Competing Roles of Ca²⁺and Nonmuscle Myosin IIA on the Dynamics of the Metastasis-Associated Protein S100A4

The calcium-binding protein S100A4 plays an important role in a wide range of biological processes such as cell motility, invasion, angiogenesis, survival, differentiation, contractility, and tumor metastasis and interacts with a range of partners. To understand the functional roles and interplay of S100A4 binding partners such as Ca2+and nonmuscle myosin IIA (NMIIA), we used molecular dynamics simulations to investigate apo S100A4 and four holo S100A4 structures: S100A4 bound to Ca2+, S100A4 bound to NMIIA, S100A4 bound to Ca2+and NMIIA, and a mutated S100A4 bound to Ca2+and NMIIA. Our results show that two competing factors, namely, Ca2+-induced activation and NMIIA-induced inhibition, modulate the dynamics of S100A4 in a competitive manner. Moreover, Ca2+binding results in enhanced dynamics, regulating the interactions of S100A4 with NMIIA, while NMIIA induces asymmetric dynamics between the chains of S100A4. The results also show that in the absence of Ca2+the S100A4-NMIIA interaction is weak compared to that of between S100A4 bound to Ca2+and NMIIA, which may offer a quick response to dropping calcium levels. In addition, certain mutations are shown to play a marked role on the dynamics of S100A4. The results described here contribute to understanding the interactions of S100A4 with NMIIA and the functional roles of Ca2+, NMIIA, and certain mutations on the dynamics of S100A4. The results of this study could be interesting for the development of inhibitors that exploit the shift of balance between the competing roles of Ca2+and NMIIA

Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results. © 2014 Das et al

Molecular dynamics study of the effect of active site protonation on Helicobacter pylori 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase

The protein 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is involved in the quorum sensing of several bacterial species, including Helicobacter pylori. In particular, these bacteria depend on MTAN for synthesis of vitamin K2 homologs. The residue D198 in the active site of MTAN seems to be of crucial importance, by acting as a hydrogen-bond acceptor for the ligand. In this study, we investigated the conformation and dynamics of apo and holo H. pylori MTAN (HpMTAN), and assessed the effect of protonation of D198 by use of molecular dynamics simulations. Our results show that protonation of the active site of HpMTAN can cause a conformational transition from a closed state to an open state even in the absence of substrate, via inter-chain mechanical coupling

Molecular dynamics investigation of Helicobacter pylori chemotactic protein CheY1 and two mutants

CheY is a chemotactic response regulator protein modulating the rotation direction of bacterial flagellar motors. It plays an important role in the colonization and infection of Helicobacter pylori (H. pylori), which is a common pathogen. Recently, the structure of CheY1 of H. pylori (HpCheY1) was solved, showing similarities and differences with CheY from E. coli. Here, we report 200 ns atomistic molecular dynamics (MD) simulations of HpCheY1 and two mutants. The results suggest that the surface of HpCheY1 has regions with increased affinity for Mg²⁺. In addition, wildtype HpCheY1 (WT HpCheY1) shows characteristic dynamics in helix 4, which is involved in FliM binding. This dynamics is altered in the D53A mutant and completely suppressed in the T84A mutant. The results are discussed in relation to the binding and function of HpCheY1

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.</div

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.</div