STUDIES ON THE S2-S3 LOOP OF THE 20S PROTEASOME: A LINK BETWEEN ASSEMBLY AND AC

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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. The 26S proteasome (~2.5 MDa) is the central protease of the ubiquitin pathway of protein degradation. The 26S proteasomal active site carries out peptide bond hydrolysis via its N-terminal threonine. The 26S proteasome is comprised of two 19S regulatory complexes and a 20S catalytic core. The 20S core complex (~700 kDa) is an assembly of 14 ?-subunits and 14 ?-subunits, arranged into two ?-rings sandwiched by two outer ?-rings (?7?7?7?7). In the 20S catalytic core complex of the Rhodococcus erythropolis proteasome, assembly is a multilevel process initiated by the joint association of an ?-type and ?-type subunit. This process continues as the ?? subunit complexes combine to form two heptameric ring structures (?7?7), the half proteasome. Lastly, two half proteasomes coalesce, via the ?7rings, to form the pre-active 20S proteasome. Activation then proceeds by the autocleavage of the ?-subunits N-terminal propeptide. In order to determine the structural link between assembly and activation, we introduced the F145A mutation into the ?-subunit. Through structural studies, we found that this mutation critically disrupts contacts within an intersubunit hydrophobic network that acts as a seal bridging together two half proteasomes. Experimental studies have shown that the F145A mutation affects the process of assembly by increasing the population of half proteasomes, yet it crystallizes as a full proteasome. The F145A mutation also renders the proteasome inactive, but has little effect on the structure. The backbone RMSD between the wildtype and mutant structures is only 0.75 angstroms. A comparison of the crystal structures did show that the active site residues became greatly disordered upon adding the mutation. As this mutation deactivated the proteasome with minimal effects on the structure, we decided to determine its impact by conducting molecular dynamics simulations. As this mutation is far removed from the active site, we hypothesized that the F145A mutants inactivity was a result of the S2-S3 loop being destabilized. The S2-S3 loop acts as a bridge between ??subunits at the junction of two half proteasomes. Two of the three active site residues are found on opposite ends of the S2-S3 loop and thus the stability of the loop is quite necessary for coordinating the active site residues. Our structural studies suggest that the wildtype S2-S3 loop acts as a rigid beam between the active site and an opposing ?-subunit. The F145A lies directly at the point of contact between these two regions and preliminary molecular dynamics simulations show this mutation to increase the mobility of the S2-S3 loop. Thus, we propose that the mutant deactivates the proteasome by destabilizing the proteasome at the half and as a result the S2-S3 loop becomes more mobile. This increased mobility propagates through the rigid S2-S3 loop on to the active site residues rendering the F145A mutant proteasome inactive. In order to test this hypothesis, we are proposing to conduct full all atom simulations on both the wildtype and the F145A mutant proteasomes. By monitoring the RMSD of the S2-S3 loop as a function of time, we will be able to ascertain how the mobility of the S2-S3 loop changes upon mutation.