Engineered Interfaces of an AAA+ ATPase Reveal a New Nucleotide-dependent Coordination Mechanism

Abstract

Homohexameric ring AAA؉ ATPases are found in all kingdoms of life and are involved in all cellular processes. To accommodate the large spectrum of substrates, the conserved AAA ؉ core has become specialized through the insertion of specific substrate-binding motifs. Given their critical roles in cellular function, understanding the nucleotide-driven mechanisms of action is of wide importance. For one type of member AAA ؉ protein (phage shock protein F, PspF), we identified and established the functional significance of strategically placed arginine and glutamate residues that form interacting pairs in response to nucleotide binding. We show that these interactions are critical for "cis" and "trans" subunit communication, which support coordination between subunits for nucleotide-dependent substrate remodeling. Using an allele-specific suppression approach for ATPase and substrate remodeling, we demonstrate that the targeted residues directly interact and are unlikely to make any other pairwise critical interactions. We then propose a mechanistic rationale by which the nucleotidebound state of adjacent subunits can be sensed without direct involvement of R-finger residues. As the structural AAA ؉ core is conserved, we propose that the functional networks established here could serve as a template to identify similar residue pairs in other AAA ؉ proteins.

Unique ATPase Site Architecture Triggers cis-Mediated Synchronized ATP Binding in Heptameric AAA+-ATPase Domain of Flagellar Regulatory Protein FlrC

“…Considering the very high degree of sequence conservation at these two regions of 54 and the conserved nature of the GAFTGA loop of bEBPs, a similar binding stoichiometry may be expected between FlrC heptamer and RNAP-54 . Extensive studies on PspF or NtrC1, however, showed that participation of L1 loops in binding 54 at the promoter is actually guided by Nt binding, hydrolysis-induced subunit remodeling, and a spatio-chemical environment offered by the asymmetric arrangement of the L1 loops (8,44,45). Although our results on FlrC C exclude the possibility of any such Nt-dependent subunit remodeling, even in the presence of ADP.AlF x , the probability of local structural changes are not ruled out.…”

Functional Asymmetries of Proteasome Translocase Pore

“…see Ref. 28) and may form the basis of asymmetry of function in proteasomes. A recent paper (29) demonstrated that incapacitating ATP hydrolysis by mutating rpt3 alone, in proteasomes that were otherwise unchanged, abolished global ATPase activity, a result consistent with coordination of hydrolysis.…”

Single Chain Forms of the Enhancer Binding Protein PspF Provide Insights into Geometric Requirements for Gene Activation

“…However, in contrast to the current ClpX model, which is based on a stochastic ATPase activity and the requirement of only one initial interaction with the substrate (16), our previous studies on PspF strongly suggest a different mechanism of action, based on cooperative ATPase activity with more than one subunit involved in the initial substrate interaction (13,27,29).…”

“…50-l samples were then injected onto a Superdex 200 column (10 ϫ 300 mm, 24 ml; GE Healthcare) and equilibrated with the sample buffer Ϯ nucleotide (13). Chromatography was performed at 4°C at a flow rate of 0.5 ml/min, and columns were calibrated with globular proteins: apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa).…”

“…Chemical cross-linking is commonly used to track the number of subunits present in the oligomer but does not provide a good tool to address the geometrical distribution of subunits for functionality. Two additional approaches have been developed recently: (i) mixing experiments based on the reconstitution of hetero-oligomers containing different variants (13)(14)(15) and (ii) a single-chain approach based on the engineering of a polypeptide covalently linking two or more subunits of the oligomer, allowing the differentiation of subunit organization in the final oligomer (16).…”