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

Abstract

Calmodulin (CaM) is a universal calcium 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. However, how this flexible linker mediates the driving forces guiding CaM's binding to regulatory targets is not well-established. Therefore, we utilized coarse-grained (CG) Martini 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 between the N- and C-terminal domains, 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 tightly-bound complex within 2 μs 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 ka =6.3×108 M −1 s−1, which is similar to the experimentally-determined rate of 2.2×108 M −1 s−1 (Cook et al 2020). 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 and its ionic strength dependence is attenuated. These effects appear 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, given that some CaM variants found in the human population and post-translational modifications sites fall within this linker region, which may alter the protein's normal regulatory functions.

Coarse-Grained Modeling and Molecular Dynamics Simulations of Ca2+-Calmodulin

“…A myriad of computational approaches, ranging from all-atom to coarse-grained ( Weinstein and Mehler, 1994 ; Spoel et al, 1996 ; Wriggers et al, 1998 ; Vigil et al, 2001 ; Yang et al, 2001 ; Barton et al, 2002 ; Komeiji et al, 2002 ; Yang et al, 2004 ; Zuckerman, 2004 ; Monticelli et al, 2008 ; Nandigrami and Portman, 2016 ; Liu et al, 2017b ; Gong and Sun, 2017 ; Robustelli et al, 2018 ; Delfino et al, 2019 ; Sun and Kekenes-Huskey, 2021 ), have been employed to model the structural dynamics of CaM. However, these models have yet to capture the reciprocal relation between Ca 2+ binding and target binding.…”

The non-canonical interaction between calmodulin and calcineurin contributes to the differential regulation of plant-derived calmodulins on calcineurin

“…Another example is the activation of calcineurin (CaN) by CaM, in which a 'distal helix' region C-terminal to CaMBR interacts with CaM to fully remove the autoinhibitory domain of CaN [25]. Namely, studies [25][26][27][28][29] have provided a model that explains the molecular interactions between CaN and CaM that are vital to CaN's complete activation (Fig. 1) [30].…”