Designing Calcium-sensitizing Mutations in the Regulatory Domain of Cardiac Troponin C

Abstract

As anticipated, these selected hydrophobic residue substitutions increased the calcium affinity of the regulatory domain of cardiac troponin C F27W ϳ2.1-15.2-fold. Surprisingly, the increased calcium affinity caused by the hydrophobic residue substitutions was largely due to faster calcium association rates (2.6 -8.7-fold faster) rather than to slower calcium dissociation rates (1.2-2.9-fold slower). The regulatory N-domains of cardiac troponin C F27W and its mutants were also able to bind magnesium competitively and with physiologically relevant affinities (1.2-2.7 mM). The design of calcium-sensitizing cardiac troponin C mutants presented in this work enhances the understanding of how to control cation binding properties of EF-hand proteins and ultimately their structure and physiological function.

Engineered Troponin C Constructs Correct Disease-related Cardiac Myofilament Calcium Sensitivity

“…By taking advantage of these rules, TnC has been engineered to encompass a wide range of Ca 2ϩ sensitivities, which can accommodate a broad spectrum of disease-related Ca 2ϩ binding (32,33). Thus, TnC is a more versatile protein to modulate and reset disease-associated myofilament Ca 2ϩ sensitivity.…”

“…For instance, the ability of TnC to open its buried N-terminal hydrophobic pocket and bind TnI is a major determinant of its apparent Ca 2ϩ sensitivity (Fig. 8) (32,33). Modifying the network of side chain interactions involved with the opening of the TnI-bind- ing pocket (such as the M45Q mutation; Fig.…”

“…A Ca 2ϩ -sensitizing TnC will be required to correct the DCM thin filament behavior. By mutating the hydrophobic pocket of the regulatory domain of TnC, Ca 2ϩ -sensitizing TnC constructs can be engineered (32,33). For instance, the TnC M45Q mutation increased thin filament Ca 2ϩ sensitivity ϳ8-fold when compared with the control Tn IAANS T53C ( Fig.…”

“…Unfortunately, there are no pharmacological compounds that just target TnC (31). However, by directly engineering TnC, our laboratory has developed several TnC constructs, which behave as Ca 2ϩ sensitizers or desensitizers in biochemical model systems and in muscle (16,32,33). By utilizing different design principles (32,33), the intrinsic Ca 2ϩ binding properties of TnC can be finely or grossly tuned.…”

“…However, by directly engineering TnC, our laboratory has developed several TnC constructs, which behave as Ca 2ϩ sensitizers or desensitizers in biochemical model systems and in muscle (16,32,33). By utilizing different design principles (32,33), the intrinsic Ca 2ϩ binding properties of TnC can be finely or grossly tuned. We have rationally engineered TnC to test whether the Ca 2ϩ dependence of biochemical and physiological systems harboring disease-associated protein modifications could be reset.…”