Several diverse projects are being pursued. These are the major ones pursued during the past year. 1. Calcium ATPase Conformational Transition The sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1a) transport calcium ions from cytoplasm into the reticulum and relaxes the muscle cells. Many crystal structures of SERCA1 in various binding states have been determined, which provide insights into the mechanism of transport Ca2+ across the membrane. Molecular modeling and simulation studies are also devoted to the understanding of this important process. To understand the transport mechanism, it is desirable to study the dynamic process during the conformation transition. Self-guided Langevin dynamics (SGLD) is a simulation method capable of studying events with large conformational change. SGLD simulations of SERCA at different binding states produce conformational transitions between conformational states. New conformations for E1.2Ca2+ and E2.P state have been identified and at E2 state the crystal structure is a preferred conformation. 2. Simulation study of glycine receptors Both exogenous and endogenous cannabinoids can allosterically modulate glycine receptors (GlyRs). However, little is known about the molecular basis of cannabinoid-GlyR interactions. Here we report that sustained incubation with the endocannabinoid anandamide (AEA) substantially increased the amplitude of glycine-activated current in both rat cultured spinal neurons and in HEK-293 cells expressing human alpha1, rat alpha2 and alpha3 GlyRs. While the alpha1 and alpha3 subunits were highly sensitive to AEA-induced potentiation, the alpha2 subunit was relatively insensitive to AEA. Switching a serine at 296 and 307 in the TM3 (transmembrane domain 3) of the alpha1 and alpha3 subunits with an alanine (A) at the equivalent position in the alpha2 subunit converted the alpha1/alpha3 AEA-sensitive receptors to sensitivity resembling that of alpha2. The S296 residue is also critical for exogenous cannabinoid-induced potentiation of I(Gly). The magnitude of AEA potentiation decreased with removal of either the hydroxyl or oxygen groups on AEA. While desoxy-AEA was significantly less efficacious in potentiating I(Gly), desoxy-AEA inhibited potentiation produced by both Delta(9)-tetrahydrocannabinol (THC), a major psychoactive component of marijuana, and AEA. Similarly, didesoxy-THC, a modified THC with removal of both hydroxyl/oxygen groups, did not affect I(Gly) when applied alone but inhibited the potentiation of I(Gly) induced by AEA and THC. These findings suggest that exogenous and endogenous cannabinoids potentiate GlyRs via a hydrogen bonding-like interaction. Such a specific interaction likely stems from a common molecular basis involving the S296 residue in the TM3 of the alpha1 and alpha3 subunits. 3. Atomic mechanism of the kinesin walking on microtubule Kinesin is a protein belonging to the class of Cytoskeletal motor proteins. Kinesin converts the energy of ATP hydrolysis into stepping movement along microtubules, which supports several vital cellular functions including mitosis, meiosis, and the transport of cellular cargo. Because kinesin is a fundamental protein, further research on the topic will provide important information as to how it functions. Combined with low resolution electron microscopic images, self-guided Langevin dynamics simulations are performed to study molecular motion and conformational change of kinesin motor domain in water and binding with microtubule. SGLD enable simulation to reach the time scale required for conformational change to understand the role of ATP binding and interaction with microtubules. 4. Understanding the Basis of a Class of Paradoxical Mutations in AraC In Escherichia coli, AraC protein is involved in regulation of the expression of genes whose products enable the cells to take up and catabolize the sugar, L-arabinose. Upon binding of arabinose AraC undergoes a conformational change and actively represses its own synthesis. Most mutations at position 15 in the N-terminal arm of the regulatory protein AraC leave the protein incapable of responding to arabinose, that is, they are uninducible. Mutations at other positions of the arm do not have this behavior. Simple energetic analysis of the interactions between the arm and bound arabinose did not explain the uninducibility of AraC with mutations at position 15. Extensive molecular dynamics simulations, carried out largely on the Open Science Grid, were done of the wild type protein with and without bound arabinose and of all possible mutations at position 15 many of which were constructed and measured for this work. Good correlation was found for deviation of arm position during the simulations and inducibility as measured in vivo of the same mutant proteins. Analysis of the molecular dynamics trajectories revealed that critical to inducibility is the preservation of the shape of the arm. The interactions needed to maintain the correct shape of the arm were identified. 5. Computational study of nitrogen oxides Nitric oxide (NO) is one of the simplest biological molecules in nature, but also in nearly every phase of biology and medicine with its role ranging from a critical endogenous regulator to blood flow, a principal neurotransmitter, to major pathophysiological mediator of inflammation and host defense, and so on. We continued our collaboration with Dr. David Wink from NCI to apply electronic structure calculation of NO and its sequential reactions in aqueous solution. We made breakthrough in applying relatively low cost spin flip density functional theory (SP-DFT) on NO and relative reactive nitrogen species (RNS) to obtain results comparable to very high level of multireference quantum chemistry theories. This breakthrough will make our near future research on NO and relative RNS more feasible. 6. Enzymatic activity of MMP-1 on collagen type I molecule Degradation of collagen is an important process in atherosclerosis, tissue remodeling and cancer. Matrix metalloproteinases cleave triple helical collagen structure at a specific single site in a sequences of binding and conformational changes that are not clearly understood by experiments and kinetic studies. Furthermore, native collagen usually undergoes mechanical forces in working condition that is difficult to recapitulate in experiments. In this work, we have been applying molecular dynamics simulation tools structure building, docking self-guided langevin dynamics and quantum/classical molecular mechanics to study in details the process in which matrix metalloproteinase-1 binds to and causes conformational changes of a collagen segment containing the cleavage site under tensile force. Molecular dynamics simulation and docking of MMP-1 on the cleavage site segment of collagen have produced results showing that tensile force can stabilize collagen cleavage site thus can increase resistance of collagen to cleaving by MMP-1. Simulation of MMP1 in solution revealed variable width of the catalytic cleft. Docking energy and self-guided Langevin dynamics simulation of docked complexes indicated that collagenolysis by MMP1 may not rely on active unwinding and stabilizing local spontaneous unfolding by the Hpx domain. Instead, binding of MMP1 to collagen I fibril are established through inducible fitting process in which the catalytic cleft is opened wider in interaction with the triple helix and the Hpx domain helps to align the cleft along the triple helix. Further self-guided Langevin dynamics and quantum mechanics/molecular mechanics simulations of docked complexes are expected to yield details of enzymatic reaction of matrix metalloproteinases on collagen.
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