Time-resolved X-ray diffraction and solution scattering studies of Sensory Rhodopsin II in isolation and in complex with its transducer

Abstract

Light is an important source of energy for many living organisms. Many life forms have therefore evolved cellular receptors that are able to sense light and thereby optimise conditions for photosynthesis and pho- totrophy. Microbial Rhodopsins are a family of heptahelical transmem- brane proteins characterised by the presence of a retinal chromophore bound to a conserved lysine of helix seven. When the retinal absorbs a photon, it photoisomerises from an all-trans to a 13-cis conformation. Sensory Rhodopsin II (SRII) is a microbial rhodopsin identified in the halophilic archaeon Nantronomonas pharaonis. Together with its trans- ducer protein HtrII, SRII it initiates a photophobic reaction of the host in response to blue light. Conformational changes within this complex are sensed by the HAMP domain of HtrII and trigger a signalling cas- cade controlled by the so-called two-component system (TCS). The TCS is ubiquitous in prokaryotes and is present in some eukaryotes. This im- plies significant pharmaceutical interest due to the involvement of TCS in bacterial virulence, antibiotic resistance, and phototaxis. Many details concerning the mechanisms of signal transduction through the SRII:HtrII complex remain unclear. In this work I aimed to address these questions by observing the nature and extent of secondary structural rearrangements in SRII in isolation and in complex with HtrII using time-resolved serial synchrotron X-ray crystallography (TR-SSX) and time-resolved X-ray so- lution scattering (TR-XSS). In PAPER I, we collected room-temperature TR-XSS data on SRII in isolation and compared the observed structural changes with those observed in bacteriorhodopsin (bR), a heavily studied light-driven proton pump. Our observations provide structural insight into why these very similar proteins have very different photocycle duration. In both proteins, helix F undergoes an outward movement, yet structural rearrangement within helix G are suppressed in SRII, resulting in a slower photocycle and reflecting its function as a signalling receptor. In PAPER II we observed the structure of the SRII:HtrII complex at room-temperature using serial synchrotron x-ray crystallography (SSX). Our data provides the first room-temperature structure of the SRII:HtrII complex and al- lows five additional residues to be modelled on the cytoplasmic side of transmembrane helix 1 (TM1) of HtrII. In Paper III we used TR-SSX to investigate light-initiated conformational changes of within the SRII:HtrII complex. Our observations show how a structural signal originating at the retinal is transferred from SRII to HtrII. A preliminary structural analysis suggests that an outward movement of helix F of SRII is translated into a piston-like movement of transmembrane helix 2 (TM2) towards the cy- toplasm, a model that is largely consistent with the conclusions of earlier cryo-trapping studies. In Paper IV we used TR-XSS to analyse conforma- tional changes in SRII and the SRII:HtrII complex. As a solution phase method, TR-XSS is complementary to crystallography and protein mo- tions are not constrained by a crystal lattice, but the information content is lower. Our TR-XSS data were consistent with a light-induced outward movement of the cytoplasmic portions of helices E and F, and more subtle movements in helices C, D and E. Structural rearrangements in helices E and F are less extensive when the transducer binds to SRII. These results increase our understanding of how a light signal is sensed by the photo- taxis receptor SRII, and how this signal is transmitted to its transducer protein, HtrII.