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. Project 1: Mechanism of DNA helicase PcrA Over the past year, the Theoretical and Computational Biophysics Group has used the powerful Jonas cluster at PSC to investigate the mechanism of DNA unwinding in the DNA helicase PcrA from Bacillus stearothermophillus. The DNA unwinding action of PcrA is caused by helicase translocation along single stranded DNA, which is driven by the hydrolysis of adenosine tri-phosphate (ATP) in a single catalytic site. We have used combined quantum mechanical/molecular mechanical (QM/MM) simulations to study the ATP hydrolysis reaction pathway and its coupling to protein conformational changes in PcrA helicase. The simulation system consisted of about 20,000 atoms, 77 of which were treated quantum mechanically at the B3LYP/6-31G level of theory; the classical part was modeled using the AMBER94 force field. In order to simulate such a large QM/MM system, we relied crucially on the availability of the Jonas cluster at PSC with its large shared memory architecture, which allowed our code to run efficiently on up to 32 processors. Analogous to our previous studies of ATP hydrolysis in F1-ATPase, 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 in silico 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.
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