Energy landscape views for interplays among folding,binding, and allostery of calmodulin domains

Ligand binding modulates the energy landscape of proteins, thus altering their folding and allosteric conformational dynamics. To investigate such interplay, calmodulin has been a model protein. Despite much attention, fully resolved mechanisms of calmodulin folding/binding have not been elucidated. Here, by constructing a computational model that can integrate folding, binding, and allosteric motions, we studied in-depth folding of isolated calmodulin domains coupled with binding of two calcium ions and associated allosteric conformational changes. First, mechanically pulled simulations revealed coexistence of three distinct conformational states: the unfolded, the closed, and the open states, which is in accord with and augments structural understanding of recent single-molecule experiments. Second, near the denaturation temperature, we found the same three conformational states as well as three distinct binding states: zero, one, and two calcium ion bound states, leading to as many as nine states. Third, in terms of the nine-state representation, we found multiroute folding/binding pathways and shifts in their probabilities with the calcium concentration. At a lower calcium concentration, “combined spontaneous folding and induced fit” occurs, whereas at a higher concentration, “binding-induced folding” dominates. Even without calcium binding, we observed that the folding pathway of calmodulin domains can be modulated by the presence of metastable states. Finally, full-length calmodulin also exhibited an intriguing coupling between two domains when applying tension.Protein folding and conformational dynamics have often been characterized by the energy landscape of proteins (1–5). The energy landscape is dependent on the molecular physiochemistry and thus is modulated by many factors, such as chemical modification and ligand binding. Ligand binding, in turn, is dependent on the conformation of proteins. Thus, folding, binding, and allosteric conformational dynamics are mutually correlated. Despite their obvious correlation in concept, it has been very challenging to characterize how they are indeed coupled for any single proteins. Here, we address, in depth, how these three types of dynamics, folding, binding, and allosteric conformational dynamics, are coupled from the energy landscape perspective for a specific protein, calmodulin (CaM).CaM is a ubiquitous calcium-binding messenger protein involved in signal transduction (6) and, more importantly here, has been a model protein to investigate folding, binding, and allostery. Full-length CaM has two nearly symmetric globular domains connected by a flexible central helix (7, 8). Each domain is composed of paired EF hands containing two Ca²⁺-binding sites (Fig. 1A). Upon binding to Ca²⁺, each CaM domain undergoes substantial conformational change from a closed state to an open state, exposing a hydrophobic patch that can bind with target proteins and regulate downstream processes (9). CaM has been frequently used as a model in studying the folding of multidomain proteins (10, 11), allosteric transitions (12–14), slow conformational dynamics around physiological temperatures (15–18), metal ion binding (19, 20), and correlation between inherent flexibility and protein functions (21, 22). For example, using structure-based coarse-grained (CG) simulations, Chen and Hummer elucidated the coexistence of an unfolded state, a closed state, and an open state around physiological temperatures for the C-terminal domain of CaM (CaM-C) without Ca²⁺ binding (15), which reconciles some seemingly contradictory experimental observations on the slow conformational dynamics of CaM.Open in a separate windowFig. 1.(A) Three-dimensional structure of calmodulin domain at closed [Protein Data Bank (PDB) code: 1cfd] and open states (PDB code:1cll). Calcium ions are represented by yellow spheres. (B) Schematic of coupling among folding, calcium binding, and allosteric motions for the CaM domain. Due to the conformational transitions between open and closed states, in addition to the direct folding pathway (red solid arrow), folding to the most stable state may involve an alternative pathway via a metastable state (green arrow plus blue arrow). The calcium binding can modulate the relative stability of the conformational states and therefore the population of folding pathways. O, C, and U represent open, closed, and unfolded states, respectively.More recently, Rief and coworkers studied the Ca²⁺-dependent folding of CaM based on a new generation technique of single-molecule force spectroscopy, which can probe the reversible folding/unfolding transitions with near equilibrium conditions (10, 23, 24). Their results revealed that at high Ca²⁺ concentrations, the folding pathway of the CaM domain proceeds via a transition state capable of binding Ca²⁺ ions, demonstrating the coupling between Ca²⁺ binding and CaM folding. All these computational and experimental works provided unprecedented understanding of many aspects of the folding and allosteric transitions of CaM. However, a full picture of the coupling among folding, Ca²⁺ binding, and allosteric motions, as schematically shown in Fig. 1B, is still lacking. Particularly, two fundamental issues arising from the allostery and Ca²⁺-binding characteristics of CaM remain elusive: (i) How does the allosteric feature of the energy landscape contribute to the folding complexity? And (ii) how can the folding mechanism of CaM be modulated by Ca²⁺ binding?Motivated by previous computational and experimental studies (15, 23), in this work we investigated the folding coupled with Ca²⁺ binding and allosteric motions of the isolated CaM domains as well as the full-length CaM. To do so, we first integrated computational tools developed for folding, ligand binding, and allosteric motions together. The proposed CG protein model was used for the subsequent series of molecular dynamics (MD) simulations. First, corresponding to Rief’s experiments, we performed MD simulations of isolated CaM domains with pretensions, which gave consistent results with the experiments and, in addition, provided the direct structural assignment on the experimentally observed states. Second, at a higher temperature, without pretension we performed reversible folding/unfolding simulations for a wide range of Ca²⁺ concentrations. The conformational and ligand-binding energy landscape revealed as many as nine distinctive states. Then, we analyzed the binding-coupled folding reactions in terms of the nine states, finding multiple routes and their modulation by Ca²⁺ concentrations. Interestingly, as the Ca²⁺ concentration increases, the CaM domain folding mechanism switches from “combined spontaneous folding and induced fit” to “binding-induced folding,” which accords with the scenario deduced from single-molecule force spectroscopy experiments. Finally, the effects of tension on the conformational fluctuations of the full-length CaM are discussed.