Linker Region of a Halobacterial Transducer Protein Interacts Directly with Its Sensor Retinal Protein

Abstract

pHtrII, a pharaonis halobacterial transducer protein, possesses two transmembrane helices and forms a signaling complex with pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, NpSRII) within the halobacterial membrane. This complex transmits a light signal to the sensory system located in the cytoplasm. It has been suggested that the linker region connecting the transmembrane region and the methylation region of pHtrII is important for binding to ppR and subsequent photosignal transduction. In this study, we present evidence to suggest that the linker region itself interacts directly with ppR in addition to the interaction in the membrane region. An in vitro pull-down assay revealed that the linker region bound to ppR, and its dissociation constant (K(D)) was estimated to be approximately 10 microM using isothermal titration calorimetry (ITC). Solution NMR analyses showed that ppR interacted with the linker region of pHtrII (pHtrII(G83)(-)(Q149)) and resulted in the broadening of many peaks, indicating structural changes within this region. These results suggest that the pHtrII linker region interacts directly with ppR. There was no demonstrable interaction between the C-terminal region of ppR (ppR(Gly224)(-)(His247)) and either the linker region (pHtrII(G83)(-)(Q149)) or the transmembrane region (pHtrII(M1)(-)(E114)) of pHtrII. On the basis of the NMR, CD, and photochemical data, we discuss the structural changes and role of the linker region of pHtrII in relation to photosignal transduction.

Early Photocycle Structural Changes in a Bacteriorhodopsin Mutant Engineered to Transmit Photosensory Signals

“…The next steps in signal relay and signal propagation through HtrII require further study. According to data from several laboratories (3), these structural changes may induce outward F-helix tilting of SRII (48), structural changes of binding surfaces between SRII and HtrII (16,43,44), rotation of TM2 of HtrII, and structural changes in the membrane proximal domain (HAMP) of HtrII (45,49) to relay the signal to the phosphorylation cascade controlling the flagellar motor switch.…”

Functional Importance of the Interhelical Hydrogen Bond between Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration during the Signaling Process

“…The photochemical reaction cycle (6) and atomic structure of SRII (7)(8)(9) are well characterized. SRII bound to an N-terminal fragment of HtrII have provided atomic details of the two proteins' interaction surface in the periplasm and within the membrane (10), and interaction of the HtrII membrane-proximal domain with the cytoplasmic domain of the receptor has been demonstrated by fluorescent probe accessibility and Förster resonance energy transfer measurements (11), EPR of spin-labels (12), and in vitro binding of HtrII peptides to SRII (13). The signal relay mechanism from SRII to HtrII in the complex has become a focus of interest in part because of its importance to the general understanding of interaction between integral membrane proteins.…”

Shape and oligomerization state of the cytoplasmic domain of the phototaxis transducer II from Natronobacterium pharaonis

“…These structures and EPR spectroscopic studies (8) suggest that conformational changes in pSRII induce a 15°rotation of pHtrII helix II and a 0.9-Å displacement at its cytoplasmic end (7). However, it is not known how this conformational signal is transmitted to the cytoplasmic signaling domain but it likely involves conformational changes in the domain for histidine kinases, adenylyl cyclases, methyl binding proteins, and phosphatases (9). The latter domain is often present in bacterial sensor and chemotaxis proteins and eukaryotic histidine kinases (10).…”