The signal transfer from the receptor NpSRII to the transducer NpHtrII is not hampered by the D75N mutation.

Sensory rhodopsin II (NpSRII) is a phototaxis receptor of Natronomonas pharaonis that performs its function in complex with its cognate transducer (NpHtrII). Upon light activation NpSRII triggers by means of NpHtrII a signal transduction chain homologous to the two component system in eubacterial chemotaxis. The D75N mutant of NpSRII, which lacks the blue-shifted M intermediate and therefore exhibits a significantly faster photocycle compared to the wild-type, mediates normal phototaxis responses demonstrating that deprotonation of the Schiff base is not a prerequisite for transducer activation. Using site-directed spin labeling and time resolved electron paramagnetic-resonance spectroscopy, we show that the mechanism revealed for activation of the wild-type complex, namely an outward tilt motion of the cytoplasmic part of the receptor helix F and a concomitant rotation of the transmembrane transducer helix TM2, is also valid for the D75N variant. Apparently, the D75N mutation shifts the ground state conformation of NpSRII-D75N and its cognate transducer into the direction of the signaling state.

Complex formation and light activation in membrane-embedded sensory rhodopsin II as seen by solid-state NMR spectroscopy.

Microbial rhodopsins execute diverse biological functions in the cellular membrane. A mechanistic understanding of their functional profile is, however, still limited. We used solid-state NMR (ssNMR) spectroscopy to study structure and dynamics of a 2 x 400 amino acid sensory rhodopsin/transducer (SRII/HtrII) complex from Natronomonas pharaonis in a natural membrane environment. We found a receptor-transducer binding interface in the ground state that significantly extends beyond the available X-ray structure. This binding domain involves the EF loop of the receptor and stabilizes the functionally relevant, directly adjacent HAMP domain of the transducer. Using 2D ssNMR difference spectroscopy, we identified protein residues that may act as a functional module around the retinal binding site during the early events of protein activation. These latter protein segments, the inherent plasticity of the HAMP domain, and the observation of an extended SRII/HtrII membrane-embedded interface may be crucial components for optimal signal relay efficiency across the cell membrane.

Single-molecule force spectroscopy measures structural changes induced by light activation and transducer binding in sensory rhodopsin II.

Microbial rhodopsins are a family of seven-helical transmembrane proteins containing retinal as chromophore. Sensory rhodopsin II (SRII) triggers two very different responses upon light excitation, depending on the presence or the absence of its cognate transducer HtrII: Whereas light activation of the NpSRII/NpHtrII complex activates a signalling cascade that initiates the photophobic response, NpSRII alone acts as a proton pump. Using single-molecule force spectroscopy, we analysed the stability of NpSRII and its complex with the transducer in the dark and under illumination. By improving force spectroscopic data analysis, we were able to reveal the localisation of occurring forces within the protein chain with a resolution of about six amino acids. Distinct regions in helices G and F were affected differently, depending on the experimental conditions. The results are generally in line with previous data on the molecular stability of NpSRII. Interestingly, new interaction sites were identified upon light activation, whose functional importance is discussed in detail.

Salt-driven equilibrium between two conformations in the HAMP domain from Natronomonas pharaonis: the language of signal transfer?

HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII157, a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a "two-state" equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus.

Interaction of a G protein-coupled receptor with a G protein-derived peptide induces structural changes in both peptide and receptor: a Fourier-transform infrared study using isotopically labeled peptides.

G protein-coupled receptor signaling involves productive interaction between agonist-activated receptor and G protein. We have used Fourier-transform infrared difference spectroscopy to examine the interaction between the active Meta II state of the visual pigment rhodopsin with a peptide analogue corresponding to the C terminus of the alpha-subunit of the G protein transducin. Formation of the receptor-peptide complex evokes a spectral signature consisting of conformationally sensitive amide I and amide II difference bands. In order to distinguish between amide backbone contributions of the peptide and of the receptor moiety to the vibrational spectra, we employed complete (13)C,(15)N-labeling of the peptide. This isotopic labeling downshifts selectively the bands of the peptide, which can thus be extracted. Our results show that formation of the complex between the activated Meta II receptor state and the peptide is accompanied by structural changes of the peptide, and of the receptor, indicating that the conformation of the Meta II.peptide complex is different from that of Meta II. This result implies that the activated receptor state has conformational flexibility. Binding of the peptide to the activated receptor state stabilizes a substate that deviates from that stabilized only by the agonist.

Analysis of light-induced conformational changes of Natronomonas pharaonis sensory rhodopsin II by time resolved electron paramagnetic resonance spectroscopy.

The nature and kinetics of the conformational changes leading to the activated state of NpSRII/NpHtrII157 were investigated by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy in combination with site-directed spin labeling (SDSL) on a series of spin labeled mutants of NpSRII. A structural rearrangement of the cytoplasmic moiety of NpSRII upon light activation was detected (helices B, C, F and G). The increase in distance between helices C and F in the M-trapped state of the complex observed in one double mutant is in line with the notion that an outward movement of helix F occurs upon receptor activation. The data obtained from the NpSRII/NpHtrII157 complex reconstituted in purple membrane lipids are compared with those obtained from the X-ray structure of the late M-state of the complex which shows some discrepancies. The results are discussed in the context also of other biophysical and EPR experimental evidences.

Structural analysis of a HAMP domain: the linker region of the phototransducer in complex with sensory rhodopsin II.

Sensory rhodopsin II, the photophobic receptor from Natronomonas pharaonis (NpSRII)5, forms a 2:2 complex with its cognate transducer (N. pharaonis halobacterial transducer of rhodopsins II (NpHtrII)) in lipid membranes. Light activation of NpSRII leads to a displacement of helix F, which in turn triggers a rotation/screw-like motion of TM2 in NpHtrII. This conformational change is thought to be transmitted through the membrane adjacent conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (HAMP domain) to the cytoplasmic signaling domain of the transducer. The architecture and function of the HAMP domain are still unknown. In order to obtain information on the structure and dynamics of this region, EPR experiments on a truncated transducer (NpHtrII(157)) and NpSRII, site-directed spin-labeled and reconstituted into purple membrane lipids, have been carried out. A nitroxide scanning involving residues in the transducer helix TM2, in the predicted AS-1 region, and at selected positions in the following connector and AS-2 regions of the HAMP domain has been performed. Accessibility and dynamics data allowed us to identify a helical region up to residue Ala(94) in the AS-1 amphipathic sequence, followed by a highly dynamic domain protruding into the water phase. Additionally, transducer-transducer and transducer-receptor proximity relations revealed the overall architecture of the AS-1 sequences in the 2:2 complex, which are suggested to form a molten globular type of a coiled-coil bundle.