Dissecting the N-terminal Myosin Binding Site of Human Cardiac Myosin-binding Protein C

Abstract

Myosin-binding protein C (MyBP-C) binds to myosin with two binding sites, one close to the N terminus and the other at the C terminus. Here we present the solution structure of one part of the N-terminal binding site, the third immunoglobulin domain of the cardiac isoform of human MyBP-C (cC2) together with a model of its interaction with myosin. Domain cC2 has the β-sandwich structure expected from a member of the immunoglobulin fold. The C-terminal part of the structure of cC2 is very closely related to telokin, the myosin binding fragment of myosin light chain kinase. Domain cC2 also contains two cysteines on neighboring strands F and G, which would be able to form a disulfide bridge in a similar position as in telokin. Using NMR spectroscopy and isothermal titration calorimetry we demonstrate that cC2 alone binds to a fragment of myosin, S2Δ, with low affinity (kD = 1.1 mm) but exhibits a highly specific binding site. This consists of the C-terminal surface of the C′CFGA′ β-sheet, which includes Glu301, a residue mutated to Gln in the disease familial hypertrophic cardiomyopathy. The binding site on S2 was identified by a combination of NMR binding experiments of cC2 with S2Δ containing the cardiomyopathy-linked mutation R870H and molecular modeling. This mutation lowers the binding affinity and changes the arrangement of side chains at the interface. Our model of the cC2-S2Δ complex gives a first glimpse of details of the MyBP-C-myosin interaction. Using this model we suggest that most key interactions are between polar amino acids, explaining why the mutations E301Q in cC2 and R870H in S2Δ could be involved in cardiomyopathy. We expect that this model will stimulate future research to further refine the details of this interaction and their importance for cardiomyopathy. Myosin-binding protein C (MyBP-C) binds to myosin with two binding sites, one close to the N terminus and the other at the C terminus. Here we present the solution structure of one part of the N-terminal binding site, the third immunoglobulin domain of the cardiac isoform of human MyBP-C (cC2) together with a model of its interaction with myosin. Domain cC2 has the β-sandwich structure expected from a member of the immunoglobulin fold. The C-terminal part of the structure of cC2 is very closely related to telokin, the myosin binding fragment of myosin light chain kinase. Domain cC2 also contains two cysteines on neighboring strands F and G, which would be able to form a disulfide bridge in a similar position as in telokin. Using NMR spectroscopy and isothermal titration calorimetry we demonstrate that cC2 alone binds to a fragment of myosin, S2Δ, with low affinity (kD = 1.1 mm) but exhibits a highly specific binding site. This consists of the C-terminal surface of the C′CFGA′ β-sheet, which includes Glu301, a residue mutated to Gln in the disease familial hypertrophic cardiomyopathy. The binding site on S2 was identified by a combination of NMR binding experiments of cC2 with S2Δ containing the cardiomyopathy-linked mutation R870H and molecular modeling. This mutation lowers the binding affinity and changes the arrangement of side chains at the interface. Our model of the cC2-S2Δ complex gives a first glimpse of details of the MyBP-C-myosin interaction. Using this model we suggest that most key interactions are between polar amino acids, explaining why the mutations E301Q in cC2 and R870H in S2Δ could be involved in cardiomyopathy. We expect that this model will stimulate future research to further refine the details of this interaction and their importance for cardiomyopathy. Regulation of myogenesis and muscle contraction are based on a complex network of protein-protein interactions. The importance of large multidomain proteins such as Nebulin, Titin, and MyBP-C 2The abbreviations used are: MyBP-C, myosin-binding protein C; FHC, familial hypertrophic cardiomyopathy; LMM, light meromyosin; ITC, isothermal titration calorimetry; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence.2The abbreviations used are: MyBP-C, myosin-binding protein C; FHC, familial hypertrophic cardiomyopathy; LMM, light meromyosin; ITC, isothermal titration calorimetry; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear single quantum coherence. for the correct assembly of muscle proteins is well established (1Labeit S. Barlow D.P. Gautel M. Gibson T. Holt J. Hsieh C.L. Francke U. Leonhard K. Wardale J. Trinick J. Nature. 1990; 345: 273-276Crossref PubMed Scopus (175) Google Scholar, 2Trinick J. Trends. Biochem. Sci. 1994; 19: 405-409Abstract Full Text PDF PubMed Scopus (179) Google Scholar), and for at least one of them, MyBP-C, there is also evidence for its contribution to the regulation of muscle contraction via an influence on myosin head group arrangement (3Gautel M. Zuffardi O. Freiburg A. Labeit S. EMBO J. 1995; 14: 1952-1960Crossref PubMed Scopus (324) Google Scholar, 4Weisberg A. Winegrad S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8999-9003Crossref PubMed Scopus (147) Google Scholar, 5Winegrad S. Circulation. 2000; 86: 6-7Crossref Scopus (42) Google Scholar, 6McClellan G. Kulikovskaya I. Winegrad S. Biophys. J. 2001; 81: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7Kunst G. Kress K.R. Gruen M. Uttenweiler D. Gautel M. Fink R.H.A. Circulation. 2000; 86: 51-58Crossref Scopus (179) Google Scholar). MyBP-C is a large (∼140 kDa) multidomain protein (8Bennett P.M. Fürst D.O. Gautel M. Rev. Physiol. Biochem. Pharmacol. 1999; 138: 204-234Google Scholar, 9Winegrad S. Circulation. 1999; 84: 1117-1126Crossref Scopus (127) Google Scholar) that is located in the thick filament (10Offer G. Moos C. Starr R. J. Mol. Biol. 1973; 74: 653-676Crossref PubMed Scopus (489) Google Scholar), where it is attached to myosin via two binding sites: one in its C terminus for the LMM subfragment (11Miyamoto C.A. Fishman D.A. Reinach F.C. J. Muscle Res. Cell. Motil. 1999; 20: 703-716Crossref PubMed Scopus (39) Google Scholar, 12Freiburg A. Gautel M. Eur. J. Biochem. 1996; 235: 317-323Crossref PubMed Scopus (222) Google Scholar) and the other one at its N terminus for the S2 subfragment (13Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (198) Google Scholar) (Fig. 1, A and B). MyBP-C binding to titin is expected to account for its regular appearance in muscle in 11 transverse stripes in intervals of 43 nm throughout the A-band. MyBP-C is made of 11 domains, 8 immunoglobulin, and 3 fibronectin type III and several linkers (14Carrier L. Bonne G. Bahrend E. Yu B. Richard P. Niel F. Hainque B. C. F. Labeit S. O. M. M. M. K. Circulation. Scopus Google Scholar) (Fig. in and as well as The cardiac isoform is highly from the by several in in the of the immunoglobulin domain Gautel M. M. J. Mol. Biol. PubMed Scopus Google Scholar), and an domain at the N terminus The N-terminal myosin binding site contains two immunoglobulin by a of amino as the (3Gautel M. Zuffardi O. Freiburg A. Labeit S. EMBO J. 1995; 14: 1952-1960Crossref PubMed Scopus (324) Google Scholar). The part of the binding site in cardiac MyBP-C contains several (3Gautel M. Zuffardi O. Freiburg A. Labeit S. EMBO J. 1995; 14: 1952-1960Crossref PubMed Scopus (324) Google Scholar). of this is by protein G. Kress K.R. Gruen M. Uttenweiler D. Gautel M. Fink R.H.A. Circulation. 2000; 86: 51-58Crossref Scopus (179) Google Scholar) a M. S. Gautel M. 1999; PubMed Scopus Google Scholar) and the interaction of the N-terminal MyBP-C binding site with myosin S2 G. Kress K.R. Gruen M. Uttenweiler D. Gautel M. Fink R.H.A. Circulation. 2000; 86: 51-58Crossref Scopus (179) Google Scholar). of MyBP-C in a of the myosin and the structure of the thick filament A. Winegrad S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8999-9003Crossref PubMed Scopus (147) Google Scholar, R. A. Kulikovskaya I. G. Winegrad S. Biophys. J. 2001; 81: Full Text Full Text PDF PubMed Scopus Google Scholar). an on muscle G. Kulikovskaya I. Winegrad S. Biophys. J. 2001; 81: 1083-1092Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The importance of this interaction is further by the of mutations to familial hypertrophic an of mutations related to an of mutations related to in the S2 subfragment that the structure of the but with binding to MyBP-C (13Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (198) Google Scholar). mutations are located in the first of S2 This part of S2Δ, was to be for binding to MyBP-C (13Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (198) Google Scholar). part of a to the of MyBP-C and are related to we the of the N-terminal myosin S2 binding site in human cardiac We the NMR structure of one of the two domains, that together with the the binding site for the S2 We also the binding site for the myosin S2 on cC2 by NMR spectroscopy and a model for the complex cC2-S2Δ based on binding experiments the mutation R870H in S2 and the between the cC2 of human cardiac was in and and as for domain Gautel M. M. J. Mol. Biol. PubMed Scopus Google Scholar). of cC2 by the in with as the of and and S2Δ from human cardiac muscle in and as (13Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (198) Google Scholar). the of cC2 in from the as by the used for human cardiac MyBP-C, for cardiac MyBP-C, for cardiac MyBP-C, for human MyBP-C, for human MyBP-C, for MyBP-C, and for the amino to for and and from human cardiac and the amino of was from human telokin. The of cC2 in this is based on the (13Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (198) Google Scholar) in to of this residue is the first residue of domain in the which is residue in the the first residue of domain cC2 has the Gibson Res. 1994; PubMed Scopus Google Scholar). used The was by for the by from structure and the The is in Gibson F. F. Res. Scopus Google Scholar). of is based on residue and specific = = = = and = titration was on a S2Δ solution was cC2 solution A in The and its to a J. PubMed Scopus Google Scholar). for the expected low protein and the was to in the The titration of a in by between The was with account for of and a titration was by of the in binding by of the to binding the by NMR with protein containing at and was in of NMR at on a an with F. S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and J. Scholar). The and made HSQC, and of cC2 in the was by and heteronuclear experiments D.A. A. PubMed Scopus Google Scholar) on cC2 protein at and protein at a of a of and of was with of and was with of and and the heteronuclear was with a of 3 from the by in used to the from a of to be least by large and A. J. 1995; Scopus Google Scholar). Using the of a G. A. J. Scopus Google Scholar, G. A. J. Scopus Google Scholar) was for the in and for the was where and are the changes in The in was for identified with the of in the NMR The with was also is based on molecular M. Scopus Google Scholar) to the for the of S. M. 2001; PubMed Scopus Google Scholar). used with the of the of to be in which was from to with the of to be to and the of for the was to A of from used as a of together with for in the and and from a by G. F. A. J. 1999; PubMed Scopus Google Scholar). for that a of and to be in a well from and experiments as 8 of of the that of A of this The structure was in the protein the NMR structure of MyBP-C domain as in this together with the structure of the S2Δ fragment of myosin J. Gautel M. I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) model the complex we the surface between cC2 and We the of protein with a with a model for the effect J. Scopus Google Scholar, J. 1999; Scopus Google Scholar) a of for and for the The was for S2Δ its which amino of human cardiac for A and for B. to to A based on the of in and D. D. C. C. T. K. R. A. J. M. R. J. I. G. U. P. P. of Scholar). The of the proteins was on their molecular and A. B. PubMed Scopus Google Scholar). The surface of proteins was to a of this we that the binding site of cC2 to S2Δ will be in the of to of the with This was and and a model for the complex with cC2 was for The of the was the C. R. J. PubMed Scopus Google Scholar), which of of and in and in with The of this consists of the where side chains and at the are to to for the used in this the in C. R. J. PubMed Scopus Google Scholar). we the interaction from the involved in the binding site. The are and and the are and for S2Δ with the of the are and are and in the of the are and the are and the of the are and the are and The are in further which complex of is most and most we the binding of as with is the molecular in the the of of the D. D. C. C. T. K. R. A. J. M. R. J. I. G. U. P. P. of Scholar), is the the and the contribution the J. 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Biol. 1994; PubMed Scopus Google Scholar). is by strands and the other by strands The of the β-sandwich is from other of the in that the strands a in to the strands of the other The are but the in the related an of a a This to one of the most of an domain with a of of with and for and as well as for the NMR structure of of from in most in in in in a The is by the which is by other amino such as and other are well such as the made of and and the in the with one from to from the the is from the the domain a cysteines and in strands F and G, are in close for an interaction of their side chains the of the of the of the protein where a with the of with low the group of a The between the in the two cysteines is with for disulfide in an a is of the is as to in of will position the close to a an could a disulfide bridge could the was throughout the NMR and changes in the NMR the of this it is that the is the of the two interactions. The of to a of of structure is in The to an domain of structure for of cC2 to to to and to the the key for the are well to other from MyBP-C there are isoform specific that the of the domain is expected to be very similar in cysteines involved in the disulfide are in cardiac and are in other with the of the The in the part of the of the is in in of of titin and telokin, this a in the to the of the is in titin but in a of the structure of cC2 to with the PubMed Scopus Google Scholar) of cC2 with titin and telokin, and titin domain in of and and of and is the to cC2 by telokin, and are the based on is based on the of with the structure of The of cC2 with the other a of cC2 on telokin, gives a low and a there is a in the C-terminal of the in with to strands and the C-terminal of strands F and G, as well as the and A similar but in the C-terminal of the domain with titin domain The of the C-terminal of cC2 to and titin is in part the of the of the in telokin, and cC2 that is in and the of the is highly the N-terminal of cC2 is to its that of and and the is The with in a the N-terminal of the with the is very The in this is the of the N-terminal of and G. was that is at its N terminus Gautel M. 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The S2Δ fragment the first of the S2 from residue to of human cardiac The binding of cC2 to S2Δ was by a NMR titration of cC2 with S2Δ The and changes the of the titration from to of S2Δ are the of cC2 in are two of and A but are well the the we will to this as The and changes the are on a structure of cC2 in The of the amino acids, by a well on the surface made by G, and the close to the C terminus of the the affinity of cC2 to S2Δ we isothermal titration calorimetry experiments for low J. PubMed Scopus Google Scholar). the as the NMR titration the of was to be the of domain cC2 on which the S2Δ binding site is with a to that the of on the surface of the S2 binding site is of with the at This arrangement is well in of and in there is on the S2 binding site of cC2 (Fig. and this the importance of the of in the S2 binding site of further the cC2-S2Δ interactions by NMR we the binding with S2Δ containing the mutation R870H (13Gruen M. Gautel M. J. Mol. 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The binding site of cC2 has a that could well the of between to of S2Δ the other of S2Δ present The surface of S2Δ for binding to cC2 for one binding site, that it was binding complex was for of sites, and Using interaction and are most as by the interaction of as with for This of binding is by the of binding to account for the for their with between and the surface is similar for the in binding between on one and and on the other is as based on and suggest that there is a specific in the N terminus of S2Δ that is well for binding to this is and be of the complex cC2-S2Δ the N terminus of cC2 from S2Δ, that its C terminus the with S2Δ be in a of close on cC2 and S2Δ and has a interaction between cC2 and S2Δ a between and as in is by a between the and the and a of of This interaction in the and and and structure of cC2 it a member of the the most one identified to The be in the of the thick filament and was from an of the R. L. R. Nature. PubMed Scopus Google Scholar) that there is the close to binding site of is also that this domain is one of the in to and titin that the This is part of the of the C. J. Mol. Biol. 1994; PubMed Scopus Google Scholar) but was in a of of the for which from importance and to the was in the from it an and to the as a M. A. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) to the of the as most of by NMR spectroscopy O. J. S. Gautel M. M. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). structure of cC2 has by the be to the of it is that the could be further two on the of the to the to telokin, cC2 contains two cysteines in F and in a position to telokin. F and are of key importance for interaction with myosin for cC2 and J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). is to that which are in the cardiac isoform of MyBP-C in most be in their form for most of the this could form a the of as in cardiac a disulfide bridge form between to the as well as of This be to the structure of cC2 and the interaction with a has for titin domain M. C. R. PubMed Scopus Google Scholar). MyBP-C-myosin binding of human cardiac MyBP-C for myosin in is the fragment the low affinity and the interaction of the domain cC2 in by NMR and to a first model for an interaction of two The of the binding site on the surface of the cC2 domain in a well group of on the G, and strands and the the C terminus of The changes binding are but for an interaction of polar amino R. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. O. C. J. Biol. PubMed Scopus Google Scholar, J. T. K. O. A. L. C. J. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). is very involved in the interaction. strands form an surface that be well to binding to a the of S2Δ, the binding site is a a surface is as in the model of together with the highly this is with the low binding affinity of 1.1 as by The interactions in the model are well in with the from the and the expected amino bridge in cC2 with and in S2Δ (cC2) with The interaction is by the of (cC2) with is to that a of interactions that changes of their close amino side chains are part of the of cC2 changes to the The is the between the of proteins is large and most interactions are made by the of amino side and The changes in the titration be from the side chains to the The of the is by a complex of from to of the as well as the residue in the E. J. 1994; PubMed Scopus Google Scholar). This the of with and changes in the side chains of and at the with S2Δ, are for and side chains the of the of the changes in the of the group in two amino are changes in the amino the titration with the S2Δ R870H the are to an of binding affinity by a between 3 and in with the (13Gruen M. Gautel M. J. Mol. Biol. 1999; 286: 933-949Crossref PubMed Scopus (198) Google Scholar). that the changes in the titration of S2Δ and S2Δ R870H to a in specific we the of S2Δ and S2Δ R870H and a titration of S2Δ and S2Δ R870H with of cC2 as well as for S2Δ and S2Δ for of in with S2Δ and S2Δ R870H are in the The of of S2Δ is with a in of well The titration of with S2Δ and S2Δ R870H This that of the R870H mutation are to the binding site of This NMR of the of cC2-S2Δ complex The most of the titration is the of binding affinity but the specific of a of to the binding of the R870H of The on the and and a in a in in at the of the binding site and is able to several that interactions with S2Δ in an that the model of is a and of the it an interaction of with This interaction is in the the side chain of a is that of an The and bridge interaction between two amino in the model of for a part of the of interactions between cC2 and a binding is as the interactions from amino the and and the are but the in complex with the interactions to be is expected that could a interaction which are that mutations of in this to as well as as a for R. C. 2000; PubMed Scopus Google Scholar). This arrangement the of is to be the of the in The interactions of on cC2 with on S2Δ are also is the amino in the cC2 used in this a its side chain is very well in the and the in the of cC2 as well as for S2Δ and S2Δ for of in with S2Δ and S2Δ R870H are in the the domain suggest that there is expected between the two we that the C-terminal of cC2 the N-terminal of the domain as in the G. G. 1994; Full Text Full Text PDF PubMed Scopus Google Scholar). in MyBP-C is expected to a well structure that binding to The interaction of cC2 with myosin is expected to be similar to that of telokin. The contribution for myosin light chain binding to myosin from the between and the the domain binds to myosin, the binding is part of the C terminus of is to a similar binding surface as in cC2 J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The in myosin binding on cC2 and is further by the of key surface for the myosin and in cC2 their in and on telokin, We used NMR spectroscopy to the first structure of domain of human cardiac MyBP-C, the most structure to the structure of cC2 with and NMR interaction of cC2 with and myosin S2Δ and molecular with a model of the cC2-S2Δ This is the first an of the interaction of the MyBP-C N terminus with myosin. This model the interaction of an immunoglobulin domain from MyBP-C in the of other myosin binding light chain and for the first the importance of amino at the and could be to of We expect that will stimulate further research on the of the structure and of MyBP-C and the mutations that to