Abstract
Calmodulin (CaM) is a highly-expressed Ca binding protein known to bind hundreds of protein targets. Its binding selectivity to many of these targets is partially attributed to the protein’s flexible alpha helical linker that connects its N- and C-domains. It is not well established how its linker mediates CaM’s binding to regulatory targets yet. Insights into this would be invaluable to understanding its regulation of diverse cellular signaling pathways. Therefore, we utilized Martini coarse-grained (CG) molecular dynamics simulations to probe CaM/target assembly for a model system: CaM binding to the calcineurin (CaN) regulatory domain. The simulations were conducted assuming a ‘wild-type’ calmodulin with normal flexibility of its linker, as well as a labile, highly-flexible linker variant to emulate structural changes that could be induced, for instance, by post-translational modifications. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 μs of simulation time; of these, 1.7% sampled the fully-bound state observed in the experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be 8.7 × 10 M s, which is similar to the diffusion-limited, experimentally-determined rate of 2.2 × 10 M s. Furthermore, our simulations recapitulated its well-known inverse relationship between the association rate and the solution ionic strength. In contrast, although over 97% of the labile linker simulations formed tightly-bound complexes, only 0.3% achieved the fully-bound configuration. This effect appears to stem from a difference in the ensembles of extended and collapsed states which are controlled by the linker flexibility. Therefore, our simulations suggest that variations in the CaM linker’s propensity for alpha helical secondary structure can modulate the kinetics of target binding.
Highlights
Calmodulin (CaM) is a ubiquitously-expressed, 16.7 kDa globular protein that regulates hundreds of protein targets [1] in a Ca2+-dependent manner
These studies have generated valuable insights into factors contributing to its binding selectivity, which includes hydrophobic residues in targets that interact with CaM, CaM’s conformational heterogeneity at the binding surface, and its Ca2+-binding sensitivity
We propose a mechanistic basis for how CaM linker flexibility shapes the kinetics of target binding and its dependence on the solvent ionic strength
Summary
Calmodulin (CaM) is a ubiquitously-expressed, 16.7 kDa globular protein that regulates hundreds of protein targets [1] in a Ca2+-dependent manner. A key basis for CaM’s selectivity is attributed to the variety of binding modes it adopts when bound to its targets (reviewed in [20]) These include an extended conformation, a collapsed conformation in which its N- and C-domains wrap around the target, and intermediate configurations that permit CaM/target stoichiometries of 1:1, 2:2, or 2:1. CaM’s ability to bind a variety of targets in part stems from the linker’s ability to assume different conformations [21,22] This flexible linker is believed to exert an entropic role in tuning target affinity. Target-binding induced mobility changes in the linker residues correlated with the conformational entropy measured for the entire protein [22] As another example, Katyal et al [23] demonstrated that tethering the N- and C-CaM termini via a disulfide bond decreased the entropic penalty of binding by reducing the ensemble of thermodynamically-accessible states available to the linker
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