Three-dimensional structure of the Na +/H + antiporter from Methanococcus jannaschii

Na+/H+ antiporters are ubiquitous membrane proteins involved in ion homeostasis and pH sensing. The amino acid sequence of one such antiporter, MjNhaP1, from Methanococcus jannaschii, shows a significant homology to eukaryotic sodium proton exchangers like NHE1 from Homo sapiens and SOS1 of Arabidopsis thaliana than to the well-characterized Escherichia coli NhaA or NhaB. MjNhaP1 shows activity at acidic pH unlike NhaA, which is active at basic pH. 13 transmembrane helices have been predicted to be present in NhaP1. A projection map, calculated by Cryo-EM of 2D crystals of MjNhaP1 grown at pH 4, showed it to be a dimer containing elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core (Vinothkumar et al., 2005). Incubation of 2D crystals at pH 8 on the EM grid resulted in well-defined conformational changes, clearly evident in a difference map as a major change in density distribution within the helix bundle (Vinothkumar et al., 2005). The aim of this dissertation is to understand the working mechanism of MjNhaP1 by determining its three-dimensional structure. The aim was initially approached by structure determination by X-ray crystallography. The limitation for this method was the low expression yield, which was 0.5–0.7mg/ml (Vinothkumar et al., 2005). After various optimization trials, the expression yield of the recombinant protein could be elevated to 2-2.5mg of pure protein per litre of culture by the method of autoinduction (Studier et al., 2005). To obtain well diffracting 3D crystals, purification conditions (Vinothkumar et al., 2005) were modified. 3D crystals were obtained under various conditions, which has so far not diffracted X-Ray beyond 8Å. Parallely, optimization of parameters (Vinothkumar et al., 2005) for 2D crystals formation was carried out. A combination of 1% DDM used for lipid solubilization, and 1% OG in the buffer of the purified protein produced 1-2 μm wide tubular 2D crystals of NhaP1. This batch of crystal proved to be the optimal for data collection at higher tilt angle with the electron microscope. A 3D map showed p22121 symmetry and revealed a tight dimer with an oval shape. The region in the central part of the dimer is composed of several tilted helices forming an interface between both monomers. On either side of the dimer interface, a group of six tightly packed helices form a bundle. This bundle contains three straight helices in the centre of the monomer and three helices in the periphery. Comparison of the structures of E.coli NhaA and M. jannaschii NhaP1 show substantial differences in length and slope of corresponding helices between both antiporters. A 3D model of NhaP1 based on the 3D map revealed 13 helices, which has been named as A-M to distinguish it from the NhaA helices. Overlaying the X-ray structure onto the 3D map revealed that the disrupted helices IV and XI of NhaA superimpose two central helices at similar position in the 3D map of NhaP1. The disrupted helices IV and XI in the X-ray structure of NhaA have been proposed as the putative ion-binding and translocation site (Hunte C et al, 2005; Arkin IT et al, 2007; Screpanti & Hunte (2007). This motif appears to be present also in NhaP1, as suggested by the close fit of NhaA helices IV and XI on the putative helices E and L of the NhaP1 model. These two putative helices E and L in NhaP1 contain the highly conserved TDP and GPRVVP motif, which are crucial for antiporter activity (Hellmer et al., 2002, Hellmer et al., 2003). In the overlay, helix V of NhaA containing the two essential, conserved aspartates D163 and D164 fits the density of the putative helix F of NhaP1, which contains the conserved motif FNDP. The homologous D161 in the FNDP motif of NhaP1 is essential for transport activity as show by mutagenesis (Hellmer at al., 2003). Significant differences are visible in the region of the dimer interface of the 3D map of NhaP1 occupied by helices VI, VII, and VIII in NhaA. This region shows an extra helical density (A) in the 3D map of NhaP1. By alignment of MjNhaP1 sequence with the amino acid sequences of several Na+/H+ exchangers, it was evident that the additional helix (A) is located in the N terminus of NhaP1. In our sequence alignment, a putative hydrophobic segment corresponding to this additional helix A is present in other archaeal and eukaryotic antiporters but not in any of the bacterial ones. The N-terminus of the human Na+/H+ exchanger NHE1 has been predicted to contain a highly hydrophobic signal peptide. This indicates the probability of the N-terminal helix A of NhaP1 to be an uncleaved signal peptide. Besides being a signal sequence targeting NhaP1 to the membrane, the map suggests that this helix might be involved in the formation of dimer contacts between both monomers. A gene duplication event is evident in the 3D map of NhaP1, as not only the helices D, E, F and K, L, M are related by an inverted repeat but also the helices B, C and I, J are related. We present here the three-dimensional architecture of a Na+/H+ antiporter from archaea. The presence of the 13th helix suggests the location of the N-terminus to be located in the cytosol and the C-terminus in the periplasm. This would orient NhaP1 in an inverted manner in the membrane in comparison to NhaA. Further structural information at higher resolution and biochemical and biophysical investigations are required to confirm the topology.Die Einstellung und Aufrechterhaltung des Gleichgewichts von Na+-Ionen und Protonen wird bei lebenden Zellen - sowohl bei Pflanzen als auch bei Tieren und Mikroorganismen – hauptsächlich über Na+/H+ Austauscher gewährleistet. Na+-Ionen und Protonen sind in Physiologie und Bioenergetik von lebensnotwendiger Bedeutung. Es wird davon ausgegangen, dass Na+/H+ Austauscher pH Sensoren besitzen, die bei Änderung des intrazellulären pH-Wertes die Austauschaktivität regulieren. Zudem leisten sie einen erheblichen Anteil bei der Regulation des Natriumflusses und des Zell-Volumens nach osmotisch bedingtem Zellschrumpfen (Pavel, 1998). Zusätzlich zu ihrer physiologischen Bedeutung sind eukaryotische Na+/H+ Austauscher ein wichtiges Angriffsziel für Medikamente, da sie in Pathologie und bei menschlichen Krankheiten eine entscheidende Rolle spielen. Beispielsweise konnte in einer Studie gezeigt werden, dass die Verabreichung von Cariporiden, einem Hemmstoff des menschlichen Na+/H+ Austauschers NHE1, nach dem Einsetzen eines Koronararterien-Bypasses eine signifikante Reduktion von Myokardinfarkten zur Folge hatte (Mentzer et al., 2003). Na+/H+ Austauscher ermöglichen Prokaryoten eine gewisse Salztoleranz und das Wachstum bei extremen pH-Werten, da Mikroorganismen ständig wechselnden Umweltbedingungen ausgesetzt sind (Padan, 2005). Erste Einblicke in die Struktur von Na+/H+ Austauschern gingen aus der dreidimensionalen Elektronendichtekarte des Escherichia coli Austauschers NhaA hervor, welche über Elektronenkristallographie erhalten wurde (Williams, 2000). Es konnte gezeigt werden, dass NhaA 12 Transmembransegmente (TMS) besitzt und in der Lipidumgebung der 2D Kristalle ein Dimer bildet. Die später erhaltene Röntgenstruktur der inaktiven Konformation von NhaA bei pH 4,0 zeigte eine bislang einzigartige Faltung (Hunte et al., 2005): Die TMS IV und XI bestehen jeweils aus zwei halben Helices, die mit einem kurzen ungewundenem Abschnitt verbunden sind Diese unterbrochenen Helices kreuzen sich in der Mitte der Membran in entgegengesetzter Richtung. Allgemein ist die Insertion von polaren Helix-Termini energetisch ungünstig, da dieser Zustand eine Ladungskompensation benötigt und damit als ideal betrachtet wird, um Ionen anzuziehen und zu binden (Toyoshima et al., 2000),(Dutzler et al., 2002),(Yernool et al., 2004),(Hunte et al., 2005),(Yamashita et al., 2005). ...