Abstract
Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.
Highlights
Sarcomeres contract owing to the translocation of the thick filament, comprised of myosin, along actin chains constituting the thin filament (TF)
The mutations included the GOF-like mutants V44Q and L48Q, as well as the LOF-like mutants V79Q and E40A. These mutants induced significant changes in the polar character of wildtype Troponin C (TnC), with V44Q, L48Q, and V79Q representing apolar to polar alterations and E40A, a charged to neutral substitution
While the Ca RMSD does not strongly distinguish between GOF and LOF mutants, differences were noted at LCD, the physiologically active Ca2z binding domain, with increasing deviations noted for V44Q, E40A, V79Q, and L48Q (RMSD from 1.0 to 3.5 A, (Fig. S2c)
Summary
Sarcomeres contract owing to the translocation of the thick filament, comprised of myosin, along actin chains constituting the thin filament (TF). A number of human cardiac diseases including hypertrophic cardiomyopathy (HCM) [2], restrictive cardiomyopathy (RCM) [3] and dilated cardiomyopathy (DCM) [4] have been attributed to mutations in thin filament, thick filament and associated proteins of the sarcomere. Mutation studies of full-length TnC have revealed engineered variants that shift the Ca2z equilibrium constant, Keq (or pCa50), leading to altered force development akin to GOF and LOF [8]. The E40A and V79Q mutations examined by [8] present LOF-like alterations in force generation with pCa50 values of 5.16 and 5.30, respectively. While these studies have implicated Ca2z binding as the primary factor in reshaping
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