Strategies for identifying dynamic regions in protein complexes: Flexibility changes accompany methylation in chemotaxis receptor signaling states

Abstract

Highlights• Receptors exhibit greater ns timescale dynamics in unmethylated vs methylated state • Methylation helix 2 likely involved in increased flexibility of unmethylated state • Dynamics occur on multiple timescales in both states of the receptor

Concerted Differential Changes of Helical Dynamics and Packing upon Ligand Occupancy in a Bacterial Chemoreceptor

“…The pair of C-helices was significantly closer to each other than the N-helix pair, and the N-helices were significantly more dynamic than the C-helices (Figure ). These observations complement and expand on previous observations of differences in dynamics between the two companion helices in studies of intact, functional chemoreceptor homodimers in native lipid bilayers using continuous wave electron paramagnetic resonance (EPR) and of receptor cytoplasmic domain fragments incorporated into functional signaling complexes using hydrogen exchange and mass spectroscopy or solid-state NMR. ,,− Taken together, these data provide compelling evidence that along a significant portion of the chemoreceptor helical coiled coil, the N-helices are more dynamic than the C-helices over time regimes from nanoseconds to seconds. Differential separation and thus differential packing for the two helical pairs add a previously uncharacterized aspect to the asymmetry within the four-helix coiled coil.…”

“…Coiled-coil proteins exhibit a seven-residue heptad repeat in which positions are denoted from a through g. Residues 319 and 456 are at heptad positions f and c, respectively, which are side chains not involved in helical packing but instead surrounded by the solvent. The positions are next to each other in the three-dimensional structure of the coiled coil, approximately three helical turns membrane-distal from the methyl-accepting sites on the companion helices of the two protomers of the homodimer (Figure A), and are near a region that other techniques have implicated in helical flexibility. − ,, We used thiol-maleimide chemistry to link donor (Atto 594) and acceptor (Atto 647N) fluorophores to purified Tar homodimers with a single cysteine at residue 319 or 456 in each protomer, resulting in a sample with fluorophores on either the two N-helices or the two C-helices, respectively.…”

“…In the receptor periplasmic and transmembrane regions, ligand-induced conformational signaling involves mechanical movements of helices. − For the chemoreceptors of Escherichia coli ( E. coli ) and Salmonella enterica, the movement is piston-like axial sliding of an extended helix a few Ångströms toward the cell interior. ,,,− That helix begins at the membrane-distal ligand-binding site of the periplasmic domain and extends through the periplasmic and the transmembrane domains to the HAMP (Histidine kinases, Adenyl cyclases, Methyl-accepting chemotaxis proteins, and Phosphatases) domain (Figure B) . Movement of the piston helix induces a different conformational change in the HAMP domain that has been suggested to be an alteration in the helical packing or dynamics. , Changes induced in the HAMP domain generate subsequent conformational changes in the cytoplasmic domain four-helix coiled coil. ,,, The nature of conformational changes generated in the coiled coil of the cytoplasmic domain by ligand occupancy has not been identified, but characterizations of signaling states generated by modifications or mutations in the cytoplasmic domain suggest involvement of helical dynamics. − …”

“…However, no significant changes were detected as a function of ligand occupancy . Other studies of intact receptors have identified changes in helical packing as a function of signaling states generated by mutational or biochemical perturbations of the cytoplasmic domain. − Several studies probing the helical dynamics of chemoreceptor cytoplasmic fragments lacking periplasmic and transmembrane domains have compared the kinase-on and kinase-off states generated by varying the adaptational modification or HAMP signaling state. − These studies found that protein dynamics varied as a function of signaling state, including increased dynamics within the region that contains the sites of adaptational modification for the kinase-off state − (Figure B). Thus, differences in the cytoplasmic domain helical dynamics and packing have been correlated with different signaling states generated by local manipulation but not with the different signaling states generated by occupancy of the ligand-binding site >200 Å away in the periplasmic domain …”

Foreword to: Biophysical studies of membrane systems and interactions - Commemorative issue in honour of Professor Michèle Auger

“…De Vlugt et al employ through-bond SS-NMR spectroscopy to identify the flexible portions of the non-annular lipids that co-purify with the Anabaena Sensory ) and find the presence of phosphatidyl ethanolamine lipids that are tightly bound to ASR[32].Thompson and Baenziger review the effects of lipids on the function of pentameric ligand-gated ion channels[33]. Malik et al investigate the differences in dynamics between the methylated and unmethylated states of a bacterial chemoreceptor using SS-NMR[34]. In their paper, Mark et al use a sophisticated EPR experiment, called 2D HYSCORE, to determine the electronic structure of two redoxactive tyrosine residues of photosystem II, which play an important role in water oxidation driven by light energy[35].…”