Article

Salt-driven equilibrium between two conformations in the HAMP domain from Natronomonas pharaonis: the language of signal transfer?

Fachbereich Physik, Universität Osnabrück, Barbarastrasse 7, 49076 Osnabrück, Germany.
Journal of Biological Chemistry (Impact Factor: 4.65). 09/2008; 283(42):28691-701. DOI: 10.1074/jbc.M801931200
Source: PubMed

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.

0 Bookmarks
 · 
78 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Available structures of HAMP domains suggest rotation as one potential mechanism in intraprotein signal transduction. It has been proposed that in poly-HAMP modules the signal sign is inverted with each additional HAMP. We examined signal transduction through the HAMP tandem domain from the phototaxis transducer of the halophilic archaeon Natronomonas pharaonis in membrane-bound chimeras consisting of the E. coli chemotaxis receptor for serine, Tsr, as an input and the mycobacterial adenylyl cyclase Rv3645 as an output domain; i.e. the basic chimera was 'Tsr - NpHAMP tandem - Rv3645 cyclase'. Neither of the NpHAMP units alone nor the NpHAMP tandem transduced a serine signal. After five targeted point mutations in the first α-helix of NpHAMP1 , the non-functional NpHAMP modules combined into a functional HAMP tandem. 1 mM serine significantly inhibited cyclase activity (-35%; IC50 = 30 μM) in disagreement with the structure-based predictions. Surprisingly, replacement of NpAS11 in the tandem by the respective AS1 from HAMPTsr resulted in signal inversion, i.e. serine activated cyclase (+129%; EC50 = 10 μM). Examination of 48 mutants of AS11 in the HAMP tandem including two residues of a putative N-terminal control cable identified five residues in NpAS11 which probably define different ground states of the output domain and, thus, affect the sign of signal output. The data question the predicted HAMP rotation as the predominant mechanism of intraprotein signal transduction and point to as yet unrecognized conformational motions of HAMP domains in intraprotein signaling.
    FEBS Journal 05/2014; · 4.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as recent progress in SDSL and EPR methods especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author's lab in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, e.g. the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.
    Biological Chemistry 08/2013; 394(10):1281-1300. · 2.68 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: HAMP domain is a ubiquitous module of bacterial and archaeal two-component signaling systems. Considerable progress has been made recently in studies of its structure and conformational changes. However, the mechanism of signal transduction through the HAMP domain is not clear. It remains a question whether all the HAMPs have the same mechanism of action and what are the differences between the domains from different protein families. Here, we present the results of unbiased molecular dynamics simulations of the HAMP domain from the archaeal phototaxis signal transducer NpHtrII. Two distinct conformational states of the HAMP domain are observed, that differ in relative position of the helices AS1 and AS2. The longitudinal shift is roughly equal to a half of an α-helix turn, although sometimes it reaches one full turn. The states are closely related to the position of bulky hydrophobic aminoacids at the HAMP domain core. The observed features are in good agreement with recent experimental results and allow us to propose that the states detected in the simulations are the resting state and the signaling state of the NpHtrII HAMP domain. To the best of our knowledge, this is the first observation of the same HAMP domain in different conformations. The simulations also underline the difference between AMBER ff99-SB-ILDN and CHARMM22-CMAP forcefields, as the former favors the resting state and the latter favors the signaling state.
    PLoS ONE 01/2013; 8(7):e66917. · 3.53 Impact Factor