Valentin I. Gordeliy

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 Å resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.

Development of the signal in sensory rhodopsin and its transfer to the cognate transducer

The microbial phototaxis receptor sensory rhodopsin II (NpSRII, also named phoborhodopsin) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behaviour of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain homologous with the two-component system of eubacterial chemotaxis. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes as shown by electron paramagnetic resonance and X-ray structure analysis. Here we present X-ray structures of the photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. The formation of late M has been correlated with the formation of the signalling state. The observed structural rearrangements allow us to propose the following mechanism for the light-induced activation of the signalling complex. On excitation by light, retinal isomerization leads in the K state to a rearrangement of a water cluster that partly disconnects two helices of the receptor. In the transition to late M the changes in the hydrogen bond network proceed further. Thus, in late M state an altered tertiary structure establishes the signalling state of the receptor. The transducer responds to the activation of the receptor by a clockwise rotation of about 15° of helix TM2 and a displacement of this helix by 0.9 Å at the cytoplasmic surface.

The archaeal sensory rhodopsin II/transducer complex: a model for transmembrane signal transfer

Archaebacterial photoreceptors mediate phototaxis by regulating cell motility through two‐component signalling cascades. Homologs of this sensory pathway occur in all three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsin II/transducer complex mediating the photophobic response of Natronomonas pharaonis have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data and the atomic resolution structure of the receptor molecule in complex with the transmembrane segment of its cognate transducer provided a model for signal transfer from the receptor to the cytoplasmic side of the transducer. This mechanism might also be relevant for eubacterial chemoreceptor signalling.

Scientific Reviews: Two-Detector System for Small-Angle Neutron Scattering Instrument

Most of the objects of small-angle neutron scattering (SANS) experiments require the measurements of a studied sample in a wide range of momentum transfer (Q-range). Larger Q-range means more reliable determination of a model of the investigated material as well as higher accuracy of its calculated structural parameters. The dynamic range of SANS instruments is normally determined by the size of the detector, which is limited mainly by technical reasons and by the wavelength range of available thermal neutrons in the neutron beam. Even in the case of the largest detector (1 m2) at one of the best beam lines—the D22 instrument at ILL—the dynamic range is about 50. Usually the problem of the Qrange is solved by a sequence of measurements with the detector at different positions. However, it leads to considerable increase in the data acquisition time. Moreover, the problem becomes critical when it is necessary to study processes in real time, especially irreversible processes.

Crystal structure of a light-driven sodium pump

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Structural changes in dipalmitoylphosphatidylcholine bilayer promoted by Ca2+ ions: a small-angle neutron scattering study.

Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60mM CaCl2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 degrees C, the lipid bilayer thickness, dL, goes through a maximum as a function of CaCl2 concentration (dL=54.4A at approximately 2.5mM of CaCl2). Simultaneously, both the area per DPPC molecule AL, and the number of water molecules nW located in the polar head group region decrease (DeltaAL=AL(DPPC))-AL(DPPC+Ca)=2.3A2 and Deltan=n(W(DPPC))-n(W(DPPC+Ca))=0.8mol/mol at approximately 2.5mM of CaCl2). In the fluid phase, at 60 degrees C, the structural parameters d(L), A(L), and n(W) show evident changes with increasing Ca2+ up to a concentration C(Ca)(2+) < or = 10mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60mM CaCl2 (at 20 and 60 degrees C) are nearly the same as those for unilamellar vesicles without Ca2+.

Structural insights into the proton pumping by unusual proteorhodopsin from nonmarine bacteria

Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESRs proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 310- and π-helix–like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.

A neutron diffraction study of the influence of ions on phospholipid membrane interactions

Neutron diffraction is used to examine the effects of Ca2+ and ClO4- ions on interactions and some structural features of dipalmitoylphosphatidylcholine membranes in both solid and fluid lamellar phases. The results are described within the framework of Derjaguin-Landau-Verwey-Overbeek (DLVO) theory with reference to electrostatic, van der Waals, and hydration components of disjoining pressure. The Hamaker constants are evaluated under equilibrium conditions. Addition of 100 mM CaCl2 to the aqueous phase substantially increases the lamellar repeat spacing (d), which is interpreted in terms of adsorption of Ca2+ ions to bilayers followed by electrostatic repulsion between membranes. The rise of NaClO4 concentration in the presence of 100 mM CaCl2 leads to gradual decrease in d, evidently resulted from the diminution of Ca(2+)-induced positive surface potential by both electrostatic screening and binding of ClO4- ions. In the absence of CaCl2, elevation of NaClO4 concentration to 100-300 mM drastically enhances the repeat spacing and then dramatically decreases d at about 1 M NaClO4. Estimation of the hydration coefficients showed that the pronounced decrease of the repeat spacing at high NaClO4 concentrations was resulted mainly from the (partial) disruption of the structure of intermembrane bound water by chaotropic ClO4- ions and subsequent decrease in hydration repulsive pressure. Moreover, in the case of solid membranes (20 degrees C) high concentrations of ClO4- induced formation of interdigitated phase paralleled with marked reduction in bilayer thickness and corresponding increase in the effective cross-sectional area per lipid molecule.

Meshandcollect: An Automated Multi-Crystal Data-Collection Workflow for Synchrotron Macromolecular Crystallography Beamlines.

The fully automated collection and merging of partial data sets from a series of cryocooled crystals of biological macromolecules contained on the same support is presented, as are the results of test experiments carried out on various systems.

Active state of sensory rhodopsin II: structural determinants for signal transfer and proton pumping.

The molecular mechanism of transmembrane signal transduction is still a pertinent question in cellular biology. Generally, a receptor can transfer an external signal via its cytoplasmic surface, as found for G-protein-coupled receptors such as rhodopsin, or via the membrane domain, such as that in sensory rhodopsin II (SRII) in complex with its transducer, HtrII. In the absence of HtrII, SRII functions as a proton pump. Here, we report on the crystal structure of the active state of uncomplexed SRII from Natronomonas pharaonis, NpSRII. The problem with a dramatic loss of diffraction quality upon loading of the active state was overcome by growing better crystals and by reducing the occupancy of the state. The conformational changes in the region comprising helices F and G are similar to those observed for the NpSRII-transducer complex but are much more pronounced. The meaning of these differences for the understanding of proton pumping and signal transduction by NpSRII is discussed.