Assessing the Role of Calmodulin’s Linker Flexibility in Target Binding

1 Abstract Calmodulin (CaM) is a universal Ca 2+ binding protein known to bind at least 300 targets. The selectivity and specificity towards these targets are partially attributed to the protein’s flexible alpha-helical linker that connects its N- and C-domains. How this flexible linker mediates the driving forces guiding CaM’s binding to regulatory targets is not well-established. Therefore, we utilized the Martini coarse-grained (CG) molecular dynamics simulations to probe interrelationships between CaM/target assembly and the role of its linker region. As a model system, we simulated the binding of CaM to the CaM binding region (CaMBR) of calcineurin (CaN). The simulations were conducted assuming a ‘wild-type’ calmodulin with normal flexibility of its linker, as well as a labile, highly flexible linker variant. For the wild-type model, 98% of the 600 simulations across three ionic strengths adopted a bound complex within 2 µ s of simulation time; of these, 1.7% sampled the fully-bound state observed in experimentally-determined crystallographic structure. By calculating the mean-first-passage-time for these simulations, we estimated the association rate to be k a = 5.9 × 10 8 M − 1 s − 1 , which is similar to the experimentally-determined rate of 2.2 × 10 8 M − 1 s − 1 [1]. Further, our simulations recapitulated the inverse relationship between the association rate and solution ionic strength reported in the literature. 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 controlled by the linker properties. Specifically, the labile linker variant samples fewer extended states compatible with target peptide binding. Therefore, our simulations suggest that variations in the CaM linker’s propensity for alpha-helical secondary structure can modulate the kinetics of target binding. This finding is important, as the linker region houses several CaM variants sites for post-translational modifications, that may alter the protein’s normal regulatory functions.