NUCLEOTIDE DEPENDENT CONFORMATIONAL CHANGES IN S54-DEPENDENT AAA+ATPASES

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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. Why do defects in molecular machines (AAA+ ATPases) cause disease? These protein machines convert ATP hydrolysis into mechanical work. It is clear that both human cells and disease causing pathogens use this work to physically manipulate proteins or DNA to dismantle and reassemble membranes or other organelles, to replicate DNA and traverse cell division, to repair damaged proteins, or to regulate gene expression. But, we do not know how these molecular machines convert ATP hydrolysis into mechanical work. A major impediment to defining these mechanisms has been our inability to probe detailed conformational changes that are related to controlling these ATPases as they step through ATP binding, hydrolysis and product release. Our project focused on defining structural changes that occur to regulate assembly of s54-dependent AAA+ ATPases, and that accompany the ATP hydrolysis cycle. These activators remodel the s54-form of bacterial RNA polymerase to enable it to initiate transcription in bacteria. Previous solution structures derived from small angle scattering data (published in 2005) revealed that in one case a regulatory domain adopts two homo-dimeric forms, alternately repressing or derepressing motor assembly by adjacent ATPase domains; in another case, regulatory and ATPase domains were found to cooperate to stabilize the assembled motor. Similar data collected in 2005 revealed a) conformational changes that are coupled with nucleotide status and that mediate two distinct interactions with the target protein, s54, and b) structure of one of those complexes. These latter results are being submitted for publication.