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. DNA helicases are ATP driven molecular motors involved in all aspects of DNA metabolism, such as replication, transcription, and repair. They catalyze the separation of double stranded DNA into its constituent single stranded DNA (ssDNA) components. PcrA is a monomeric 3 5 helicase for which several atomic resolution x-ray structures have been reported [177]. The DNA unwinding action of PcrA is caused by helicase translocation along ssDNA, which is driven by the hydrolysis of ATP in a single catalytic site. The translocation proceeds at a rate of approximately 50 nucleotides per second [178]. The Resource has used three complementary approaches to investigate PcrA s overall function, the combination of which is novel and required the use of quantum chemistry, classical molecular modeling, and non-equilibrium statistical mechanics simulations. At the catalytic site level, combined quantum mechanical/molecular mechanical (QM/MM) simulations were used to study the ATP hydrolysis reaction pathway and its coupling to protein conformational changes. The simulation system consisted of about 20,000 atoms, 77 of which were treated quantum mechanically at the B3LYP/6-31G level of theory. Analogous to the Resource s previous studies of ATP hydrolysis in F1-ATPase [179, 180], a proton relay mechanism was identified as the physiologically relevant proton transfer pathway during nucleophilic attack. The arginine finger residue R287 from a protein domain neighboring the catalytic site was found to be crucial for transition state stabilization, thereby, adding to the list of similarities between PcrA and F1-ATPase at the catalytic site level. Employing computational mutation studies, it could be shown that the position of Q254 with respect to the terminal phosphate group of ATP greatly influences the energy of the ATP hydrolysis product state, since a slight decrease in side chain length (mutation Q254N) changed the reaction energy profile from endothermic in the wild type to almost equi-energetic. This suggests a mechanism by which protein conformational changes induced by DNA translocation are coupled to the catalytic reaction in the binding site of PcrA. The ATP hydrolysis powered ssDNA translocation of PcrA was then studied by molecular dynamics (MD) simulations. Using the program NAMD [44], MD simulations were performed on a fully solvated PrcA-DNA complex with and without ATP bound to the catalytic site. The system contained approximately 110,000 atoms and the simulations lasted for several nanoseconds. On the basis of these simulations, interaction energies between the protein and the ssDNA nucleotides were evaluated, resulting in effective potentials governing the domain movements of PcrA. The simulations revealed that during the ATP hydrolysis cycle the domains 1A and 2A alternatively become weakly and strongly bound to ssDNA in such a manner that unidirectional translocation of PcrA along ssDNA results. The alternating effective potentials were then utilized in a coarse grained Langevin dynamics description that corroborated an inchworm mechanism of PcrA translocation that had previously been proposed based on x-ray crystallography studies [177]. The combined approaches of MD simulation and Langevin dynamics description identified key amino acids affecting the unidirectional translocation of PcrA, suggesting candidate residues for mutations of PcrA that may achieve reverse translocation.
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