This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: 91/8/704    most recent 01.RES.0000036750.81083.83v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Moolman-Smook, J. Articles by Watkins, H. Search for Related Content PubMed PubMed Citation Articles by Moolman-Smook, J. Articles by Watkins, H. Related Collections Structure Contractile function Myocardial cardiomyopathy disease Genetics of cardiovascular disease

(Circulation Research. 2002;91:704.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Identification of Novel Interactions Between Domains of Myosin Binding Protein-C That Are Modulated by Hypertrophic Cardiomyopathy Missense Mutations

Johanna Moolman-Smook, Emily Flashman, Willem de Lange, Zhili Li, Valerie Corfield, Charles Redwood, Hugh Watkins

From the US/MRC Centre for Molecular and Cellular Biology (J.M.-S., W.d.L., V.C.), University of Stellenbosch, Tygerberg, South Africa; and the Department of Cardiovascular Medicine (E.F., Z.L., C.R., H.W.), University of Oxford, Oxford, UK. Present address for Z.L. is the Department of Cardiology, Fourth Military Medical University, Xi’an City, People’s Republic of China.

Correspondence to Hugh Watkins, MD, PhD, Dept of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, UK OX28AF. E-mail hugh.watkins{at}cardiov.ox.ac.uk

Cardiac myosin binding protein-C (cMyBPC) is a modular protein consisting of 11 domains whose precise function and sarcomeric arrangement are incompletely understood. Identification of hypertrophic cardiomyopathy (HCM)–causing missense mutations in cMyBPC has highlighted the significance of certain domains. Of particular interest is domain C5, an immunoglobulin-like domain with a cardiac-specific insert, which is of unknown function yet is the site of two HCM-causing missense mutations. To identify interactors with this region, a human cardiac cDNA library was screened in a yeast two-hybrid (Y2H) assay using the C5 sequence as bait. Screening >7x10⁶ clones surprisingly revealed that domain C5 preferentially bound to clones encoding C-terminal fragments of cMyBPC; the interacting region was narrowed to domain C8 by deletion mapping. A surface plasmon resonance assay using purified recombinant cMyBPC domains was used to measure the affinity of C5 and C8 in vitro (K_a=1x10⁵ mol/L^-1). This affinity was decreased about 2-fold by the HCM mutation R654H, and by at least 10-fold by the mutation N755K. Further Y2H assays also demonstrated specific binding between domains C7 and C10 of cMyBPC. Based on these novel interactions, and previous biochemical and structural data, we propose that cMyBPC molecules trimerize into a collar around the thick filament, with overlaps of domains C5-C7 of one cMyBPC with C8-C10 of another. We speculate that this interaction may be dynamically formed and released, thereby restricting or favoring cross-bridge formation, respectively. We suggest that the HCM mutations act by altering the cMyBPC collar, indicating its importance in thick filament structure and regulation.

Key Words: cardiac myosin binding protein-C • hypertrophic cardiomyopathy • missense mutations • ligand binding

This article has been cited by other articles:

				J. L. Theis, J. M. Bos, J. D. Theis, D. V. Miller, J. A. Dearani, H. V. Schaff, B. J. Gersh, S. R. Ommen, R. L. Moss, and M. J. Ackerman Expression Patterns of Cardiac Myofilament Proteins: Genomic and Protein Analysis of Surgical Myectomy Tissue From Patients With Obstructive Hypertrophic Cardiomyopathy Circ Heart Fail, July 1, 2009; 2(4): 325 - 333. [Abstract] [Full Text] [PDF]

				B. A. Colson, T. Bekyarova, M. R. Locher, D. P. Fitzsimons, T. C. Irving, and R. L. Moss Protein Kinase A-Mediated Phosphorylation of cMyBP-C Increases Proximity of Myosin Heads to Actin in Resting Myocardium Circ. Res., August 1, 2008; 103(3): 244 - 251. [Abstract] [Full Text] [PDF]

				R. D. S. Dixon, D. K. Arneman, A. S. Rachlin, N. R. Sundaresan, M. J. Costello, S. L. Campbell, and C. A. Otey Palladin Is an Actin Cross-linking Protein That Uses Immunoglobulin-like Domains to Bind Filamentous Actin J. Biol. Chem., March 7, 2008; 283(10): 6222 - 6231. [Abstract] [Full Text] [PDF]

				M. E. Zoghbi, J. L. Woodhead, R. L. Moss, and R. Craig Three-dimensional structure of vertebrate cardiac muscle myosin filaments PNAS, February 19, 2008; 105(7): 2386 - 2390. [Abstract] [Full Text] [PDF]

				T. Nagayama, E. Takimoto, S. Sadayappan, J. O. Mudd, J.G. Seidman, J. Robbins, and D. A. Kass Control of In Vivo Contraction/Relaxation Kinetics by Myosin Binding Protein C: Protein Kinase A Phosphorylation-Dependent and -Independent Regulation Circulation, November 20, 2007; 116(21): 2399 - 2408. [Abstract] [Full Text] [PDF]

				F. S. Korte and K. S. McDonald Sarcomere length dependence of rat skinned cardiac myocyte mechanical properties: dependence on myosin heavy chain J. Physiol., June 1, 2007; 581(2): 725 - 739. [Abstract] [Full Text] [PDF]

				I. Kulikovskaya, G. B. McClellan, R. Levine, and S. Winegrad Multiple Forms of Cardiac Myosin-binding Protein C Exist and Can Regulate Thick Filament Stability J. Gen. Physiol., April 30, 2007; 129(5): 419 - 428. [Abstract] [Full Text] [PDF]

				S. Brickson, D. P. Fitzsimons, L. Pereira, T. Hacker, H. Valdivia, and R. L. Moss In vivo left ventricular functional capacity is compromised in cMyBP-C null mice Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1747 - H1754. [Abstract] [Full Text] [PDF]

				J. E. Stelzer, S. B. Dunning, and R. L. Moss Ablation of Cardiac Myosin-Binding Protein-C Accelerates Stretch Activation in Murine Skinned Myocardium Circ. Res., May 12, 2006; 98(9): 1212 - 1218. [Abstract] [Full Text] [PDF]

				B. M. Palmer, T. Noguchi, Y. Wang, J. R. Heim, N. R. Alpert, P. G. Burgon, C. E. Seidman, J.G. Seidman, D. W. Maughan, and M. M. LeWinter Effect of Cardiac Myosin Binding Protein-C on Mechanoenergetics in Mouse Myocardium Circ. Res., June 25, 2004; 94(12): 1615 - 1622. [Abstract] [Full Text] [PDF]

				E. Flashman, C. Redwood, J. Moolman-Smook, and H. Watkins Cardiac Myosin Binding Protein C: Its Role in Physiology and Disease Circ. Res., May 28, 2004; 94(10): 1279 - 1289. [Abstract] [Full Text] [PDF]

				B. M. Palmer, D. Georgakopoulos, P. M. Janssen, Y. Wang, N. R. Alpert, D. F. Belardi, S. P. Harris, R. L. Moss, P. G. Burgon, C. E. Seidman, et al. Role of Cardiac Myosin Binding Protein C in Sustaining Left Ventricular Systolic Stiffening Circ. Res., May 14, 2004; 94(9): 1249 - 1255. [Abstract] [Full Text] [PDF]

				I. Kulikovskaya, G. McClellan, J. Flavigny, L. Carrier, and S. Winegrad Effect of MyBP-C Binding to Actin on Contractility in Heart Muscle J. Gen. Physiol., November 24, 2003; 122(6): 761 - 774. [Abstract] [Full Text] [PDF]

				J. Flavigny, P. Robert, J.-C. Camelin, K. Schwartz, L. Carrier, and I. Berrebi-Bertrand Biomolecular interactions between human recombinant {beta}-MyHC and cMyBP-Cs implicated in familial hypertrophic cardiomyopathy Cardiovasc Res, November 1, 2003; 60(2): 388 - 396. [Abstract] [Full Text] [PDF]

				F. S. Korte, K. S. McDonald, S. P. Harris, and R. L. Moss Loaded Shortening, Power Output, and Rate of Force Redevelopment Are Increased With Knockout of Cardiac Myosin Binding Protein-C Circ. Res., October 17, 2003; 93(8): 752 - 758. [Abstract] [Full Text] [PDF]

				J. Robbins and D. W. Benson Structure-Function Relationships in Myosin Binding Protein-C: Taking Off the Blinders and Collaring Hypertrophic Cardiomyopathy Circ. Res., October 18, 2002; 91(8): 656 - 658. [Full Text] [PDF]