This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract: Despite our growing body of knowledge on enzymes, many questions still remain and MD simulations are the only way to provide answers to some of them. The particular class of enzyme we have chosen to study is dihydrofolate reductase (DHFR). A form of this enzyme exists in almost every living organism and is important for cellular metabolism. 1 While many issues are still unresolved, we plan to study two specific problems that are idea catalysis.lly suited for investigation by MD simulations. The first is a comprehensive look at the relationship between ligand binding and the M20 loop conformation of chromosomal E. coli DHFR. The second involves deciphering the cause of binding cooperativity in a R-plasmid-encoded R67 DHFR. DHFR M20 Loop Conformations Because DHFR is such an important cellular protein, an extensive amount of research has focused on this enzyme.2 These experiments have greatly increased our knowledge of DHFR. Yet, some aspects of this protein are still not understood. In particular, the function of the M20 loop and how it relates to catalysis has not been successfully determined. This research proposes a series of molecular dynamics simulations that will investigate this relationship. The results will provide insight into the M20 loop conformers and determine which are present at different points of the catalytic cycle. The main objectives of this project are listed below. Perform molecular dynamics on conformations of each complex formed during the catalytic cycle and of each complex studied experimentally. For each complex, several simulations will be performed starting from different M20 loop conformations. Calculate the relative free energies of the various conformations of each complex using the MM FPBSA method. Examine the above calculations to try and relate the relative free energies to specific effects, such as solvation or electrostatics, and if possible, to locate the specific amino acid or ligand interactions involved. Determine the contribution, if any, of the M20 loop conformation to catalysis. 0 %2 0 %2 0 %2 0 %2 Not only will these calculations provide additional insight into the catalytic cycle of DHFR, but they will also indicate how the M20 loop potential energy surface varies for different complexes of this enzyme. In addition, these results will indicate the ability of the experimental complexes to represent the catalytic cycle complexes. R67 DHFR Binding Properties: R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that catalyzes the same reaction as chromosomal DHFR. Unlike most plasmid enzymes, R67 DHFR shares no structural similarity with its chromosomal counterpart and thus must rely on different methods for achieving binding and catalysis. The structure consists of four identical monomers that assemble to form a D2 symmetric tetramer. 3 The active site binds two ligands and exhibits binding cooperativity.4 We propose to study how the active site of R67 DHFR operates by performing molecular dynamics simulations on several complexes and mutants. Comparison of these simulations should provide useful insights into the unique binding properties of this enzyme. The main objectives of this project are listed below. Perform initial molecular dynamics simulations on several complexes, using the truncated ligands. This removes the disordered chain of the substrate and the chain of the cofactor for which no binding information is available. This will help simplify the preliminary calculations. Analyze the trajectories of the different complexes to determine the effect of ligands on protein structure, the significance of protein-ligand or ligand-ligand interactions, and the importance of protein dynamics. Perform several molecular dynamics simulations on each of the complexes containing the complete ligands to determine the binding modes of the chains of the ligands and perform analysis. In the future, perform molecular dynamics simulations on mutants of R67 DHFR that mutagenesis studies have implicated in binding. Then analyze their effect on the binding properties of the enzyme. These comparisons will provide insights into how the protein contributes to binding cooperativity. They will also provide information on how identical binding sites can be used to bind both NADPH and DHF. The two sets of calculations will also supply a unique opportunity to compare two enzymes that catalyze the same reaction through very different means. Presumably, the chromosomal DHFR has evolved many ways to enhance catalysis. Yet, R67 DHFR, which most likely evolved from an enzyme with a very different function, has not had the same opportunity for improvement. Despite this, R67 DHF R is a reasonably efficient enzyme. 4 These calculations will help to probe some of the differences and similarities in how these two disparate enzymes achieve catalysis. Blakley, R. L. in Chemistry and Biochemistry, Vol. I, Folates and Pterins, ed. R. L. Blakley and S. J. Benkovic, John Wiley & Sons, New York, 1984, pp 191-253. Sawaya, M. R., Kraut, J. Loop and subdomain movements in the mechanism of Escherichia coli dihydrofolate reductase, Crystallographic Evidence, Biochemistry 1997, 36, 586-603 and references therein. Schnell, J. R., Dyson, H. J., Wright, P. E., Structure, dynamics, and catalytic function of dihydrofolate reductase, Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 119-140. Narayana, N., Matthews, A., Howell, E. E., Xuong, N.-H., A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site, Nat. Struct. Biol. 1995, 2, 1018-1025.
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