.............................................................. Molecular basis of transmembrane signalling by sensory rhodopsin II– transducer complex Valentin I. Gordeliy*†, Jo ¨ rg Labahn*, Rouslan Moukhametzianov*†, Rouslan Efremov*†, Joachim Granzin*, Ramona Schlesinger*, Georg Bu ¨ ldt*, Tudor Savopol§k, Axel J. Scheidig§, Johann P. Klare§ & Martin Engelhard§ * Research Centre Ju ¨lich, Institute of Structural Biology (IBI-2), 52425 Ju ¨lich, Germany † Centre for Biophysics and Physical Chemistry of Supramolecular Structures, MIPT, 141700, Moscow District, Russia § Max-Planck-Institut fu ¨r Molekulare Physiologie, Otto Hahn Str. 11, 44227 Dortmund, Germany k Present address: Carol Davila Medical and Pharmaceutical University, POB15- 205 Bucharest, Romania. ............................................................................................................................................................................. 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 1–3 . 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 4 . The link between the photoreceptor and the follow- ing cytoplasmic signal cascade is formed by a transducer mol- ecule that binds tightly and specifically 5 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 transdu- cer (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 A ˚ resol- ution, 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. Crystallization of the receptor–transducer complex, a member of the two-component signalling cascade (Fig. 1), has been achieved successfully using a shortened transducer (residues 1–114; N. pharaonis HtrII 114 ) comprising the two transmembrane helices (TM1 and TM2) and an additional small cytoplasmic fragment. This construct satisfies the properties of an appropriate model system for the native receptor–transducer complex as indicated by a low dissociation constant (K d < 100 nM, S. Hippler-Mreyen, unpublished data) and by its capability to inhibit the inherent proton pump activity of SRII, as was shown for a larger transducer fragment (G. Schmies, unpublished data) and the full- length transducer 8,9 . These data and those establishing a functional signal transfer from receptor to transducer 7 indicate that HtrII 114 forms a functionally unimpaired complex with its cognate receptor SRII. The thin orange crystals of SRII in complex with HtrII 114 grown in lipidic cubic phase 10 displayed an orthorhombic shape of about 140 mm in size and diffracted to 1.8 A ˚ . The asymmetric unit contains one complex. The expected dimer of the complex is formed by a crystallographic two-fold rotation axis, which is located in the middle of four transmembrane helices: TM1, TM2, TM1 0 , TM2 0 (where a prime indicates the right-hand complex; Fig. 2a). The transmembrane helices F and G of the receptor are in contact with the helices of the transducer. The overall X-ray structure is in good agreement with a recently published model of the receptor–trans- ducer complex deduced from electron paramagnetic resonance (EPR) measurements 7 . The structure of SRII complexed with its transducer is markedly similar to that of the receptor alone including the retinal confor- mation 11,12 . Obviously, the binding of the transducer to helices F and G hardly interferes with the side-chain arrangement of the receptor. A notable exception is found for Tyr 199. The aromatic plane of Tyr 199 has turned in the complex by about 908 and is now pointing into the direction of TM2 where its phenolic group forms a hydrogen bond to Nd(2)-Asn 74 (2.8 A ˚ ). An interaction of Tyr 199 with the transducer has been proposed previously 12 . It should be mentioned that a chloride ion, identified by ref. 11, close to the guanidinium group of Arg 72 is clearly absent in the present structure. The crystallization conditions used by this study (low pH and high NaCl concentration) favour the uptake of a chloride ion and therefore can explain the differences. The natural habitat of N. pharaonis (pH . 9) does not support the binding of a chloride ion to SRII. The interface between receptor and transducer is formed mainly by van der Waals (vdW) contacts and only a few hydrogen bonds. Whereas the straight TM2 is oriented parallel to helix G of the receptor, TM1 is kinked at Gly 37 and bends away from the receptor. Thus, a crevice (formed by helices A, G, TM2 and TM1) opens to the cytoplasmic surface (Fig. 2a), which might harbour the back-folded amino terminus of TM1, as residual electron densities suggest. Although only van der Waals contacts are observed between the four transducer helices themselves, defined cross-connections are observed between receptor and transducer. The F–G loop region affixes the transducer by several contacts as well as by three hydrogen bonds between Thr 189 (SRII), Glu 43 (TM1) and Ser 62 (TM2) (Fig. 3). A second anchor point is observed in the middle of the membrane where, as mentioned above, the phenolic hydroxyl of Tyr 199 (helix G) bridges to Asn 74 (TM2). A view from the cytoplasm down the binding domain (Fig. 4a) reveals that closer contacts are between helix G and TM2, fixating these two trans- membrane helices to one another. There are twice as many van der Waals contacts between helices G and TM2 than between F and TM2. The closer packing between G and TM2 can be quantified by an average van der Waals distance of 4.06 A ˚ in comparison to a value of 4.22 A ˚ between F and TM2 (Fig. 4b). The four helices of the Figure 1 Two-component signalling cascade. The activation of the transducer HtrII by its receptor SRII leads to a conformational change of TM2 that propagates to the tip of the coiled-coil cytoplasmic domain (structure taken from ref. 31). The next steps in the signalling cascade involve—in analogy to the bacterial sensory system 20 —the homodimeric histidine kinase CheA, the coupling protein CheW, and the response regulators/aspartate kinases CheY and CheB. Phosphorylated (P) CheY functions as a switch factor of the flagellar motor. CheB (a methylesterase) together with CheR (a methyltransferase) are involved in the adaptation processes of the bacteria. The box highlights the receptor–transducer complex reported in the present study. SR, sensory rhodopsin. letters to nature NATURE | VOL 419 | 3 OCTOBER 2002 | www.nature.com/nature 484 © 2002 Nature Publishing Group