HtrI Is a Dimer Whose Interface Is Sensitive to Receptor Photoactivation and His-166 Replacements in Sensory Rhodopsin I

Abstract

Single cysteine substitutions were introduced into three positions of otherwise cysteineless HtrI, a phototaxis transducer found in Halobacterium salinarum that transmits signals from the photoreceptor sensory rhodopsin I (SRI) to a cytoplasmic pathway controlling the cell's motility. Oxidative cross-linking of the monocysteine HtrI mutants in membrane suspensions resulted in dimer forms evident in SDS-polyacrylamide gels. The rate of cross-linking of I64C on the cytoplasmic side of HtrI was accelerated by SRI binding in the dark and further increased by SRI photoactivation. Several residue replacements of His-166 in SRI accelerated the cross-linking rate of I64C in the dark and His-166 mutants that exhibit "inverted signaling" (mediating repellent instead of the normally attractant response to orange light) inverted the light effect on the cross-linking rate of I64C. Secondary structure prediction of HtrI indicates a coiled coil structure in the cytoplasmic region following TM2, a dimerization domain found in a diverse group of proteins. We conclude that 1) HtrI exists as a dimer both in the absence of SRI and in the SRI-HtrI complex, 2) binding of SRI in the dark increases reactivity of the two cysteines at position 64 in the dimer by increasing their proximity or mobility, 3) light activation of wild-type SRI further increases their reactivity, 4) His-166 replacements in the SRI receptor have conformational effects on the structure of HtrI at position 64, and 5) inverted signaling by His-166 mutants likely results from an inverted conformational change at this region induced by SRI photoactivation.HtrI is a transducer protein found in the archaeon Halobacterium salinarum (1, 2), which is homologous to eubacterial methyl-accepting chemotaxis proteins (MCPs), 1 such as the aspartate receptor (Tar) in Escherichia coli (3-5). It contains two transmembrane segments, connected by a 220-residue portion to methylation regions and a His-kinase binding domain that share high sequence identity with those of the MCP family. Together with its membrane partner, sensory rhodopsin I (SRI), HtrI mediates phototaxis. HtrI and SRI physically interact and form a tight complex in the membrane (6 -8). Signaling by the complex starts from the photoactivation of SRI, which is structurally similar to the visual pigment rhodopsin.The E. coli Tar also contains two transmembrane segments and exists as a dimer both in its ligand-free state and activated, ligand-occupied state (9). Extensive site-directed disulfide cross-linking studies of Tar and related MCPs have revealed a four-helix bundle of the transmembrane helices in the membrane and details of helix packing (10 -12). Activation of these types of receptors occurs when a new conformation is assumed either within a single subunit (13)(14)(15) or between subunits within a dimer (11, 12, 16 -18).In this study, we applied site-directed disulfide cross-linking to monocysteine HtrI mutants to examine whether HtrI exists as a dimer, to detect a conformational change in HtrI during SRI act...

Five Residues in the HtrI Transducer Membrane-proximal Domain Close the Cytoplasmic Proton-conducting Channel of Sensory Rhodopsin I

“…Residue 64 is shown here to be critical for phototaxis signaling, perhaps because of its involvement in HtrI homodimer interaction in this region (34).…”

The Cytoplasmic Membrane-proximal Domain of the HtrII Transducer Interacts with the E-F Loop of Photoactivated Natronomonas pharaonis Sensory Rhodopsin II

“…6) predicts to be a turn and a flexible region. Also, a second turn and flexible region is predicted at positions 101-104, which would serve to bring the two post-turn helices of the HtrII monomers together in the coiled coil dimer motif expected from sequence algorithms for HtrII (11) and observed in the homologous Tsr transducer (24). In the model (Fig.…”

“…A suppressor mutation analysis of randomly mutagenized SRI-HtrI complex revealed a cluster of functionally important residues ranging from positions 53 to 96 in HtrI (Asn-53 corresponds in HtrII to Leu-82 at the membrane/cytoplasm interface), and also 3 residues in the cytoplasmic regions of helices F and G of SRI (10). Also a decreased phototaxis response occurs in HtrI-I64C (11). Moreover, cytoplasmic extension of HtrI TM2 by 13 residues is necessary and sufficient for HtrI-dependent properties of SRI in the SRI-HtrI molecular complex, including the HtrI inhibition of opening of its protonconducting cytoplasmic channel during the photocycle (12, 13).…”

Electrophysiological characterization of specific interactions between bacterial sensory rhodopsins and their transducers

“…The recent model of the signal relay involves a light-induced tilt of the cytoplasmic region of the receptor helix F, which is detected by the second transmembrane helix (TM2) of the transducer (3,23). Indeed, from mutant analysis in the SRI͞HtrI complex, it is assumed that a physical contact of HtrI TM2 with SRI blocks the cytoplasmic part of the SRI proton pathway (24)(25)(26). Consistent with these models, our data point to interaction sites located in the cytoplasmic part of the receptors as well.…”