ASYMMETRY IN THE OLIGOMERIC NTRC1 AAA+ MOTOR: NEW IMPLICATIONS FOR THE MECHANISM
Open Access
- Author:
- Sysoeva, Tatyana A
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 07, 2011
- Committee Members:
- B Tracy Nixon, Dissertation Advisor/Co-Advisor
B Tracy Nixon, Committee Chair/Co-Chair
Kathleen Postle, Committee Member
Ming Tien, Committee Member
Hemant P Yennawar, Committee Member
Howard M Salis, Committee Member - Keywords:
- sigma54-dependent transcription
transcription activator
SAXS
NtrC1
AAA+ ATPase
protein motor - Abstract:
- In all living organisms ATP fuels chemical reactions, ligand transport, heat release, and mechanical work. ATPases Associated with various cellular Activities (AAA+ ATPases) are ubiquitous and abundant in all kingdoms of life, where they convert ATP binding and hydrolysis energy into various forms of mechanical work to reshape other macromolecules. Dysfunctions in AAA+ ATPases lead to numerous human diseases, and many of these proteins are crucial to the functioning of pathogenic bacteria. Despite the great variety, number, and importance of the AAA+ ATPases, there is not enough structural information available to understand the mechanism of the AAA+ ATPase work cycle. An AAA+ ATPase is required to start transcription by sigma54-RNA polymerase in bacteria. This sigma54-dependent transcription activator changes the conformation of the sigma54-holoenzyme by an unknown mechanism, leading to melting of the promoter DNA duplex and thus transcription initiation. In this work modern methods of structural biology were applied to study the NtrC1 ATPase – a sigma54 activator from the extreme thermophile Aquifex aeolicus. Prior electron microscopy and small-angle X-ray scattering (SAXS) studies showed that upon activation NtrC1 forms homo-heptamers that undergo large-scale conformational changes upon ATP binding. As a result the conserved stem loops that interact with the sigma54-holoenzyme are exposed from the NtrC1 surface. The present study continues to concentrate on the structural changes within the AAA+ activator upon its binding to nucleotide. This study presents two new states of the NtrC1 AAA+ ATPase ensemble at resolutions of 2.8 and 3.6 Angstroms. It was further shown that consecutive nucleotide binding events cause a series of large-scale conformational changes in the NtrC1 oligomer. Applying a novel time-resolved SAXS approach revealed how the conformational changes develop in time. These results showed that intersubunit interactions within the homo-oligomeric ring-shaped ATPase cause strong cooperativity for nucleotide binding and complex conformational shifts. These changes lead to asymmetry and alter subunit stoichiometry in the NtrC1 AAA+ ATPase ring. Based on these results, a sequential mechanism of ATP hydrolysis was proposed for the work cycle of the NtrC1 ATPase.