Salt-driven Equilibrium between Two Conformations in the HAMP Domain from Natronomonas pharaonis

Abstract

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. 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. Negative phototaxis in the haloarchaeum Natronomonas pharaonis is mediated by a transducer molecule (NpHtrII) that shares structural analogies and functional similarities with a variety of chemoreceptors reactive to attractant and repellent stimuli mediating the bacterial positive and negative taxis (1Klare J.P. Gordeliy V.I. Labahn J. Büldt G. Steinhoff H.J. Engelhard M. FEBS Lett. 2004; 564: 219-224Crossref PubMed Scopus (91) Google Scholar, 2Jung K.H. Trivedi V.D. Spudich J.L. Mol. Microbiol. 2003; 47: 1513-1522Crossref PubMed Scopus (211) Google Scholar, 3Trivedi V.D. Spudich J.L. Biochemistry. 2003; 42: 13887-13892Crossref PubMed Scopus (18) Google Scholar). Different from chemoreceptors the repellent signal is not sensed by a periplasmic sensor domain, but by a cognate sensor protein, sensory rhodopsin II (NpSRII), which belongs to the seven-transmembrane helices protein family. A light-induced structural rearrangement in NpSRII (4Klare J.P. Bordignon E. Engelhard M. Steinhoff H.J. Photochem. Photobiol. Sci. 2004; 3: 543-547Crossref PubMed Scopus (51) Google Scholar) at the interface with the transmembrane transducer helices TM1 4The abbreviations used are: TM, transmembrane; HAMP domain, conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases; NpSRII, Natronomonas pharaonis sensory rhodopsin II; NpHtrII, Natronomonas pharaonis halobacterial transducer of rhodopsins II; NpHtrII157, construct truncated at position 157; NpHtrII1-230, construct truncated at position 230; PML, purple membrane lipid; EPR, electron paramagnetic resonance; cw, continuous wave; DEER, double electron-electron resonance; mT, millitesla; NiEDDA, Ni(II)ethylenediamine diacetate; dHAMP, dynamic HAMP; cHAMP, compact HAMP; cp, centipoise. and TM2 causes a rotation and displacement of helix TM2, which can be considered as the starting event for signal transduction (5Wegener A.A. Klare J.P. Engelhard M. Steinhoff H.J. EMBO J. 2001; 20: 5312-5319Crossref PubMed Scopus (151) Google Scholar, 6Moukhametzianov R. Klare J.P. Efremov R. Baeken C. Göppner A. Labahn J. Engelhard M. Büldt G. Gordeliy V.I. Nature. 2006; 440: 115-119Crossref PubMed Scopus (150) Google Scholar, 7Falke J.J. Hazelbauer G.L. Trends Biochem. Sci. 2001; 26: 257-265Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Attractant or repellent signals are then further transmitted to the cytoplasmic signaling domain through a linker region, the latter comprising either one or two HAMP domains (8Appleman J.A. Chen L.L. Stewart V. J. Bacteriol. 2003; 185: 4872-4882Crossref PubMed Scopus (67) Google Scholar, 9Aravind L. Ponting C.P. FEMS Microbiol. Lett. 1999; 176: 111-116Crossref PubMed Google Scholar). HAMP modules are found in a wide variety of proteins connecting signal input with signal output. This diversity in the context of function and environment implies common principles of signal relay. A breakthrough in the mechanistic picture of the HAMP domain was the solution NMR structure of the HAMP domain from Archaeoglobus fulgidus showing a homodimeric, four helical, parallel coiled coil with an unusual interhelical packing (10Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). The authors suggested a model for the propagation of the signal transduction, in which “the HAMP domain alternates between the observed conformation and a canonical coiled coil” via concerted helix rotations (10Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Recent results on the intact membrane-bound aspartate receptor of bacterial chemotaxis (11Swain K.E. Falke J.J. Biochemistry. 2007; 46: 13684-13695Crossref PubMed Scopus (77) Google Scholar) and on the aerotaxis receptor Aer (12Watts K.J. Johnson M.S. Taylor B.L. J. Bacteriol. 2008; 190: 2118-2127Crossref PubMed Scopus (43) Google Scholar) are in agreement with the NMR structure of A. fulgidus (10Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Despite these clear evidences for defined structural elements, indications for an intrinsic fragility of the HAMP domain are obvious. For example, the HAMP domain from NpHtrII was suggested to be in a molten-globule-like state at low salt concentration (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google that from the histidine from was shown to be to a structure by R. L. M. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the dynamic and structural properties of the first HAMP domain of NpHtrII are investigated in of salt concentration and pharaonis is an in 3: Scopus Google Scholar) at M. F. V. J. PubMed Scopus Google Scholar) and of in the V. The of Scholar, J.L. E. K.J. J. Bacteriol. PubMed Scopus Google Scholar). and salt can be to be with to Microbiol. PubMed Scopus Google Scholar). to on and Microbiol. Mol. Biol. Scholar) the structure and dynamic properties of the HAMP domain are to be by The and the of proteins are by an on the complex for a conformational in the transducer HAMP domain the to Biochemistry. 2006; PubMed Scopus Google Scholar). is shown that the HAMP domain of pharaonis an equilibrium between a conformation (dHAMP) and a more compact structure The latter conformation is shown to be in agreement with the four helical bundle structure by NMR for the HAMP domain from A. fulgidus. was used as a for was in E. or E. were by the as PubMed Scopus Google Scholar) starting with the Wegener A.A. Engelhard M. FEBS Lett. 1999; PubMed Scopus Google truncated (5Wegener A.A. Klare J.P. Engelhard M. Steinhoff H.J. EMBO J. 2001; 20: 5312-5319Crossref PubMed Scopus (151) Google truncated the by and the by Klare J.P. R. R. Engelhard M. FEBS Lett. 2007; PubMed Scopus Google Scholar). The were the and were by and truncated at position truncated at position and as as their mutants were either in E. to M. M. FEBS Lett. PubMed Scopus Google Scholar) in the of to Klare J.P. R. R. Engelhard M. FEBS Lett. 2007; PubMed Scopus Google and as Wegener A.A. Engelhard M. FEBS Lett. 1999; PubMed Scopus Google Scholar, Klare J.P. R. R. Engelhard M. FEBS Lett. 2007; PubMed Scopus Google Scholar). The spin was to the of the or as by M. Steinhoff H.J. J. Mol. Biol. 1999; PubMed Scopus Google Scholar). The is as in the was by For of the were and in a For as in M. Scopus Google Scholar) from purple membrane the were at in the in containing either a not in or a of as as of the proteins were by at and in continuous were a with a with the to and to of at protein of were For salt the were at the was and the was and in containing the salt concentration in the to with of or For a with a was used in with a containing to the in the The was to and the to The for paramagnetic in or were by the of C. PubMed Scopus Google Scholar, C. Sci. S. A. PubMed Scopus Google Scholar) and are as between the spin and the paramagnetic or the The from of the were by the to to C. R. J. Full Text Full Text PDF PubMed Scopus Google A with a was used with the in the A of was a For the was by a the that was by for For of the spin from the a solution of was to the to a concentration of or and the was to the was with the for for distance were at a with an The was to and the to A and a continuous of the and of the temperature, of at protein of were were at with a with a and a continuous by an were the M. S. A. G. J. PubMed Scopus Google Scholar). For the at the the were A is on is and are and the is by were only for The was to the was to the of the and with the of the the was and with the low of the were at a of with of for and for and a of was by at starting at and by the of used for on the was by at starting at and by The for was between and The data of the was with the G. V. A. J. 2006; Scopus Google Scholar). of of to the at the distance to the H.J. V. A. G. A. J. Full Text PDF PubMed Scopus Google Scholar). for a of to from spin a of with a of is the the the of and of the and the and were to the found for the of the labeled The used were by the low at Bordignon E. L. Klare J.P. Engelhard M. Steinhoff H.J. 2006; Scopus Google Scholar). and were to and mT, was to for in the of the spin environment Bordignon E. L. Klare J.P. Engelhard M. Steinhoff H.J. 2006; Scopus Google Scholar, M. Steinhoff H.J. Wegener C. A. Mol. Scopus Google Scholar). The were with a function of a of and of and The for the of the are the and the of the spin labeled The spin labeling was in the for the investigated of to the between and was to a model of of the and Google Scholar). 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Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) with four of the investigated The in the A. fulgidus HAMP model were to the for the HAMP namely the the region, and the at and in the transducer protein and at position in the receptor of and on the HAMP in were for spin labeled in the transducer which only one HAMP the of the on the HAMP were engineered and spin labeled in the HAMP and in the transducer of the for a position in the the in are in 2. low salt are by the of the two a of the the spin is engaged in and a of the the spin is by The is used in a and on the to and of the the of the in the The between the two was found to be on the the salt concentration to an of for This that the salt are and the of the is data in the following were in the truncated construct are in the of two by at low salt The can be with an of showing a is by of The latter the observed for highly dynamic R. Biochemistry. PubMed Scopus Google Scholar, Biochemistry. 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The induced the of the dynamic the observed changes a conformational in the HAMP domain induced by salt of the was induced by low The for position at is in the is to be one of the for signal transduction PubMed Scopus Google the for pharaonis is the of low in the NpHtrII structure are not further investigated in of on TM2, NpSRII, and a the salt was investigated for a spin labeled position in transmembrane helix TM2 of as as for a spin labeled position in the of sensory rhodopsin at the interface with TM2 the salt concentration the of these spin that the transmembrane of the transducer more compact as on the labeled that the distance to with salt not in the NpSRII cytoplasmic a in the of the changes observed with position in the of are with the changes in the in the HAMP domain, that only the signal transduction domain a between two were to the of the at salt to the of the structural rearrangement of the HAMP domain with to changes in the were the purple membrane and their properties investigated at and salt The are in the salt concentration to a in the of the in the at and in the of the protein the of the of the at salt the and the salt are the of the protein in the membrane The data that the changes observed for spin labeled in the HAMP domain of the complex in be by a rearrangement of the membrane of and in the of the HAMP domain at salt concentration was and with the data at low salt concentration (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) The of the was via the of the low salt a in the of the was only to position the of an by Bordignon E. L. Klare J.P. Engelhard M. Steinhoff H.J. 2006; Scopus Google Scholar). from position the two are the data are dynamic (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The of in the and the found suggested a dynamic of the investigated in the and in the a of The observed not to via between a dynamic helix or a structure and E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). salt concentration an in in the as as in the and in is found in the the of of the agreement with the complex for investigated at salt concentration to the of two spin a defined structure Sci. 2007; PubMed Scopus Google Scholar, Sci. 2008; PubMed Scopus Google Scholar). the of a dynamic domain at low salt and a more compact structure at salt The data were by to between spin to the the or in the protein of and at low and salt are in with or or low for are for the protein The at low salt concentration for to the at salt concentration a with the of a compact HAMP agreement with the a in the of the position is to defined and in the The from the was with at and were to For the investigated in the HAMP domain at low salt the at are at The that the are not to the concentration to the of two by from the This can be by of two at and the of with the that the spin labeled in the are The by in the of are then with the the at concentration the is by the at the of the in the The at at low salt are for and to the at salt (cHAMP) of the spin This that at low salt and the at salt the cHAMP low were at salt concentration for for to be in the NMR HAMP This can be by the packing of the protein at salt in the of of of on the to the of the cHAMP a of the by at salt concentration with the of the HAMP domain from A. fulgidus was The of the and of the pharaonis HAMP domain are in agreement with the of the NMR structure of the HAMP domain from A. fulgidus from K.E. Falke J.J. Biochemistry. 2007; 46: 13684-13695Crossref PubMed Scopus (77) Google to the investigated in the region, an not be The and data that the in the cHAMP by salt are in and from the The cHAMP conformation is shown to be in agreement with the four helical bundle structure by NMR for the HAMP domain of A. fulgidus. the the dynamic and to in the conformation. The the of the two and to their in of a with NiEDDA, and were the of the was with the paramagnetic and the at low salt concentration are in A and a of to with the investigated spin the of spin the only for spin from the The of is shown in the of to the of a of is not be The of is shown in the is the between the two of the at and results the of an equilibrium between the and with the were with to further the of the conformation low and salt of was by an in the of 2. at low salt of on the are This that the HAMP domain is at low salt concentration and that the spin with of the is by to of a at salt concentration the on the is is that is not to the protein at low at salt For of the of the by the at salt concentration from the one at low salt concentration is The dynamic of (dHAMP) is The results the to to in that and in a the of the for at low and salt to the to the (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). The observed are by of in the the or the salt in the equilibrium the cHAMP conformation. the equilibrium was not by the in the from to the of to the and was to the of the two in and their The of for the of the for and in the and was by of and found to be in agreement with data H.J. J. C. PubMed Scopus Google Scholar). The of the with at low and salt is shown in low salt concentration the for were found to be the for position (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) was from the of the not from their The are: the of the were found to be with and The agreement of the equilibrium for in HAMP the of a equilibrium between cHAMP and distance in the complex is in of spin labeled at low and salt were not and in of and were on for the of the of and and are for of the distance as can be in the between the and distance for position in the are in the is to or The at low salt concentration were (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Despite the changes in the and data observed salt at temperature, the at low not changes in the This can be by the for the of the two in and the to a of the equilibrium at low salt the for distance only of the cHAMP conformation. The distance were with the of for in the NMR model of the HAMP domain from A. fulgidus to A. M. J. 2008; Full Text Full Text PDF PubMed Scopus Google The the distance the the of a dynamic (dHAMP) and compact (cHAMP) conformation as in the spin at the HAMP domain are by mobility, for to of proteins or to in which a in the of the for that position in the dynamic conformation is to the for position The of the conformation from the of comprising the functional signaling found in as the HAMP domain, the of protein in equilibrium with the cHAMP conformation. to be that were either the for or transducer proteins, the intrinsic or a attractant signaling shown that the conformation from of the protein and that is observed with the the two are for in of the HAMP their is from position in to position which the that an to the of the spin that the and cHAMP conformational of the NpHtrII HAMP For the cHAMP conformation a parallel four helical bundle the and data to a defined of properties in and the of the found with the in for the in the HAMP NMR model (10Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google the not a clear is in with the a four helical for and an was found between the data and the NMR model of the HAMP domain from A. fulgidus (10Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google not be in the region, only a were the four helical bundle structure by NMR on an unusual membrane protein of function to the cHAMP conformation of the transducer from Recent data by and Falke (11Swain K.E. Falke J.J. Biochemistry. 2007; 46: 13684-13695Crossref PubMed Scopus (77) Google Scholar) on the HAMP domain of the from E. in membrane-bound chemoreceptors that the HAMP NMR model the of the conserved HAMP domain in the The authors found that conformational changes were induced by the agreement between the A. fulgidus and the of the in the aspartate receptor is in in a more dynamic structure of the first of the HAMP domain from the aspartate A in by Taylor and (12Watts K.J. Johnson M.S. Taylor B.L. J. Bacteriol. 2008; 190: 2118-2127Crossref PubMed Scopus (43) Google Scholar) that the HAMP domain of the E. aerotaxis the four helical bundle the NMR model is a for HAMP The authors suggested a of a to the the domain is to for a dynamic in the and of in the Aer HAMP domain (12Watts K.J. Johnson M.S. Taylor B.L. J. Bacteriol. 2008; 190: 2118-2127Crossref PubMed Scopus (43) Google Scholar). Despite on HAMP structural the of for the negative for the positive taxis the of a model for the of chemoreceptors by Sci. 3: PubMed Scopus Google with the signaling domains in two by of is in the of on bacterial the that a equilibrium between with of can be the of the of the cytoplasmic domain was by Hazelbauer G.L. Falke J.J. Trends Biochem. Sci. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). for a for the bacterial signal transduction were in L.L. Biol. 2008; 3: PubMed Scopus Google Scholar). The found for the NpHtrII HAMP domain is that two are found to at low and salt which the two by their dynamic properties are found to be in a which can be at by changes of or salt equilibrium to one the of The HAMP domain to be engaged in a between that to a the equilibrium the of receptor The HAMP domains be to their a variety of attractant the and repellent the by to periplasmic conformational changes of in the or in the of the aerotaxis receptor sensor domains in to the HAMP The of the equilibrium between the two HAMP be an to an of the function of and S. M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Biol. 2007; PubMed Scopus Google Scholar). for a dynamic conformation an intrinsic of the HAMP domain in (13Bordignon E. Klare J.P. Doebber M. Wegener A.A. Martell S. Engelhard M. Steinhoff H.J. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, R. L. M. J. Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). the NMR model was the of the A. which the HAMP structure in one of the HAMP function by the HAMP was shown to be highly at to (10Hulko M. Berndt F. Gruber M. Linder J.U. Truffault V. Schultz A. Martin J. Schultz J.E. Lupas A.N. Coles M. Cell. 2006; 126: 929-940Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). at that the HAMP was a HAMP domain from a and to be functional in E. is an This to a signaling only the of the equilibrium between two proven by and that a rotation of TM2 of NpHtrII light of NpSRII (5Wegener A.A. Klare J.P. Engelhard M. Steinhoff H.J. EMBO J. 2001; 20: 5312-5319Crossref PubMed Scopus (151) Google Scholar, 6Moukhametzianov R. Klare J.P. Efremov R. Baeken C. Göppner A. Labahn J. Engelhard M. Büldt G. Gordeliy V.I. Nature. 2006; 440: 115-119Crossref PubMed Scopus (150) Google Scholar). A rotation event in the transmembrane of the protein the rotation of the HAMP which in the equilibrium between the two HAMP the on or the signal the of the HAMP domain be an to the transducer signaling domain This to changes in the between the chemoreceptors A. J. Mol. Biol. 2007; PubMed Scopus Google Scholar) and in the suggested for the and an for which L.L. Biol. 2008; 3: PubMed Scopus Google Scholar). NMR and site-directed spin labeling the functional and of the between dynamic of HAMP M. E. A. Göppner for