Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding

Abstract

In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well-characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca²⁺, leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca²⁺ and target proteins.

Introduction

Since the pioneering work of Laurent and Ogston, thermodynamic analyses have been applied to investigate the effect of volume interactions on proteins.¹ Most nonideal thermodynamic behaviors of biomolecules in biochemical reactions that depend on available space can be explained by modeling crowding agents as impenetrable hard-core models.2, 3 The role of density, geometry, and amount of crowding agents in the displacement of thermodynamic equilibrium can be analyzed through the theoretical framework of the scaled particle theory.⁴ These studies, however, assumed that the native folded proteins are hard-core objects, disregarding their inherent conformational flexibility. In this regard, molecular simulation provides an excellent approach to studying the dynamics of proteins by probing their statistical information.⁵ For example, certain proteins that exhibit structural plasticity in the folded state were shown to change their structural characteristics in the presence of crowding agents.⁶ As such, the conditions imposed by the surrounding environment of a protein could be critical to its function, as some characteristics may only be induced under crowded cell-like conditions when only rarely present in dilute solutions.7, 8, 9, 10

Here, we apply experimental and theoretical approaches to address how macromolecular crowding impacts the structural characteristics of the essential and ubiquitous protein calmodulin (CaM). CaM is the predominant Ca²⁺-sensing protein in cells responsible for regulating a wide variety of biological functions including muscle contraction, cell division, and neuronal signaling, among many others (for reviews, see Refs. 11, 12, 13). CaM is a dumbbell-shaped protein composed of 148 amino acids that contain four EF hands, two in each half of the dumbbell, and two Ca²⁺ ions bind cooperatively to each lobe. What is critical to the present study is that there is a wealth of structural and kinetic information available for both the Ca²⁺-free and the Ca²⁺-saturated states14, 15, 16, 17, 18 on which simulation and experiments can be based to probe the impact of crowding. Interestingly, the structures of CaM solved by different methods and under different conditions vary drastically, implying the molecule exhibits a range of metastable conformations. Results from NMR show that in the Ca²⁺-free state, the C-lobe of CaM exhibits significantly greater backbone flexibility than does the N-lobe.¹⁴ The interlobe linker also plays an important role in CaM function. Although in some crystal structures it is resolved as an extended alpha helix, NMR studies have shown this central helix to be highly flexible.¹⁹ This flexibility is critical in allowing the lobes of CaM to adjust in the recognition of over 300 target proteins. The remarkable promiscuity in molecular recognition is due to the structural plasticity exhibited by CaM in its capacity to sample a wide range of conformational substates.²⁰

The inherent flexibility of CaM has been investigated with the use of computer modeling and simulations. Fluctuations in lobes and helices associated with conformational changes were investigated by all-atomistic molecular dynamics simulations.21, 22, 23 The differentiable intrinsic flexibility of the C-lobe compared with that of the N-lobe was suggested by using the normal-mode analysis²⁴ and low-resolution protein models.25, 26 How such inherent flexibility in CaM's globular lobes relates to its ability in target binding are discussed for various substrates.27, 28 Nevertheless, the plasticity of CaM has not been investigated under crowded conditions that are most relevant to the determination of the functional states of CaM inside cells.

A fundamental challenge to studying protein dynamics in vivo is to quantitatively dissect protein structure and dynamics²⁹ within a complex environment. The surrounding macromolecules, such as proteins, nucleic acids, lipids, and cytoskeletons, exert volume exclusions, and the volume fractions can reach as high as 40% of the total cellular volume.³⁰ To circumvent the physical and chemical complexities presented by polydisperse macromolecules, experimentalists mimic crowding effects by adding inert synthetic polymers such as polyethylene glycol, dextran, or Ficoll as crowding agents.31, 32 With this framework, studies of protein folding,33, 34, 35, 36, 37 stability,35, 38, 39, 40, 41 and interaction⁴² under crowded conditions suggested that reactions in a crowded milieu are indeed different from that in dilute solution. The effects of excluded volume of crowding agents on the kinetics of protein folding mechanism and stability have also been studied by computer modeling and simulations5, 6, 10, 43, 44, 45, 46, 47 and analytical calculations using different polymer models.3, 48, 49

Our approach consisted on the one hand of the use of fluorescence spectroscopy to measure the distance between N- and C-lobes of CaM in the absence and presence of Ficoll 70. On the other hand, we use coarse-grained molecular simulations that adequately probe large-scale structural fluctuations of CaM in a spatiotemporally complex system providing complementary analysis to the experimental findings. Together with the reconstruction of protein models, we found an increased probability of compacted conformations with an enhanced flexibility of the EF hands when CaM is placed in a crowded environment. Our interpretation of these results is that crowding changes the free-energy barrier between the basins corresponding to different compact CaM conformations. As a consequence, there is a shift in the distribution of compact substates to those more likely to bind Ca²⁺ and/or target enzymes. This suggestion is consistent with recent results describing that the conformational entropy of CaM contributes in a significant way to the free energy of binding.⁵⁰