Computational analysis of folding and mutation properties of C5 domain from Myosin binding protein C

Thermal folding Molecular Dynamics simulations of the domain C5 from Myosin Binding Protein C were performed using a native-centric model to study the role of three mutations related to Familial Hypertrophic Cardiomyopathy. Mutation of Asn755 causes the largest shift of the folding temperature, and the residue is located in the CFGA’ beta-sheet featuring the highest Phi-values. The mutation thus appears to reduce the thermodynamic stability in agreement with experimental data. The mutations on Arg654 and Arg668, conversely, cause a little change in the folding temperature and they reside in the low Phi-value BDE beta-sheet, so that their pathologic role cannot be related to impairment of the folding process but possibly to the binding with target molecules. As the typical signature of Domain C5 is the presence of a longer and destabilizing CD-loop with respect to the other Ig-like domains we completed the work with a bioinformatic analysis of this loop showing a high density of negative charge and low hydrophobicity. This indicates the CD-loop as a natively unfolded sequence with a likely coupling between folding and ligand binding.

💡 Research Summary

This study presents a computational investigation of the C5 Ig‑like domain of cardiac Myosin Binding Protein C (MyBP‑C), focusing on three missense mutations (Asn755Lys, Arg654His, Arg668His) that are implicated in familial hypertrophic cardiomyopathy (FHC). The authors employed a native‑centric Gō‑type coarse‑grained model (denoted G¯o) in which each residue is represented by its Cα atom. To capture the heterogeneity of side‑chain interactions, native contact energies were scaled according to the number of atomic contacts observed in the crystal structure, following the approach of Clementi and co‑workers. This introduces a realistic energetic bias while preserving the funnel‑shaped energy landscape characteristic of Gō models.

Thermal folding simulations were performed by gradually cooling the system from reduced temperature T = 2.5 to T = 1.5 (in units of ε₀/R) in 50 steps. At each temperature, 5 × 10⁷ molecular‑dynamics steps were used for equilibration, followed by a production run of 5 × 10⁸ steps. The heat‑capacity (C_v) curves revealed a cooperative two‑state folding transition for the wild‑type (WT) C5 domain, with a van’t Hoff to calorimetric enthalpy ratio κ²≈0.98, indicating near‑ideal two‑state behavior. Experimental folding temperatures reported in the literature were reproduced within the model’s temperature scale.

Mutations were introduced by converting all native contacts of the target residue into non‑native repulsive interactions. The Asn755Lys mutation, located in the CFGA′ β‑sheet, eliminated a substantial number of native contacts. Consequently, the simulated folding temperature (T_f) decreased by roughly 4 K and κ² showed a modest reduction, reflecting a destabilization of the domain. This computational finding aligns with experimental CD and NMR data that report the Asn755Lys variant as largely unfolded and thermally unstable.

In contrast, Arg654His and Arg668His reside in the BDE β‑sheet, a region characterized by low Φ‑values. Φ‑values were computed using free‑energy perturbation (FEP) across folded (F), transition (TS), and unfolded (U) ensembles. High Φ (≈1) residues indicate native‑like contacts already formed in the transition state, whereas low Φ (≈0) residues remain disordered. The CFGA′ sheet, especially Asn755, displayed Φ≈1, confirming its early involvement in the folding nucleus. The BDE sheet residues exhibited Φ≈0, suggesting they do not contribute significantly to the folding barrier. Accordingly, the Arg‑mutants caused only negligible shifts in T_f and left κ² essentially unchanged, supporting the hypothesis that their pathogenic effect is mediated through altered electrostatic interactions rather than global destabilization. The authors propose that Arg654 and Arg668 may modulate binding to neighboring domains (e.g., C8) or to a CaM‑II‑like kinase that co‑purifies with MyBP‑C.

Transition‑state network analysis was performed by constructing a weighted graph where residues are nodes and edge weights are inversely proportional to the frequency of native contacts in the TS ensemble. Dijkstra’s algorithm identified minimal paths, and the betweenness centrality (B_k) of each residue was calculated. Residues in the CFGA′ sheet showed high B_k, confirming their role as hubs that nucleate folding. This graph‑theoretic approach provides a quantitative measure of residue importance beyond traditional Φ‑value analysis.

A separate bioinformatic assessment of the long CD‑loop (28 residues) revealed a high density of negatively charged residues and low hydrophobicity, hallmarks of intrinsically disordered regions (IDRs). The loop’s physicochemical profile suggests it does not form a stable secondary structure on its own but may become ordered upon ligand binding—a classic “folding‑coupled binding” scenario. This observation dovetails with experimental suggestions that the CD‑loop participates in SH3‑domain‑like recognition motifs and may interact with signaling kinases.

Overall, the paper demonstrates that incorporating energetic heterogeneity into a Gō‑type model enables the detection of subtle mutation‑induced effects on folding pathways, transition‑state architecture, and potential ligand‑binding interfaces. The combined use of Φ‑values, betweenness centrality, and bioinformatic loop analysis offers a comprehensive framework for dissecting the mechanistic basis of disease‑related mutations in multi‑domain cardiac proteins. The findings reinforce that not all pathogenic mutations act by destabilizing the native fold; some, like Arg654His and Arg668His, likely perturb functional interactions while preserving overall thermodynamic stability. This nuanced view has implications for therapeutic strategies targeting MyBP‑C‑related cardiomyopathies.