Structural studies of conformational changes of proteins upon phosphorylation: Structures of activated CheY, CheY-N16-FliM complex, and AAA(+) ATPase domain of NtrC1 in both inactive and active states

Protein phosphorylation is a general mechanism for signal transduction as well as regulation of cellular function. Unlike phosphorylation in eukaryotec systems that uses Ser/Thr for the sites of modification, two-component signal transduction systems, which are prevalent in bacteria, archea, and lower eukaryotes, use an aspartate as the site of phosphorylation. Two-component systems comprise a histidine kinase and a receiver domain. The conformational change of the receiver domain upon phosphorylation leads to signal transfer to the downstream target, a process that had not been understood well at the molecular level. The transient nature of the phospho-Asp bond had made structural studies difficult. The discovery of an excellent analogue for acylphosphate, BeF₃ −, enabled structural study of activated receiver domains. The structure of activated Chemotaxis protein Y (CheY) was determined both by NMR spectroscopy and X-ray crystallography. These structures revealed the molecular basis of the conformational change that is coupled to phosphorylation. Phosphorylation of the conserved Asp residue in the active site allows hydrogen bonding of the T87 Oγ to phospho-aspartate, which in turn leads to the rotation of Y106 into the “in” position (termed Y-T coupling). The structure of activated CheY complexed with the 16 N-terminal residues of FliM (N16-FliM), its target, was also determined by X-ray crystallography and confirmed the proposed mechanism of activation (Y-T coupling). First, N16-FliM binds to the region on CheY that undergoes a significant conformational change. Second, the “in” position of Y106 presents a better binding surface for FliM because the sidechain of Y106 in the inactive form of CheY (“out” position) sterically interferes with binding of N16-FliM.; Nitrogen Regulatory protein C (NtrC) is the response regulator that is involved in σ54 dependent transcription upon nitrogen depletion in enteric bacteria. It comprises a receiver, a central AAA + ATPase, and a DNA binding domains. An NMR study of the BeF ₃−-activated receiver domain of NtrC (NtrC R) showed that there was a large conformational change including secondary structure rearrangement in H4 and the loop between H4 and β4 unlike that seen in activated CheY. The high-resolution solution structure of BeF ₃−-activated NtrCR showed that Y-T coupling may also be the key to trigger the large conformational change, extending the notion that basic Y-T coupling can cause different structural changes in different receiver domains, allowing fidelity in signaling pathways. (Abstract shortened by UMI.)...