striated muscle cells contain a complex , highly ordered cytoskeleton , mainly composed of interdigitating thick myosin and thin actin filaments . muscle contraction and relaxation occurs by the sliding of myosin filaments past actin filaments under the strict regulation of their accessory proteins , which are responsible for their assembly and maintenance as well as the regulation of contractile activity . myosin - binding protein - c ( mybp - c ) comprises a family of accessory proteins of the thick myosin filaments that encompasses ~2% of the total myofibrillar protein . to date , two major roles have been attributed to mybp - c ; it contributes to the regular organization and stabilization of thick filaments and modulates the formation of cross - bridges between myosin and actin , via direct interactions with both filamentous systems ( as reviewed in [ 3 , 4 ] ) . within the sarcomere , mybp - c localizes to the c - zone , the cross - bridges containing region of the a - band , in 79 transverse stripes that are ~43  nm apart [ 57 ] . three mybp - c isoforms have been identified ; cardiac , slow skeletal , and fast skeletal ( cmybp - c , smybp - c , and fmybp - c ) , encoded by different genes localizing to human chromosomes 11 , 12 , and 19 , respectively [ 8 , 9 ] . the core structure of mybp - c is composed of seven immunoglobulin ( ig ) domains and three fibronectin type iii ( fn - iii ) repeats , numbered from the nh2-terminus as c1c10 ( figure 1 ; ) . a proline / alanine- ( pro / ala- ) rich motif and a conserved linker region , termed mybp - c or m - motif , flank the first ig domain , c1 . cmybp - c possesses three unique features , including an additional ig domain at the extreme nh2-terminus ( termed c0 ) , a 9-residue long insertion within the m - motif containing consensus phosphorylation sites and a 28-amino acid long loop in the middle of domain c5 [ 8 , 11 ] . conversely , smybp - c comprises a subfamily of four alternatively spliced variants ( v ) , v1v4 , differing from one another due to the retention or exclusion of select exons encoding three novel insertions . these are located at the very nh2-terminus within the pro / ala- rich motif , the middle of domain c7 , and the very cooh - terminus of the molecule , following the c10 domain ( figure 1 ; ) . the presence of these insertions may result in different topographies and possibly functions of the smybp - c variants that contain them [ 12 , 13 ] . consistent with this , v1 , which carries the cooh - terminal insertion preferentially localizes to the periphery of the m - band , rather than the c - zone , where it codistributes with its binding partners obscurin   and titin ( our unpublished observations ) and plays key roles in the structural integrity of m- and a - bands . the last ig domain ( c10 ) of smybp - c v1 interacts with the second ig domain ( ig2 ) of obscurin . although the presence of the novel cooh - terminal insertion further strengthens this interaction , it is not required for binding . however , this novel motif is both necessary and sufficient to support binding of smybp - c v1 to the last ig domain of titin , m10 , which is a hotspot for mutations associated with tibial muscular dystrophy [ 14 , 15 ] . moreover , our laboratory has recently identified novel phosphorylation sites within the pro / ala- rich motif of the nh2-terminus of smybp - c , specifically ser-59 , located in the novel nh2-terminal insertion and ser-62 are targets of pka , while ser-83 and thr-84 are targets of pkc ; ser-204 is a substrate for both pka and pkc . similar to its cardiac counterpart , phosphorylation of these sites may be essential in regulating the activities of smybp - c . nevertheless , despite the differences in the core structure of the cardiac , slow , and fast mybp - c proteins , they share a ~50% homology and a ~65% identity . although mybp - c was identified over forty years ago as a contaminant of purified myosin , a better understanding of its physiology has emerged in the last two decades , paralleling the discovery of mutations in cmybp - c that result in the development of familial hypertrophic cardiomyopathy ( fhc ; [ 18 , 19 ] ) . consequently , most of our knowledge originates from studies focusing on the cardiac form of the protein . this review will focus on the role of mybp - c in regulating the formation of actomyosin cross - bridges via its direct interaction with both myosin and actin filaments . myosin is a hexameric protein consisting of two heavy chains ( mhc ) and two light chains ( regulatory and essential , rlc and elc , resp . ) . a dimer of heavy chains forms a coiled - coil helix that constitutes the rod or light meromyosin ( lmm ) segment of myosin . toward its nh2-terminus , the helix unwinds and each mhc gives rise to a catalytic head , referred to as subfragment 1 ( s1 ) that has atpase activity and participates in the formation of cross - bridges with filamentous actin [ 21 , 22 ] . the lever arm , referred to as subfragment 2 ( s2 ) , separates the s1 segment from the rod portion , and transduces the chemical energy from the hydrolysis of atp into mechanical movement along the thin filament . a pair of rlc and elc binds in tandem to each head of the s1 segment modulating the speed and force of contraction [ 24 , 25 ] . in addition to myosin light chains and actin , myosin is intimately associated with mybp - c ( table 1 and figure 2 ; ) . the cooh - terminal c10 domain of all three mybp - c isoforms harbors binding sites for the lmm portion of myosin . charged residues r1064 , e1079 , n1083 , r1100 , and r1101 present on the surface of the c10 domain of mybp - c support an electrostatic interaction with residues 15541581 of the rod domain of myosin that has a modest affinity of ~3.5  m [ 28 , 29 ] . moreover , through domains c8c10 , mybp - c interacts with titin , a giant sarcomeric protein spanning half a sarcomere , which is also tightly bound to the thick myosin filament . specifically , mybp - c binds to the first ig domain of an 11-domain superrepeat present in the a - band portion of titin . thus , it has been speculated that the repetitive binding of mybp - c to the a - band portion of titin likely contributes to its periodicity within the c - zone . consistent with these findings , a number of studies have further shown that the cooh - terminal c8c10 domains are necessary and sufficient to target mybp - c to the a - band [ 44 , 45 ] . while binding of the cooh - terminus of mybp - c to the rod domain of myosin may contribute to the maintenance and stability of the thick filament , ( reviewed in [ 3 , 4 ] ) , binding of the nh2-terminus of mybp - c to myosin may mediate contractile regulation a number of biochemical and structural studies have implicated the nh2-terminal portion of cmybp - c , containing domains c0c2 , in binding to the s2 region of myosin . specifically , the m - motif located between the c1 and c2 ig domains has been shown to bind directly to the nh2-terminal 126 residues of the s2 fragment with an affinity of ~4.3  m ; this interaction is abolished by phosphorylation of specific ser residues within the m - motif of cmybp - c via camp - dependent protein kinase [ 4649 ] . similarly , the c2 domain has been also shown to interact with the same 126 residues of the nh2-terminus of the s2 fragment , albeit with considerably lower affinity ( ~1.1  mm ) , compared to the m - motif . interestingly , molecular modeling has suggested that the interaction between the s2 portion of myosin and the c2 domain of cmybp - c is mediated by polar residues . on the other hand , the c1 domain of cmybp - c has been reported to interact with the hinge region between the s1 heads and the s2 fragment , in close proximity to the myosin light chains , while a recent study reported that the first ig domain of cmybp - c , c0 , binds to the rlcs with an affinity of ~3.2  m . in support of this , overexpressed c0 targets to the a - band independently of the rest of the molecule , likely through its interaction with the rlcs .   taken together , these findings strongly suggest that cmybp - c may regulate the position and thus proximity of the myosin s1 heads relative to the actin filament , through its interaction with the s2 region and the rlcs , therefore affecting the formation of actomyosin cross - bridges . in addition to the mounting experimental support indicating the intimate association of the nh2-terminus of cmybp - c with the s2 region of myosin , there is also significant evidence supporting its direct interaction with actin thin filaments . early studies have shown that purified full - length cmybp - c binds to filamentous actin ( f - actin ) [ 36 , 50 ] . however , reconstituted thin filaments ( i.e. preincubated with troponin and tropomyosin ) fail to bind purified full length cmybp - c in the presence of edta , a ca chelating agent , but binding is restored upon addition of ca [ 36 , 50 ] . further studies have recently begun to identify the actin - binding domain(s ) of cmybp - c through biochemical and structural approaches . neutron scattering experiments have indicated that recombinant c0c2 binds to f - actin in a repetitive manner , stabilizing it , while biochemical studies have shown that recombinant c0 is capable of directly interacting with f - actin . moreover , bacterially expressed c1-c2 associates with naked f - actin as well as reconstituted thin filaments , likely through the m - motif , exhibiting a binding affinity of ~10  m to naked f - actin [ 40 , 41 ] . this finding has been also substantiated by a recent study that used negative stain electron microscopy and three - dimensional reconstruction to show that bacterially expressed c0c3 is well ordered on actin filaments near subdomain 1 . noticeably , some of the above studies report linear , nonsaturating binding , suggesting the presence of weak electrostatic interactions , as exemplified by the calculated micromolar affinity . consistent with this , these binding reactions depend largely on ph and ionic strength and are regulated by phosphorylation of cmybp - c . thus , an increase in ph or ionic strength significantly decreases the capacity of the aforementioned cmybp - c peptides to bind to f - actin [ 4042 ] . similarly , pretreatment of these recombinant peptides with pka results in a dramatic reduction of their ability to associate with f - actin . importantly , a recent study by rybakova and colleagues investigated the actin - binding capabilities of cmybp - c across its entire length . in support of previously published studies discussed above , the authors found that the nh2-terminal domains of cmybp - c bind to f - actin in a linear , nonsaturating manner , likely mediated by weak electrostatic interactions . however , recombinant full - length cmybp - c , expressed in the baculovirus system , supported a direct and saturating interaction with f - actin with a calculated kd of ~4.3  m . moreover , constructs lacking domains c0c5 exhibited similar binding properties as full - length cmybp - c ; this observation prompted the authors to conclude that the weak actin binding mediated by the nh2-terminus of the molecule does not contribute significantly to the overall affinity of cmybp - c for f - actin . moreover , the authors further showed that the actin binding supported by the cooh - terminal c6c10 domains , unlike the nh2-terminal c0c2 domains , is independent of the regulatory elements , troponin and tropomyosin , of thin filaments , the levels of ca , and the phosphorylation status of the m - motif . although the cooh - terminus of cmybp - c exhibits a higher affinity for f - actin , the minimal binding region and its physiological relevance are still elusive . these open questions are especially important because the cooh - terminal domains c6c10 also harbor binding sites for myosin and titin filaments ( discussed above ) . indeed , space constraints would favor dynamic rather than simultaneous interactions of the cooh - terminus of cmybp - c with the actin , myosin , and titin filaments . however , three - dimensional reconstruction of thick filaments demonstrated that domains c7c10 of cmybp - c run along the length of the myosin rod , suggesting that its interaction with the lmm portion of myosin is not of dynamic nature . collectively these studies suggest the presence of various but weak binding sites for f - actin in the nh2-terminus of cmybp - c as well as the presence of a high affinity , saturating binding site in the cooh - terminus of the molecule . obviously , more work is required before we can obtain a clear understanding of the physiological relevance of these interactions during muscle contraction and relaxation . early work has shown that the skeletal isoforms of mybp - c also interact directly with both thick and thin filaments ; however , their binding is much less characterized ( figure 3 and table 2 ) . similar to cmybp - c , the skeletal isoforms bind to the rod portion of sarcomeric myosin through their c10 domain and to the s2 region through their nh2-terminus . further characterization of the nh2-terminal binding has shown that the first two ig domains of smybp - c ( c1-c2 ) bind to the s2 region with an affinity of ~2.2  m [ 26 , 34 ] . in addition , native skeletal mybp - c ( presumably containing a mixture of the slow and fast isoforms ) coaggregates with f - actin in a ca sensitive manner . it becomes apparent from the above studies that our knowledge on the interaction of slow or fast mybp - c with myosin and actin filaments is limited . the identification of multiple mutations in the cardiac isoform that have been causally linked to the development of fhc is a major contributing factor that has shifted the interest of researchers towards cmybp - c ; however , this has recently changed , as mutations in smybp - c were identified in patients suffering from distal arthrogryposis type 1 ( da1 ) , a disorder characterized by congenital contractures of the hands and feet (; please see below ) . more importantly , the molecular diversity of the skeletal isoforms further complicates the relevant studies as there are at least five different skeletal forms of mybp - c ( one fast and four slow variants ) that share homologous or common sequences , contain novel insertions in the nh2- and cooh - termini , and may coexist in the same muscle , fiber , or even sarcomere . further detailed investigation is , therefore , necessary to evaluate the ability of each isoform to bind to myosin and actin filaments as well as the physiological significance of these interactions . during the last decade , an overwhelming number of mutations ( ~200 ) have been identified in the mybpc3 gene , which encodes the human cardiac mybp - c protein . these include missense , nonsense , deletion / insertion , and frame - shift mutations and have been causally linked to the development of modest and late onset familial hypertrophic cardiomyopathy ( fch ) [ 5658 ] . the majority of the nonsense and frame - shift mutations result in truncated forms of the protein , that lack the cooh - terminus harboring binding sites for lmm , actin and titin , and thus negate the ability of cmybp - c to associate with these filaments and regulate contractile activity . interestingly , these truncated peptides are often undetectable in patient biopsies , possibly due to transcriptional misregulation or rapid degradation . contrary to non - sense and frame shift mutations , missense mutations are generally associated with a less severe cardiomyopathic phenotype   and do not affect the structure or stability of the protein although some of them have been suggested to weaken myosin binding . moreover , recent genome wide linkage analysis revealed that mutations in the mybpc1 gene that encodes the human skeletal mybp - c slow protein lead to the development of distal arthrogryposis type 1 , an autosomal dominant disorder characterized by congenital contractures of the hands and feet . two missense mutations have been identified to date , w236r and y856h , which are present in the m - motif and c8 domain , respectively . evaluation of skeletal muscle biopsies obtained from affected individuals revealed that slow twitch fibers were significantly smaller than fast twitch fibers . importantly , the locations of these two mutations indicate possible alterations in the ability of smybp - c to interact with myosin or actin via its nh2-terminus and to associate with the thick and titin filaments through its cooh - terminus . the causal involvement of the mybp - c family of proteins in the development of cardiac and skeletal myopathies , as exemplified by the aforementioned studies , clearly indicates that the members of this multifaceted and complex family are essential components and key regulators of muscle structure and function . through its dynamic interactions with myosin and actin filaments , mybp - c affects the formation and cycling of cross - bridges in three distinct ways : ( i ) by maintaining the normal structure of myosin and actin filaments , ( ii ) by regulating the rate at which myosin and actin interact , and ( iii ) by modulating the atpase activity of myosin . consistent with this , the addition of skeletal mybp - c reduces the critical concentration necessary for myosin polymerization in vitro and results in the formation of longer and more uniform thick filaments [ 61 , 62 ] . in addition , the nh2-terminal region c0c2 of cmybp - c supports actin bundling in vitro , as evidenced by the increased turbidity of f - actin in the solution and the formation of significantly thicker actin filaments , as visualized by electron microscopy . notably , the effect of cmybp - c on actin bundling is dependent on ph and regulated by phosphorylation events mediated by pka . moreover , in vitro motility assays , examining the sliding of actin filaments over myosin heads in the presence of full - length or truncated forms of cmybp - c revealed a significant reduction in their sliding velocity [ 40 , 63 , 64 ] . notably , the degree of reduction was also dependent on the presence of the actin regulatory proteins , troponin , and tropomyosin ; in their presence , there was a lesser effect on the decrease of actin motility over full - length myosin [ 63 , 65 ] , hmm   or s1 heads . mybp - c also modulates the atpase activity of myosin , though in an indirect way , via interactions with both myosin and actin filaments supported by its nh2-terminus . in agreement with this , purified smybp - c inhibited the atpase activity of actin - activated myosin ; however , native cmybp - c moderately enhanced the actin - activated atpase activity of myosin [ 66 , 67 ] . surprisingly though , recombinant cmybp - c containing the c0c2 region inhibited the atpase activity of myosin in the presence of actin . the majority of the aforementioned studies have been performed with native proteins obtained from different species or types of muscles , presumably due to ease of purification . however , alyonycheva et al . have convincingly shown that the strength of the interaction between myosin and mybp - c is considerably increased when both proteins are purified from the same source . as such , cmybp - c and skeletal mybp - c bound with higher affinity to cardiac and skeletal myosin , respectively .   as our knowledge about the molecular and functional properties of mybp - c broadens , it becomes apparent that through interactions supported by its cooh and nh2 termini , mybp - c contributes to the maintenance of the normal structure of thick filaments , and the regulation of cross - bridges formation and cycling . it has , therefore , been suggested that mybp - c may serve as a linker , whereby its cooh - terminus is necessary for anchoring it to lmm in the a - band , leaving the nh2-terminus available to modulate contractility through dynamic interactions with the head region of myosin and actin . consequently , calaghan and colleagues proposed the  tether model  to explain how mybp - c modulates the formation of actomyosin cross - bridges . this model suggests that both myosin binding sites on mybp - c act together by limiting the movement of the s1 heads relative to lmm and actin , thereby restricting the formation of actomyosin cross - bridges . phosphorylation of select ser residues within the m - motif of cmybp - c , mediated mainly by pka , regulates the interaction between cmybp - c and myosin by acting as an  on - off  switch . consequently , when in the  on  or dephosphorylated state , the nh2-terminus of cmybp - c binds to myosin and limits its interaction with actin , when in the  off  or phosphorylated state , myosin is free to interact with actin and form cross - bridges .    the tether model is centered on the dynamic and highly regulated interaction of mybp - c with myosin and actin filaments . however , it is necessary to understand the orientation of mybp - c within the sarcomere , as well . two proposed models for the topology of mybp - c have been suggested : a  collar model  in which a trimer of mybp - c molecules wraps around the rod domain of myosin , and an  axial model  , where the cooh - terminus of mybp - c runs along the length of the rod domain . a stipulation of the collar model is that mybp - c is required to dimerize . in support of this , it has been shown that mybp - c dimerizes through interactions mediated by domains c5 and c7 with c8 and c10 , respectively [ 43 , 72 ] . on the other hand , x - ray diffraction in combination with molecular and computational modeling studies support the axial model , suggesting that the last three domains of mybp - c lay along the length of the myosin rod [ 71 , 73 ] . recently , a study using electron microscopy and three - dimensional reconstructions of cardiac thick filaments further demonstrated that the cooh - terminus of cmybp - c runs along the rod domain surface of thick filaments . although mybp - c dimerization is not essential for the arrangement of mybp - c along the length of the myosin rod , the axial model does not negate its dimerization . nevertheless , in both models the nh2-terminus of mybp - c extends into the interfilament space where it is accessible to bind to the s2 portion of myosin and actin . physiological dynamic interactions of mybp - c with myosin and actin are possible as the distance between thick and thin filaments is ~916  nm   and the length of extended mybp - c is ~40  nm . therefore , mybp - c is capable to span the interfilament space , enabling interactions with both actin and myosin filaments . it is not without precedence that regulation of contractility occurs through dynamic interactions with both actin and myosin filaments , as the essential and regulatory light chains bind tightly to myosin , but also interact with actin [ 25 , 76 , 77 ] . additionally , twitchin , a protein similar to titin , characterized in c. elegans as a thick filament regulatory protein , interacts with both myosin and actin in a phosphorylation - dependent manner to modulate cross - bridges formation [ 78 , 79 ] . however , current models ( as described above ) to explain the mechanism of contractile regulation through dynamic binding to myosin and actin are lacking . undoubtedly , more work is necessary in order to understand the precise mechanisms by which mybp - c modulates contractility .
myosin - binding protein - c ( mybp - c ) is a family of accessory proteins of striated muscles that contributes to the assembly and stabilization of thick filaments , and regulates the formation of actomyosin cross - bridges , via direct interactions with both thick myosin and thin actin filaments . 
 three distinct mybp - c isoforms have been characterized ; cardiac , slow skeletal , and fast skeletal . 
 numerous mutations in the gene for cardiac mybp - c ( cmybp - c ) have been associated with familial hypertrophic cardiomyopathy ( fhc ) and have led to increased interest in the regulation and roles of the cardiac isoform . 
 this review will summarize our current knowledge on mybp - c and its role in modulating contractility , focusing on its interactions with both myosin and actin filaments in cardiac and skeletal muscles .