Study of AAA proteins by X-ray protein crystallography

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Intracellular protein degradation is a major post-translational regulatory mechanism and plays a crucial role in many vital cellular functions; it also serves to remove damaged, denatured, and other abnormal proteins. The Clp proteases, which we have chosen to study crystallographically are essential in many organisms and highly conserved. ClpA belongs to the family of AAA+ proteins, a broad class of protein conformation-transducing ATPases involved in a plethora of vital cellular functions. Clp and other ATP-dependent proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the submolecular level and would be beneficial to understanding functions of other ATP utilizing enzymes, such as ABC transporters. My group has determined the crystal structure of the full-length ClpA, the regulatory component of the ClpAP complex. As the first AAA+ structure with two AAA+ modules, the ClpA structure has provided insights into the structural basis for functional difference of the two AAA+ modules. my group has also obtained the structure of the isolated N-domain of ClpA in complex with ClpS that is known to alter the substrate selectivity in ClpA, much like the adaptor proteins used by many other AAA+ proteins. We also defined the function of the ClpA N-terminal domain by analyzing crystallographically details of its multiple protein and peptide binding sites. Having obtained structures of all components in the ClpAPS system, our group is now focusing on obtaining structures of ClpA in various conformations induced by bound nucleotide, and on crystallizing binary complexes of ClpA with either ClpS or ClpP, as well as with various substrates.In order to understand the common mechanism by which AAA proteins couple the energy released from ATP hydrolysis to mechanical work performed by these enzymes, we are also looking at the structures in different states of other AAA proteins such as P97, an eukaryotic AAA protein involved in a number of important cellular functions such as protein degradation and membrane fusion. We will compare structures of different AAA proteins so that such a goal can be achieved.