1.4.1 The receptor-transducer interaction
The interaction of receptors with their cognate transducer as outlined above has been assessed by phototaxis, photocycle, and proton pump measurements. A modulating effect on the photocyle kinetics of sensory rhodopsin was frequently considered as characteristic of this interaction extensively exemplified by SRI and Htrl (for a review see ). Also for the green light receptor of H. salinarum HsSRII Sasaki & Spudich  observed significant acceleration of the "M" to "O" to ground state transition upon binding of the transducer. The authors suggest that the nearby transducer produces changes in the hydrogen network around Asp73, thereby accelerating proton transfer reactions. In contrast the photochemistry of NpSRII is not altered in the presence of the transducer .
Most of the cytoplasmic domain of Htrl can be deleted without altering the photochemical properties of the SRI/Htrl complex [89,91]. These experiments indicated that the specificity of the SRI/Htrl interaction is confined to the two transmembrane helices and a hydrophilic stretch of 90 residues subsequent to the cytoplasmic end of TM-2. As already pointed out, the specificity to recognise the cognate receptor is restricted to the two transmembrane helices of the transducers . Despite the high sequence homologies among the archaebacterial phototransducers [46,82] on the one hand and the eubacterial chemotaxis receptors  on the other hand, it is not yet possible to further narrow the interaction domain (see note on pg 39). For NpSRII the binding to NpHtrll is, with a Kv < 160 nM, quite strong. This data was obtained by titrating a solubilised truncated transducer (1-157) to the receptor and analysing the complexation by blue native gel chromatography and isothermal titration calorimetry . The stoichiometry was determined to be 1:1. The calorimetric experiments allowed the calculation of ACp according to Kirchhoffs law. The large negative value of -1.7 kJ mol-1 K"1 might be the result of the removal of protein surface area from exposure to the solvent as one would expect during a complex assembly [194-197], The binding of a truncated transducer to NpSRII induces the transition from random coil to a-helix as revealed by CD spectroscopy  which is comparable to the a-helical content of the structurally related aspartate receptor ,
Obtaining structural information at a molecular level is crucial for an understanding of the transmembrane signal transfer. To extract this data and to acquire knowledge on the dynamics of the process EPR spectroscopy has been applied. Previous studies on rhodopsin and BR have demonstrated the general applicability of the method to clarify e.g., domain fold and light-activated kinetics of mobility and/or distance changes between two spin labels (reviewed in ). The method relies on site-directed spin labeling (SDSL) of the protein under investigation. The spin-label is introduced via single cysteine mutants at positions of interest. The shape of the EPR spectrum reflects the re-orientational motion of the nitroxide side chain, which depends on the interaction with neighbouring protein structures and - if a second spin label is present - provides distances between the two paramagnetic centres [199-201]. Further information can be gained from measurements of samples in the presence of freely diffusing paramagnetic probes (e.g. oxygen or Cr3+). These experiments can differentiate between water, lipid bilayer, or protein interior accessibility of a particular protein side chain . Additionally, conformational changes can be monitored with a time resolution of about 1 ms ,
This methodology has been applied extensively to BR [204,205] and more recently to NpSRII [84,108]. Sequential spin labeling of helical turns on helices F and G of NpSRII allowed to deduce the topology of the cytoplasmic extensions of these helices . Comparing these data with those obtained for BR [202,206], it becomes evident that helices F and G are not only similarly oriented to each other and to the other parts of the protein but also have the same boundary separating cytoplasmic residues as those immersed in the membrane. Co-expression of NpSRII with a truncated fragment of NpHtrll affects the accessibility and the mobility of outward oriented spin-labeled residues on helices F and G as a result of direct physical contact with the transducer molecule. Therefore these helices are located within the binding surface of the photoreceptor with its transducer .
The crystal structure of Luecke et al.  confirmed most of the assignments made by EPR , demonstrating the potential for studying membrane proteins with this particular technique. Concerning the interaction between NpSRII and its transducer, NpHtrll, the crystal structure revealed a tyrosine residue (Tyrl99) which sticks out from the lipid-facing surface of helix G. The authors note that this Tyr is an 'excellent candidate for transducer binding in the SRII-Htrll complex in N. pharaonis membranes'.
To build up a detailed model of the membrane embedded transduction complex the SDSL approach was extended to the cytoplasmic parts of both transmembrane helices (TM1 and TM2) of the transducer . The results reveal a quaternary complex between two copies of truncated-Htr and NpSRII each with an apparent two-fold symmetry (Figure 8). The core is composed of two transmembrane helices of the transducer. This structure is in agreement with cross-linking experiments on the Htrl/SRI complex which demonstrate the dimeric nature of Htrl  and has now been confirmed by the crystal structure of the complex (see note on pg 39). Moreover, the formation of a pseudo four helix bundle in the transmembrane region proves the hypothesis that archaeal phototransducers resemble structural features of the eubacterial chemoreceptors (MCP) for which a four helical bundle was also proposed for the dimer (see e.g. ).
Figure 8. Schematic illustration of light-induced conformational changes within the transmembrane region of the 2:2-complex of NpSRII with NpHtrll viewed from the cytoplasm , According to distance changes in the signalling M-state conformation helix F moves outwardly in the direction of the neighbouring TM2, which in turn is rotated clockwise as indicated by the red arrows. Black areas represent the original positions. The inset shows a close up of the dimer interface, suggesting the relative orientations of V78R1 and L82R1 in the dark (grey) and the light-activated states (white). The numbers at the arrows depict the distances (in nm) between corresponding residues in the dark (black) and light (red) states. It should be noted that a small piston-like movement of TM2 cannot be excluded.
Was this article helpful?