Coupling of Redox and Structural States in Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation.

Cytochrome P450 reductase (CPR) is the key protein that regulates the electron transfer from NADPH to various heme-containing monooxygenases. CPR has two flavin-containing domains: one with flavin adenine dinucleotide (FAD), called FAD domain, and the other with flavin mononucleotide (FMN), called FMN domain. It is considered that the electron transfer occurs via FAD and FMN (NADPH → FAD → FMN → monooxygenase) and is regulated by an interdomain open-close motion. It is generally thought that the structural state is coupled with the redox state, which, however, has not yet been firmly established. In this report, we studied the coupling of the redox and the structural states by full-scale molecular dynamics (MD) simulation of CPR (total 86.4 µs). Our MD result showed that while CPR predominantly adopts the closed state both in the oxidized and reduced states, it exhibits a tendency to open in the reduced state. We also found a correlation between the FAD-FMN distance and the predicted FMN-monooxygenase distance, which is embedded in the equilibrium thermal fluctuation of CPR. Based on these results, a physical mechanism for the electron transfer by CPR is discussed.

Hydrophobic Surface Enhances Electrostatic Interaction in Water.

A high dielectric constant is one of the peculiar properties of liquid water, indicating that the electrostatic interaction between charged substances is largely reduced in water. We show by molecular dynamics simulation that the dielectric constant of water is decreased near the hydrophobic surface. We further show that the decrease in the dielectric constant is due to both the decreased water density and the reduced water dipole correlation in the direction perpendicular to the surface. We finally demonstrate that electrostatic interaction in water is actually strengthened near the hydrophobic surface.

Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery.

A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

Dielectric Allostery of Protein: Response of Myosin to ATP Binding.

Protein uses allostery to execute biological function. The physical mechanism underlying the allostery has long been studied, with the focus on the mechanical response by ligand binding. Here, we highlight the electrostatic response, presenting an idea of "dielectric allostery". We conducted molecular dynamics simulations of myosin, a motor protein with allostery, and analyzed the response to ATP binding which is a crucial step in force-generating function, forcing myosin to unbind from the actin filament. We found that the net negative charge of ATP causes a large-scale, anisotropic dielectric response in myosin, altering the electrostatic potential in the distant actin-binding region and accordingly retracting a positively charged actin-binding loop. A large-scale rearrangement of electrostatic bond network was found to occur upon ATP binding. Since proteins are dielectric and ligands are charged/polar in general, the dielectric allostery might underlie a wide spectrum of functions by proteins.

Piezoelectric allostery of protein.

Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

Temperature-enhanced association of proteins due to electrostatic interaction: a coarse-grained simulation of actin-myosin binding.

Association of protein molecules constitutes the basis for the interaction network in a cell. Despite its fundamental importance, the thermodynamic aspect of protein-protein binding, particularly the issues relating to the entropy change upon binding, remains elusive. The binding of actin and myosin, which are vital proteins in motility, is a typical example, in which two different binding mechanisms have been argued: the binding affinity increases with increasing temperature and with decreasing salt-concentration, indicating the entropy-driven binding and the enthalpy-driven binding, respectively. How can these thermodynamically different binding mechanisms coexist? To address this question, which is of general importance in understanding protein-protein bindings, we conducted an in silico titration of the actin-myosin system by molecular dynamics simulation using a residue-level coarse-grained model, with particular focus on the role of the electrostatic interaction. We found a good agreement between in silico and in vitro experiments on the salt-concentration dependence and the temperature dependence of the binding affinity. We then figured out how the two binding mechanisms can coexist: the enthalpy (due to electrostatic interaction between actin and myosin) provides the basal binding affinity, and the entropy (due to the orientational disorder of water molecules) enhances it at higher temperatures. In addition, we analyzed the actin-myosin complex structures observed during the simulation and obtained a variety of weak-binding complex structures, among which were found an unusual binding mode suggested by an earlier experiment and precursor structures of the strong-binding complex proposed by electron microscopy. These results collectively indicate the potential capability of a residue-level coarse-grained model to simulate the association-dissociation dynamics (particularly for transient weak-bindings) exhibited by larger and more complicated systems, as in a cell.

A simple detection method for low-affinity membrane protein interactions by baculoviral display.

BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV). In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA). Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L), and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand (GITRL). Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system.

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein alpha-subunit (Galpha(s)), G-protein beta(1)gamma(2) subunit dimer (Gbeta(1)gamma(2)), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Galpha(s), Gbeta(1)gamma(2), and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Galpha(s)-coupled receptors together with Galpha(s), Gbeta(1)gamma(2), and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Galpha(s) and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.