C, only the

2) and c-C2C5 (3). Mini®laments, 2.5 mM; MyBP-C frag-ions of the MyBP-C fragments. (c) A 2.5 mM solution ofs of c-C1C2. The mixtures were sedimented by ultracen-as described in Materials and Methods. The plot shows

s the total concentration of c-C1C2. Quadratic ®tting ofconstant of 6.8 (�1.4) mM. This suggests that C1C2 and

Figure 6. Isothermal calorimetry demonstrates the liquid-phase interaction of S2-� with C1C2. S2-� is titratedagainst C1C2 in regular time intervals and the reaction heat is monitored as a change of heating current (mcal/second;upper panel). Integration of the resulting peaks yields the titration plot (lower panel). Different ratios of C1C2 andS2-� were titrated and result in similar binding curves. (a) 20 mM c-C1C2 and 350 mM S2-�;(b) 30 mMc-C1C2 � 550 mM S2-�. The titration of slow MyBP-C s-C1C2 (23 mM s-C1C2 � 290 mM S2-�) shows comparablebinding (c). The experiments demonstrate the interaction of both proteins in a 1:1 complex and yields dissociationconstants between 2.2 mM (s-C1C2) and 4.5 mM (c-C1C2) for the interaction (see Table 1 for kinetic parameters).


role of the mutations in the myosin rod has been asubject for speculation up to now. All pointmutations in the rod that have been reported(Vikstrom & Leinwand, 1996, and references there-in) are, however, located on the S2-� fragment,and it is an obvious possibility that they mightin¯uence the binding of MyBP-C.

To test this hypothesis, two of these mutations(R870H and E924K) were mimicked by site-directed mutagenesis of the respective positions inthe S2-� fragment. The binding properties of themutants were investigated by ITC (Figure 7) andthe results are summarised in Table 2. For the
mutant R870H, the af®nity decreases by approxi-
mately one order of magnitude. Since the stoichi-ometry n of the interaction was previouslydetermined for the wild type, n was set at 1.00 toavoid multiple minima during data ®tting. A bind-ing constant of approximately 62 mM wasobtained; however, the af®nity is so weak that thevalue of Mtot � KB � 0.3 is outside the experimentalwindow that allows accurate determination of thebinding constant. For the mutant E924K, only theheat of dilution was detected, indicating that thischarge reversal mutation inhibits binding ofMyBP-C more strongly (Figure 7).

Mutations in myosin S2 could affect the stability
of the coiled-coil and thus indirectly in¯uence the

Table 1. Parameters for the binding C1C2 to S2-� at 15�C

Isoform C1C2 (mM) S2-� (mM) n �RHO (cal/mol) KB (Mÿ1) Kd (mM) SD (mM) Mtot � Kb

c-C1C2 20 350 0.96 ÿ9760 233,000 4.3 0.5 5c-C1C2 30 550 0.85 ÿ8250 220,000 4.5 0.7 7s-C1C2 23 290 1.08 ÿ9339 456,000 2.2 0.2 10


interaction with MyBP-C. We examined the wild-type S2-� and both point mutants by circulardichroism (CD) spectroscopy to detect possiblechanges in the content of a-helix that would signifya destabilisation of the coiled coil. Figure 8(a)shows the CD spectra of the three S2-� constructs.All three spectra superimpose and show the
characteristic pro®le of a-helical proteins,
Figure 7. Mutations in b-myosin S2-� associated with Fmutants (254 mM R870H and 390 mM E924 K) were titratedc-C1C2 for the mutant R870H was detected (a) with a Kd

could be detected but no interaction with c-C1C2 (b). Similthe E924 K mutant (c). These data show that no detectable b

suggesting that no major structural changes haveoccurred in any of the mutants. To verify that themutants still form homodimers, analytical gel ®l-tration was used and shows that all constructselute in symmetrical peaks that superimpose(Figure 8(b)). Again, this suggests that the FHC-associated mutations do not result in gross struc-
tural changes and destabilisation of the coiled-coil.
HC reduce or abolish the interaction with C1C2. S2-�against c-C1C2 (20 mM). A marked decrease in af®nity of� 60 mM. For the E924 K mutation, the heat of dilutionarly, the interaction of s-C1C2 with S2-� is abolished ininding occurs between the E924 K mutant and C1C2.

Table 2. Parameters for the binding of cC1C2 to S2-� (wild-type, WT) in comparison to the FHC mutation R870Hand E924K

cC1C2 (mM) S2-� (mM) n �RHO (cal/mol) KB (Mÿ1) Kd (mM) SD (mM) Mtot � Kb

Cardiac WT 0.96 ÿ9760 233,000 4.3 0.5 520 350Cardiac R870H 1.00 (ÿ28150) (16,000) (62) 19 0.320 254 (fixed)Cardiac E924K - - - - - -20 390Slow E924K - - - - - -20 390


Discussion

The function of the myosin-binding proteinfamily in vertebrate striated muscle has remainedelusive. It was suggested that MyBP-C could regu-late the assembly of thick ®laments in agreementwith the early expression of the protein duringmyo®brillogenesis (Obinata et al., 1984; BaÈhler et al.,1985; Lin et al., 1994; Koshida et al., 1995). Theobservation that the cardiac isoform can be phos-phorylated upon b-adrenergic stimulation of myo-cardium by cAMP-dependent protein kinase(cAPK), as well as by an associated Ca2�/calmodu-lin-dependent kinase, suggested a role in the regu-lation of muscle contraction (Jeacocke & England,1980; Hartzell & Titus, 1982; Hartzell & Glass,1984; Garvey et al., 1988; Schlender & Bean, 1991;Gautel et al., 1995). Mutations in a number of sar-comeric proteins, including MyBP-C, have beenidenti®ed that cause familial hypertrophic cardio-myopathy (reviewed by Vikstrom & Leinwand,1996; Towbin, 1998). Interestingly, most of theMyBP-C mutations are predicted to encode mutantproteins that are C-terminally deleted. TheseN-terminal fragments lack their myosin and titin-binding regions, and are predicted to be deloca-lised, on the basis of the earlier transfection studiesby Gilbert et al. (1995). Our myocyte transfectionand in vitro assays show a clear association of theN-terminal MyBP-C fragment C1C2 with theA-bands of cardiac or skeletal myo®brils. This is incontrast to the observations by Gilbert et al. (1995),who reported that N-terminal fragments show nosarcomeric association. Only the C-terminal frag-ment �l-6 (residues 761-1132) of the fast MyBP-Cisoform was competent for correct A-band target-ing. The difference between the observations ofGilbert et al. (1995) and ours may reside in differentexpression levels of the vectors used in transfectionexperiments, due to different promoter strengths,or the protein concentrations that can be usedin vitro which are favourably close to the Kd thatwe determined experimentally.

Previous investigations suggested that thecAPK-mediated phosphorylation of c-MyBP-C isassociated with structural changes of the myosincrossbridges (Weinberg & Winegrad, 1996). Ourobservation that the N-terminal phosphorylationdomain of MyBP-C binds to thick ®laments points
to an interaction with myosin itself. We mapped
this interaction to the �100 residue MyBP-C motif.This domain is highly conserved between speciesand isoforms, with only two exceptions: thecardiac-speci®c phosphorylation loop insertion andan isoform-speci®c region rich in charged residues(Figure 1(c)). Both cardiac and skeletal C1C2 frag-ments show similar binding behaviour in thecosedimentation assay and in ITC. We thereforeconclude that this interaction is a general feature ofthe regulatory domain of MyBP-C. This ®nding isfurther supported by the work of Okagaki et al.(1993), who reported the interaction of a chymo-tryptic 100 kDa fragment of chicken skeletalMyBP-C (lacking the major LMM binding site inthe C10 domain but containing the regulatorydomain) with myosin.

Our results also reveal the existence of a secondbinding site for MyBP-C on myosin, different fromthat on the LMM subfragment. This second bind-ing site resides within the proximal 126 amino acidresidues of myosin S2, immediately following thehead-tail junction and adjacent to the binding sitefor the regulatory myosin light chains. The af®nityof the interaction is in the low micromolar rangeand thus in a reasonable range for a sarcomericprotein interaction. Our observations are in excel-lent agreement with the results of Starr & Offer(1978), who observed S2 binding of nativeMyBP-C. It is interesting to note that the dimericcoiled-coil of myosin S2 interacts with the MyBP-Cmotif as a complex of one myosin dimer to oneMyBP-C motif. How this molecular interaction isbrought about at the molecular level remains to beelucidated.

Mutations in b-MHC and MyBP-C account forabout two-thirds of all cases of FHC. In MyBP-C,predominantly mutations that encode a C-termin-ally truncated protein were detected (Bonne et al.,1995; Watkins et al., 1995; Carrier et al., 1997).Some point mutations, however, are not thought tolead to a truncated protein, but can be explainedneither mechanistically nor, in all cases, structu-rally (Carrier et al., 1997; Yu et al., 1997; Niimuraet al., 1998). In the case of b-MHC, at least 40mutations have been associated with FHC(Vikstrom & Leinwand, 1996, and references there-in). The majority of them can be explained by com-parison with the atomic structure of myosin S1:they occur in clusters around speci®c sites associ-
ated with actin binding, ATP binding or light

Figure 8. (a) The mutations in b-myosin S2-� do not result in detectable changes in a-helical content as measuredby CD spectroscopy. CD spectra were recorded at equimolar concentrations and superimposed; the three S2 frag-ments show no discernible difference in their helix contents. (b) The dimerisation of S2 is not impaired by FHC-associated point mutations. Gel ®ltration was carried out using identical amounts of proteins and an equimolar mix-ture of the two mutants and the wild-type S2-�. All constructs elute at identical retention volumes; the mixture ofwild-type and mutant protein shows no broadening of the peak and elutes at the same retention volume as the indi-vidual component. Note that subtle structural changes in the mutant S2 constructs would escape detection by thesemethods. Numbers above marker trace: molecular mass in kDa.


chain binding, or contain the two reactive cysteineresidues (Rayment et al., 1995). The ®ve knownmutations in the S2 portion of the rod have beenspeculated to affect assembly of the thick ®lamentor the overall stability of the protein (Raymentet al., 1995). Measurements of the in vitro actinmotility generated by a few such mutated myosins
revealed only very mild impairment of the motor
function (Cuda et al., 1997). All of these ®ve S2mutations are located in the segment of the head-tail junction that interacts with MyBP-C. A mul-tiple sequence alignment of this region, comparingmyosins from striated muscle, smooth muscle, obli-quely striated muscle and non-muscle sources,reveals that most of the mutations that can cause
FHC occur at positions speci®c for sarcomeric


myosins (Figure 9). The mutations do not follow aspeci®c pattern but occur at the positions A, c, D, e
and f of the coiled-coil heptad repeat. In fact, two
Figure 9. Multiple sequence alignment of the proximal 12isoforms. Identical residues are boxed. The Swissprot entriemuscle (top rows), sarcomeric striated muscle (middle roaligned and identical residues are boxed. The residue numbthe myosins. the positions of the coiled-coil heptad repeatmarked in capital letters. Note that S2-� is essentially identisin S2 associated with FHC are shaded in grey. These positidue, speci®c for sarcomeric myosins. Asterisks mark the twand E924K. Note that the position of E924 (second asterisk)elegans is a lysine. Mysg Chick, chicken gizzard MHC; Mhuman non-muscle MHC B; Mysn Chic, chicken non-mMyss Chick, chicken adult slow MHC. Mysa Rat, rat cardiaChick, chicken cardiac a-MHC; Mys Aeqir, Aequipeten irradA; Mysb-Caeel, C. elegans MHC-B; Mysc Caeel, C. elegans M

of the charge-reverse exchanges associated withFHC occur naturally in the myosins from obliquely
striated nematode muscle (Figure 9). One of these
6 residues of myosin S2 (S2-�) from various species ands of myosins from smooth muscle and mammalian non-ws) and nematode/mollusc or amoebic myosins wereering starts arbitrarily at 1, due to the different sizes ofare marked on top; the ®rst position of each heptad iscal between all sarcomeric myosins. Mutations in b-myo-ons are, with the exception of the conserved leucine resi-o b-myosin mutations investigated in this study, R870Hin the naturally occurring myosin-C from Caenorhabolitisyst Rabit, rabbit smooth muscle MHC; Myso Human,uscle MHC. Myse Human, human embryonic MHC;c a-MHC; Mysb Human, human cardiac b-MHC; Mysc -ians striated muscle MHC; Mysa Caeel, C. elegans MHC-HC-C; Mys2 Dicdi: Dictoystelium discoideum MHC-2.


positions, E924K, is represented in our study. Thisresidue is in an f position that is outside the coiled-coil dimerization surface, which is a position thatis particularly exposed. It is interesting that thisresidue results in no detectable structural changesbut leads to a total loss of MyBP-C binding. TheR870H mutation occurs in an A position. This pos-ition is part of the coiled-coil interface. However,histidine is found in A positions in some coiledcoils, and even in the H position of the Max leucinezipper (Muhle-Goll et al., 1995). Again, thismutation does not result in obvious structuralchanges in the S2 dimer (Figure 8); however, theeffect on MyBP-C binding is also less pronouncedthan that of the exposed E924. It therefore seemsthat a destabilisation of the coiled-coil is not gener-ally responsible for the pathogenesis of FHC bymyosin S2 mutations.

The mutation R870H reduced the af®nity of S2for MyBP-C by at least one order of magnitude,while E924K showed no detectable binding at all.The b-myosin S2 mutations impaired the motorfunction at worst slightly (Cuda et al., 1997), whilethey drastically in¯uence the interaction withMyBP-C. This interference with a crucial protein-protein interaction could make the mutated myosina true poison polypeptide. Conversely, the deloca-lised MyBP-C peptides predicted for the majorityof the FHC-associated mutant MyBP-C genes canbe envisaged to compete for the S2 binding sitewith the intact protein. Although the analysis ofcardiac biopsies of one such mutation revealed nodetectable levels of mutant protein (Rottbauer et al.,1997), very low levels of competing protein mightsuf®ce to induce long-term decompensation ofcardiac function by competing for the S2-bindingsite with wild-type MyBP-C. Our transfectionexperiments in cardiomyocytes revealed that theN-terminal MyBP-C fragments do not exert adominant-negative effect on myo®bril integrity,in agreement with the results found in skeletalmyocytes by Gilbert et al. (1996). Consequently,we suggest that the S2 mutations in b-myosin S2manifest themselves by their altered interactionwith the regulatory MyBP-C motif.

Although single-headed myosin is suf®cient togenerate movement in vitro and occurs in severalnon-muscle myosins, the naturally occurring myo-sin II in muscle is two-headed (Cope et al., 1996).The interplay between the two heads is still poorlyunderstood. This applies especially for the role ofthe S2 segment. Skinned ®bre experiments andin vitro motility assays, where the S2 portion wasbound by speci®c antibodies, were interpreted asevidence for a direct involvement of the S2 regionin the force generation by myosin (Margossianet al., 1991; Sugi et al., 1992). However, sincemyosin can be immobilised at the S1-S2 junctionwithout loss of motility in other assays, and S1alone in fact suf®ces to generate movement, theseobservations are not easily interpreted. Molecularmodelling of the junction between S1 and S2, and
investigations of the dynamic behaviour of this
region, suggest that the two heads are stericallyconstrained (Offer & Knight, 1996) and thatdynamic opening of the S2 junction could makeboth heads available for actin binding (Knight,1996). In fact, actin ®laments decorated with thetwo-headed HMM fragment show the same opticaldiffraction patterns as ®laments decorated withsingle-headed S1, indicating that the two heads inHMM are equally available for actin binding,despite their predicted steric hindrance (Craig et al.,1980). This was taken as a suggestion that a revers-ible opening of the proximal segment of the coiled-coil in S2 could occur upon actin binding (Knight,1996). In the two-headed kinesin motors, the neckforms a special coiled-coil dimer with two distinctsubsegments. The segment adjacent to the motordomains was proposed to be capable of reversiblefolding-unfolding events (ThormaÈhlen et al., 1998)that would allow the processive movement of thetwo-headed motor. By analogy, reversible confor-mational changes in the head-tail junction of myo-sin may play an important role in force generationin muscle (Knight, 1996), although the underlyingmechanics is different. It is compelling to speculatethat ligands of this region like MyBP-C may modu-late the ¯exibility of the head-tail junction. Themolecular details of how MyBP-C binding to S2could control the activity of the motor domain,and how this action is modulated by the phos-phorylation of the cardiac isoform must now beelucidated.

Materials and Methods

Neonatal cardiomyocyte culturesand immunofluorescence

Primary cultures of neonatal rat cardiomyocytes wereprepared as described (Sen et al., 1988; Komiyama et al.,1996). MyBP-C fragments were cloned into a modi®edpCMV-5 vector (Andersson et al., 1989) bearing anN-terminal phage T7 tag. Plasmid DNA was transfectedusing a modi®ed calcium phosphate protocol (Komiyamaet al. 1996). Following transfection, cells were culturedfor another 72 hours before ®xing and staining followingstandard methods (Komiyama et al., 1996). The transfectedMyBP-C fragments were detected with the mouse mono-clonal anti-T7 tag antibody (Novagen). Counterstainswere carried out using a polyclonal rabbit antibodyagainst Z-disk titin, a-Z1Z2-ra (Young et al., 1998).

Protein purification and expression

MyBP-C fragments were expressed solubly in E. coliand prepared essentially as described (Gautel et al., 1995;Freiburg & Gautel, 1996). Myosin fragments were clonedby PCR from total human cardiac cDNA. Expression ofmyosin S2-fragments in E. coli was performed using thepET expression system (Studier et al., 1990). His6-taggedprotein was puri®ed on Ni2� NTA columns followingthe manufacturer's instructions (Qiagen, Germany) andfurther puri®ed by anion exchange chromatography on aMonoQ column (Pharmacia, Sweden). Analytical gel ®l-tration was carried out on a Superose-12 column
(Pharmacia, Sweden) in buffer C at a ¯ow rate of


0.5 ml/min. In some experiments, the His6 tag wascleaved off by recombinant TEV protease without aneffect on the binding properties. Mutagenesis of S2 byPCR assembly followed standard protocols (Ausubelet al., 1987); the mutations were veri®ed by DNA sequen-cing. Myosin (rabbit, skeletal) and its proteolytic subfrag-ments, S1 and heavy meromyosin, were preparedaccording to the method by Margossian & Lowey (1982);HMM and S1 were kind gifts from Mike Geeves andNancy Adamek. Myosin mini®laments were prepared asdescribed (Reisler et al., 1986) from myosin that had beenpuri®ed by repeated precipitation. Concentration deter-mination was carried out by UV spectroscopy, andN-terminal peptide sequencing in the case of S2.

Buffer

Buffer A (myosin mini®laments): 10 mM Tris/citrate(pH 8.0). Buffer B (cosedimentation assay): 20 mM Tris-HCl (pH 7.0), 200 mM NaCl, 1 mM DTE, 1 mM EDTA.Buffer C (ITC): 20 mM MES/NaOH (pH 7.0), 50 mMNaCl, 1 mM DTE, 1 mM EDTA.

Myofibril binding assays

Recombinant MyBP-C fragment were labelled withisothiocyanate/rhodamine essentially as described(Harlow & Lane, 1988) at a calculated molar ratio of�1:1. The labelled proteins (C1C2* and C2C5*) wereadjusted to buffer C and a protein concentration of�15 mM. Myo®brils from rabbit psoas or heart musclewere prepared according to the method by Knight &Trinick (1982) and were sedimented for 30 minutes onice onto glass coverslips that had been coated with1 mg/ml poly-L-lysine solution. After immobilisation,the myo®brils were blocked for ten minutes with 1 %BSA in buffer C. The myo®brils were then incubatedwith C1C2* or C2C5* on ice. After 20 minutes of incu-bation, the myo®brils were brie¯y washed with 2 � 50 mlof buffer C, and subsequently with 50 ml of 3 % formal-dehyde in buffer C (for photography), and then mountedin buffer C, 50 % glycerol for microscopy. Formaldehyde®xation did not change the protein distribution but pre-vented diffusion of the bound protein from the myo®bril.In some cases, standard immuno¯uorescence using theanti-titin antibody TZ1-ra (Gautel et al., 1996) and FITC-labelled secondary antibody was performed prior to label-ling with C1C2* or C2C5* to obtain a counterstain forthe MyBP-C fragments. Labelled myo®brils were viewedunder a Zeiss Axiomat microscope ®tted with a CCDcamera, and ¯uorescence and phase contrast imageswere recorded.

Cosedimentation assay

All proteins (except myosin mini®laments) were dia-lysed into buffer B and centrifuged prior to use at 4 �Cfor 30 minutes at 400,000 g using a Beckman TLA 100.1rotor and an OptimaTM TL ultracentrifuge. Appropriateamounts were mixed in Beckman polycarbonate centri-fuge tubes (no. 343776) and the volume was made up to25 ml with buffer B. Myosin mini®laments (25 ml of 5 mMin buffer A) were added to the mixture to give a ®nalvolume of 50 ml, pH 7.3, and a NaCl concentration of100 mM. The mixture was incubated at 4 �C for 30 min-utes and subsequently centrifuged for 20 minutes at400,000 g. The supernatant was removed and the pellet
was washed twice with 100 ml of a 1:1 mixture of buffer
A and buffer B. The pellet was re-dissolved in 50 ml of7 M urea, and appropriate amounts of supernatant andpellets were analysed by SDS/PAGE, as described(Laemmli, 1970). Correction of the quanti®cation wasthen carried out using the volume factor of the amountof pellet loaded. Gels were stained for 24 hours in stain-ing solution (1.5 % (w/v) Coomassie brilliant blue R250,40 % ethanol, 10 % acetic acid) and subsequentlydestained for 48 hours. For quantitative analysis, the wetgel was scanned using a Mustek SP1200 scanner andquanti®cation of the bands was performed with the soft-ware Intelligent Quanti®er from Bioimage, USA. Theconcentration of bound C protein fragments was calcu-lated from the relative intensities of supernatant and pel-let, and the known total concentration. Data for thecomplex formed were ®tted using a quadratic ®t withthe equation: (Bmax[(M � C � K) ÿ p((M � C � K)2

ÿ 4MC))/2M, where Bmax is the amplitude, M the con-centration of myosin and C the concentration of C1C2.

Isothermal titration calorimetry, CD spectroscopyand analytical gel filtration

All proteins were dialysed into buffer C and centri-fuged at 100,000 g for ten minutes immediately prior touse. Calorimetric experiments were carried out using atitration calorimeter from Microcal, USA, with a 250 mlinjection syringe, while stirring at 400 rpm. The concen-trations of the myosin S2 constructs in the syringe weregenerally 10-20 times higher than the C1C2 concen-trations in the reaction cell. The reference cell was ®lledwith 1 mM sodium azide. An initial injection was per-formed with a small volume, 0.25 of the experimentalinjection volume (5-10 ml). Data analysis was performedwith the manufacturer's software. Experimental valuesfor the binding constant, heat of binding, and stoichio-metric ratio were determined from deconvolution usingnon-linear least-squares minimisation. The values forheat of dilution were determined in independent exper-iments and subtracted from the raw data before dataanalysis.

CD spectra were recorded on Jasco J710 in 50 mMsodium phosphate buffer(pH 6.8) using a 1 mm cuvette.Raw data were baseline corrected and analysed with themanufacturer's software. The spectra of S2 subfragmentsare typical for a-helical proteins.

Analytical gel ®ltration was carried out on a Pharma-cia Superdex 200 10/30 column in 20 mM sodium phos-phate (pH 7) 300 mM NaCl, 1 mM EDTA at a ¯ow rateof 1 ml/min. Protein detection was carried out at254 nm due to the absence of aromatic residues in S2.Elution pro®les were compared with a set of standardmarker proteins (BioRad).

Sequence alignment

Sequences were extracted from the Swissprot datalibrary and aligned using the GCG package and Clus-talW (Thompson et al., 1994).

Acknowledgements

We are grateful to Roger S. Goody for his generoussupport, to Christian Herrmann for valuable discussionsabout ITC, to Evelyne and Jean-Claude Perriard for
initial help with the cardiomyocyte cultures, and to


Annalisa Pastore and Belinda Bullard for critical com-ments and reading of the manuscript. We are gratefulfor the EMBL DNA and peptide sequencing services fortheir support. This work was supported by the DeutscheForschungsgemeinschaft (Ga405/3-2).

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revised form 4 January 1999; acce

Edited by J. Karn

pted 4 January 1999)

mutations in β-myosin s2 that cause familial hypertrophic cardiomyopathy (fhc) abolish the...

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