MOLECULAR DYNAMICS SIMULATIONS TO UNDERSTAND THE EFFECTS OF ACTIVE-SITE MUTATIO

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract: All type II restriction endonucleases contain a cluster of active-site Glu and Asp residues, which are responsible for coordination of the catalytic cofactor Mg2+. The site for metal ion binding is not assembled completely and precisely until the enzyme binds to its specific DNA recognition sequence. This provides a mechanism for enhancing specificity by coupling recognition to catalysis. At the specific EcoRI-DNA interface, this active-site cluster includes D91, E111, and the scissile phosphate oxygen, located at the GPAATTC position, within the EcoRI specific recognition sequence. D59 and E144 are also located in the vicinity of the acidic active-site cluster but do not play a role in coordinating Mg2+. In addition, two basic side-chain residues are located near this acidic cluster namely, R145 and K113. Apposition of the negative charges in the active-site cluster creates strong electrostatic repulsion, which can be relieved by the addition of divalent metal, or by protonation of active-site residues. Thus, large enhancements in binding affinity (~500-fold in KA) are observed upon removal of negative charge from the protein (by mutation of active-site acidic residues to alanine), from the DNA (by removal of the scissile phosphate), by addition of the divalent metal ion Ca2+, which acts as a non-catalytic mimic of Mg2+, or by titration to pH below 6. Current Interests: We want to extend our understanding of the role of active-site repulsion in the structural, dynamic, and energetic aspects of specific EcoRI-DNA complex formation. Specifically, we ask 1) what are the rotameric positions and the degree of atomic fluctuation of the charged active-site residues in the free enzyme, and in the enzyme-DNA complex in the presence and absence of divalent metal, 2) what are the electrostatic potentials at the active-site in all three of these structures, 3) how does mutation of charged residues to a neutral residue, or to a residue of the opposite charge, affect the rotameric conformation, atomic fluctuation, and electrostatic potentials of active-site residues in all three structures, and 4) how is the water structure at or near the active-site affected by electrostatic perturbation in all three structures? Justification for Allocation: In order to address the questions above, we will use the Amber suite of programs to perform MD simulations and electrostatic potential calculations on wild-type and mutant EcoRI complexes, in the presence and absence of DNA and divalent metal. Due to the size of these systems and the need to run long simulations in order to see possible conformational fluctuations, we believe the XT3 platform will suit our needs. We recognize that the XT3 Cray system will be decommissioned on March 31st, 2010, so we include in our request an allocation on the People SGI Altix 7400 system in order to transition to this platform in the future. Data obtained from these studies, along with the results of rigorous thermodynamic analyses performed in our laboratory, will allow us to better understand the structural, energetic, and dynamic role of the acidic cluster of residues at the EcoRI-DNA active-site, and allow us to compare this system with the active-site of other type II restriction endonucleases having similar but slightly different, active-site geometries.