Myosin binding protein C (MyBP-C) is a thick filament protein involved in the regulation of muscle contraction. Mutations in the gene for MyBP-C are the second most frequent cause of hypertrophic cardiomyopathy. MyBP-C binds to myosin with two binding sites, one at its C-terminus and another at its N-terminus. The N-terminal binding site, consisting of immunoglobulin domains C1 and C2 connected by a flexible linker, interacts with the S2 segment of myosin in a phosphorylation-regulated manner. It is assumed that the function of MyBP-C is to act as a tether that fixes the S1 heads in a resting position and that phosphorylation releases the S1 heads into an active state. Here, we report the structure and binding properties of domain C1. Using a combination of site-directed mutagenesis and NMR interaction experiments, we identified the binding site of domain C1 in the immediate vicinity of the S1–S2 hinge, very close to the light chains. In addition, we identified a zinc binding site on domain C1 in close proximity to the S2 binding site. Its zinc binding affinity (Kd of approximately 10–20 μM) might not be sufficient for a physiological effect. However, the familial hypertrophic cardiomyopathy-related mutation of one of the zinc ligands, glutamine 210 to histidine, will significantly increase the binding affinity, suggesting that this mutation may affect S2 binding. The close proximity of the C1 binding site to the hinge, the light chains and the S1 heads also provides an explanation for recent observations that (a) shorter fragments of MyBP-C unable to act as a tether still have an effect on the actomyosin ATPase and (b) as to why the myosin head positions in phosphorylated wild-type mice and MyBP-C knockout mice are so different: Domain C1 bound to the S1–S2 hinge is able to manipulate S1 head positions, thus influencing force generation without tether. The potentially extensive extra interactions of C1 are expected to keep it in place, while phosphorylation dislodges the C1–C2 linker and domain C2. As a result, the myosin heads would always be attached to a tether that has phosphorylation-dependent length regulation.

Calmodulin and other members of the EF-hand protein family are known to undergo major changes in conformation upon binding Ca 2+. However, some EF-hand proteins, such as calbindin D9k, bind Ca 2+ without a significant change in conformation. Here, we show the importance of a precise balance of solvation energetics to conformational change, using mutational analysis of partially buried polar groups in the N-terminal domain of calmodulin (N-cam). Several variants were characterized using fluorescence, circular dichroism, and NMR spectroscopy. Strikingly, the replacement of polar side chains glutamine and lysine at positions 41 and 75 with nonpolar side chains leads to dramatic enhancement of the stability of the Ca 2+ -free state, a corresponding decrease in Ca 2+ -binding affinity, and an apparent loss of ability to change conformation to the open form. The results suggest a paradigm for conformational change in which energetic strain is accumulated in one state in order to modulate the energetics of change to the alternative state.Keywords: Conformational change; EF-hand; calmodulin; calbindin; solvation; buried polar; calcium Conformational changes are intrinsic to the function of a variety of proteins. The changes in structure are typically triggered by a change in environment such as pH, phosphorylation state, or ligand-binding status (Schachman 1987;Ackers et al. 1992;Bullough et al. 1994;Ikura 1996). EF-hand proteins such as calmodulin are known to change conformation upon calcium (Ca 2+ ) binding and often are termed Ca 2+ -sensor proteins. However, EF-hand proteins that appear to be Ca 2+ -buffering or -transporter proteins, such as calbindin D9k, exhibit only subtle changes in conformation (Skelton et al. 1994;Chazin 1995;Ikura 1996). A number of three-dimensional structures of EF-hand proteins have been determined in the Ca 2+ -saturated and -free forms (Herzberg and James 1985;Flaherty et al. 1993;Kordel et al. 1993;Gagne et al. 1995;Kuboniwa et al. 1995;Skelton et al. 1995;Slupsky and Sykes 1995;Drohat et al. 1996Drohat et al. , 1998Sastry et al. 1998). The recent progress of atomicresolution structural analysis has provided valuable information about Ca 2+ -induced conformational change in EFhand proteins.Several groups have made important contributions in this direction through structure determination, mutational and structural analysis, and comparative studies (Skelton et al.

The market for commercially available isothermal titration calorimeters continues to grow as new applications and methodologies are developed. Concomitantly the number of users (and abusers) increases dramatically, resulting in a steady increase in the number of publications in which isothermal titration calorimetry (ITC) plays a role. In the present review, we will focus on areas where ITC is making a significant contribution and will highlight some interesting applications of the technique. This overview of papers published in 2004 also discusses current issues of interest in the development of ITC as a tool of choice in the determination of the thermodynamics of molecular recognition and interaction.

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 ␤-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, S2⌬, with low affinity (k D ‫؍‬ 1.1 mM) but exhibits a highly specific binding site. This consists of the C-terminal surface of the CCFGA ␤-sheet, which includes Glu 301 , 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 S2⌬ 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-S2⌬ 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 S2⌬ 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 2 for the correct assembly of muscle proteins is well established (1, 2), 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 (3-7). MyBP-C is a large (ϳ140 kDa) multidomain protein (8, 9) that is located in the thick filament (10), where it is attached to myosin via two binding sites: one in its C terminus for the LMM subfragment (11, 12) and the other one at its N terminus for the S2 subfragment (13) (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 (14) (Fig. 1A). It exists in three different isoforms: fast and slow skeletal as well as cardiac. The cardiac isoform is highly distinct from the skeletal isoforms by several substantial insertions in some linkers, right in the middle of the immunoglobulin domain cC5 (15...

Proteins within the EF-hand protein family exhibit different conformational responses to Ca(2+) binding. Calmodulin and other members of the EF-hand protein family undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k (Clb), bind Ca(2+) without a significant change in conformation. Here, we investigate the effects of replacement of a leucine at position 39 of the N-terminal domain of calmodulin (N-Cam) with a phenylalanine derived from Clb. This variant is studied alone and in the context of other mutations that affect the conformational properties of N-Cam. Strikingly, the introduction of Phe39, which is distant from the calcium binding sites, leads to a significant enhancement of Ca(2+) binding affinity, even in the context of other mutations which trap the protein in the closed form. The results yield novel insights into the evolution of EF-hand proteins as calcium sensors versus calcium buffers.