Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize the transport reaction. Binding of ATP to the NBD monomer results in formation of a NBD dimer. Hydrolysis of the ATP drives the dissociation of the dimer. The thermodynamics of distinct steps in the ATPase cycle of GlcV, the NBD of the glucose ABC transporter of the extreme thermoacidophile Sulfolobus solfataricus, were studied by isothermal titration calorimetry using the wild-type protein and two mutants, which are arrested at different steps in the ATP hydrolytic cycle. The G144A mutant is unable to dimerize, while the E166A mutant is defective in dimer dissociation. The ATP, ADP, and AMP-PNP binding affinities, stoichiometries, and enthalpies of binding were determined at different temperatures. From these data, the thermodynamic parameters of nucleotide binding, NBD dimerization, and ATP hydrolysis were calculated. The data demonstrate that the ATP hydrolysis cycle of isolated NBDs consists of consecutive steps where only the final step of ADP release is energetically unfavorable.

Part I: Structural framework for the mechanism of archaeal exosomes in RNA processing; Part II: Structural insights into DNA duplex separation by the archaeal superfamily 2 helicase Hel308

Part I The exosome is a conserved 3´- 5´ exoribonuclease complex involved in cellular RNA metabolic processes in eukaryotes and archaea. Its involvement in the accurate processing of nuclear RNA precursors and in the degradation of RNA in both nucleus and cytoplasm implies a central function in the eukaryotic RNA surveillance machinery. This widespread function implies the ability of the exosome to distinguish between RNA substrates that should be matured by the removal of nucleotides to a precisely defined end point, and defective RNAs that undergo rapid and complete degradation. However, the structural and molecular mechanisms of processive 3´- 5´ RNA degradation and substrate specificity remain unclear. To obtain insights into the structural and functional organization of the exosome, I determined crystal structures of two 230 kDa nine subunit exosome isoforms from Archaeoglobus fulgidus. Both exosome isoforms contain a hexameric ring of RNase PH-like domain subunits Rrp41 and Rrp42 with a central chamber. A trimer of Rrp4 or Csl4 subunits is situated on one side of the RNase PH domain ring and forms a multidomain macromolecular interaction surface with central S1 domains and peripheral KH and zinc-ribbon domains. Tungstate soaks identified three phosphorolytic active sites inside the central processing chamber. Additional structural and functional results suggest that the S1 domains of Rrp4 or Csl4 subunits and a subsequent neck in the RNase PH domain ring form an RNA entry pore that only allows access of unstructured RNA to the active sites. The structural results presented here can not only mechanistically unify observed features of exosomes, including processive 3´ RNA degradation of unstructured RNA, the requirement for regulatory factors and coactivators to degrade structured RNA, and the precision in processing RNA species to a defined length. But the high conservation of the archaeal exosome to the eukaryotic exosome and its additional high structural similarity to bacterial mRNA-degrading PNPase suggest a common basis for 3´ RNA-degradation in all kingdoms of life. Furthermore, the structure of the archaeal exosome reveals remarkable architectural and functional similarities to the protein degrading proteasome. Part II Adenosine triphosphate (ATP) dependent nucleic acid unwinding by superfamily 2 (SF2) helicases is required for numerous biological processes, including DNA recombination, RNA decay and viral replication. The structural and molecular mechanism for processive duplex unwinding of SF2 helicases is still unclear, in part due to a lack of structural insights into the actual strand separation reaction. Archaeal SF2 helicase Hel308 preferentially unwinds lagging strands at replication forks and is closely sequence related to human PolΘ and Hel308 as well as Drosophila Mus308. Furthermore, the RecA ATPase-core of archaeal Hel308 shares high sequence conservation to the SF2 RNA decay factors Ski2p and Mtr4p. Thus, archaeal Hel308 appears as representative model to understand processive 3´- 5´ DNA unwinding by SF2 helicases. During this PhD thesis crystal structures of Archaeogloubs fulgidus Hel308 (afHel308) in the absence and presence of a 15mer duplex DNA containing a 10mer 3´-overhang were determinded using X-ray crystallography. afHel308 exhibits two typical SF2 RecA-like domains at the N-terminus. The C-terminus comprises a winged-helix (WH) domain, followed by a unique seven-helix-bundle domain and a helix-loop-helix (HLH) domain. The DNA bound structure captures the initial duplex separation and argues that initial strand separation does not require ATP binding. Comparison with ATP bound SF2 enzymes suggests that ATP binding and hydrolysis promotes processive unwinding of one base pair by a ratchet like transport of the 3’ product strand. In addition, the structure suggests that unwinding is promoted by a prominent β-hairpin loop. The identification of similar β-hairpin loops in Hepatitis C virus (HCV) NS3 helicase and RNA decay factors Ski2p and Mtr4p, and consistency of the results with biochemical data on HCV NS3 helicase argue that the observed duplex unwinding mechanism is applicable to a broader subset of processive SF2 helicases. Furthermore, the interaction between afHel308 and its DNA substrate also may explain how afHel308 is targeted to branched nucleic acid substrates.The presented results provide a first structural framework for duplex unwinding by processive SF2 helicases and reveal important mechanistic differences to SF1 helicases and the SF2 helicase RecG

A new family of periplasmic-binding proteins that sense arsenic oxyanions

Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that's role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate

Strukturelle Studien von Proteinen involviert in Membrantransport

My graduate thesis is on the "Structural studies of membrane transport proteins". Transporters are membrane proteins that have multiple membrane-spanning a-helices. They are dynamic and diverse proteins, undergoing a large conformational change and transporting wide range of susbtrates. Based on their energy source they can be classified into primary and secondary transport systems. Primary transport systems are driven by the use of chemical (ATP) or light energy, while secondary transporters utilize ion gradients to transport substrates. I began my PhD dissertation on secondary transporters by two-dimensional crystallization and electron crystallographic analysis and recently my focus also has shifted towards 3D crystallization. The following projects constitute my PhD thesis: 1) 2D crystallization of MjNhaP1 and pH induced structural change: MjNhaP1, a Na+/H+ antiporter that is regulated by pH has been implicated in homeostasis of H+ and Na+ in Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C. MjNhaP1 was cloned and expressed in E. coli. Two-dimensional crystals were obtained from purified protein at pH4. Electron cryo-microscopy yielded an 8Å projection map. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1was studied in situ in 2D crystals revealing a major change in density within the helix bundle relative to the dimer interface. This change occurred at pH6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter, respectively. This is the first instance where a conformational change associated with the regulation of a secondary transporter appears to map structurally. Reconstruction of 3D map and high-resolution structure by x-ray crystallography would be necessary to understand the mechanism of ion transport and regulation by pH. 2) 2D crystallization of Proline transporter: Proline transporter (PutP) from E.coli belongs the sodium-solute symporter family that includes disease related sodium dependent glucose and iodide transporter in humans. Sodium and proline are co-transported with a stoichiometry of 1:1. Purified PutP was reconstituted to yield 2D crystals that were hexagonal in nature. The 2D crystals had tendency to stack indicating their willingness to form 3D crystals. A projection map of PutP from negatively stained crystals showed trimeric arrangement of protein. Other members of the SSF family have been shown to be monomers. My analysis of oligomeric state of PutP in detergent by blue native gel indicates a monomer in detergent solution. It is likely that PutP can function as a monomer but at higher concentration and in lipid bilayer it tends to form trimer. 3) Oligomeric state and crystallization of carnitine transporter from E.coli: E.coli carnitine transporter (CaiT) belongs to the BCCT (Betaine, Carnitine and Choline) superfamily that transports molecules with quaternary amine groups. CaiT is predicted to span the membrane 12 times and acts as a L-carnitine/g-butyrobetaine exchanger. Unlike other members in this transporter family, it does not require an ion gradient and does not respond to osmotic stress. Over-expression of the protein yielded ~2mg of protein/L of culture. The structure and oligomeric state of the protein were analyzed in detergent and lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. Gel filtration and cross-linking studies further support this. Reconstitution of CaiT into lipid bilayers resulted in 2D crystals. Analysis of negatively stained 2D crystals confirmed that CaiT is a trimer in the membrane. Initial 3D crystallization trials have been successful and currently, the crystals diffract to 6Å and are being improved. 4) Monomeric porin OmpG: OmpG is a bacterial outer membrane b-barrel protein. It is monomeric and its size (33kDa) places it as a prime candidate for a structural solution, using the recently developed method of solid state NMR (work in collaboration with Prof.Hartmut Oskinat, FMP, Berlin). A long-term aim would be to study porins as templates for designing nanopores, for DNA sequencing and identification. I have expressed OmpG in inclusion bodies and refolded at an efficiency of >90% into a functional form using detergent. OmpG was then crystallized by 2D crystallization yielding an 8Å projection map whose structure was similar to native protein. In addition, these crystals were used for structure determination by solid state NMR. An initial spectrum of heavy isotopically labeled OmpG has allowed identification of specific amino acid residues including threonine and proline. Additionally, I obtained 3D crystals in detergent that diffract to 5.5Å and are being improved.Transporter sind Membranproteine, welche mehrere, die Membran durchspannende, a-Helices aufweisen. Diese beweglichen und vielfältig vorkommenden Proteine ermöglichen durch eine Änderung ihrer Konformation den Transport eines großen Spektrums an verschiedenen Substraten. Zu Beginn meiner Dissertation habe ich an sekundären Transportern, deren 2D Kristallisation mit anschließender elektronenkristallographischer Analyse gearbeitet. Da die mittels Elektronenmikroskopie (EM) erzielte Auflösung begrenzt war, hat sich mein Fokus vor kurzer Zeit zusätzlich auch in Richtung 3D Kristallisation verschoben. Die folgenden Projekte bilden die Basis meiner Doktorarbeit. 1) 2D Kristallisation von MjNhaP1 und pH-Wert induzierte Strukturänderung Der Na+/H+ Antiporter MjNhaP1 ist pH-reguliert und an der Homöostase von H+ und Na+ im hyperthermophilen Archäon Methanococcus jannaschii, dessen optimale Wachstumstemperatur 85°C beträgt, beteiligt. MjNhaP1 wurde in E. coli kloniert und exprimiert. Zweidimensionale Kristalle konnten mit aufgereinigtem Protein bei einem pH-Wert von 4 erhalten werden. Mittels Cryo-Elektronenmikroskopie wurde eine Projektionskarte mit einer Auflösung von 8Å erstellt, welche verlängerte Elektronendichten in der Mitte und strukturierte Elektronendichten zu beiden Seiten des Dimers aufzeigte, die indikativ für ein Bündel aus 4-6 transmembran Helices sind. Der Effekt des pH-Werts auf die Struktur von MjNhaP1 wurde an den 2D Kristallen in-situ untersucht und enthüllte eine deutliche Veränderung der Elektronendichte innerhalb des Helix-Bündels relativ zur Dimergrenze. Diese Veränderung trat ab einem pH-Wert von 6 und darüber auf. Die beiden, bei hohem und niedrigem pH-Wert erhaltenen, Konformationen repräsentieren aller Wahrscheinlichkeit nach den geöffneten beziehungsweise geschlossenen Zustand des Antiporters. Dies ist das erste bekannte Beispiel für eine mit der Regulation eines sekundären Transporters einhergehenden Konformationsänderung, die strukturell dargestellt werden konnte. 2) 2D Kristallisation eines Prolin-Transporters: Der Prolin-Transporter PutP von E. coli gehört zur Familie der Natrium-abhängigen Symporter, welcher auch die für bestimmte Krankheiten beim Menschen relevanten Glukose- und Iodid-Transporter zugehörig sind. Aufgereinigtes PutP wurde rekonstituiert, um hexagonale 2D Kristalle zu erhalten, welche die Tendenz aufwiesen sich übereinander zu Stapeln anzuordnen, was ihre Bereitschaft aufzeigte 3D Kristalle zu bilden. Die Projektionskarte von negativ kontrastierten PutP Kristallen offenbarte eine trimere. Meine Analyse des oligomeren Zustands von in Detergenz gelöstem PutP mittels nativer Gelelektropherese zeigte im Gegensatz zu den 2D Kristallen einen monomeren Zustand auf. Es ist wahrscheinlich, dass PutP als Monomer funktionsfähig ist, aber in höheren Konzentrationen und der Lipiddoppelschicht dazu neigt, Trimere zu bilden. 3) Oligomerer Zustand und Kristallisation des Karnitin-Transporters von E.coli Der Karnitin-Transporter CaiT aus E. coli gehört zur BCCT (Betain, Karnitin und Cholin) Superfamilie, welche Moleküle transportiert, die quaternäre Amingruppen aufweisen. CaiT ist ein L-Karnitin/g-Butyrobetain-Antiporter. Im Gegensatz zu anderen Mitgliedern dieser Transporterfamilie benötigt es keinen Ionengradienten und reagiert nicht auf osmotischen Stress. Die Überexpression des Proteins ergab ~2mg Protein pro Liter Kulturvolumen. Die native Gelelektrophorese zeigte auf, dass CaiT in Detergenzlösung als Trimer vorliegt, was zusätzlich durch Gelfiltrations- und Cross-Linking-Experimente untermauert wurde. Die Rekonstitution von CaiT in eine Lipiddoppelschicht ergab 2D Kristalle, deren Analyse nach der negativen Kontrastierung bestätigte, dass CaiT als Trimer in der Membran vorliegt. Erste Versuche einer 3D Kristallisation waren erfolgreich und ergaben bisher Auflösungen von bis zu 6Å, welche momentan verbessert wird. 4) Monomeres Porin OmpG: OmpG ist ein aus b-Faltblättern bestehendes Protein der äußeren bakteriellen Zellmembran. Es ist monomer und seine Größe (33 kDa) macht es zu einem idealen Kandidaten für die Strukturaufklärung mittels der kürzlich entwickelten Methode der Festkörper-NMR (mit Prof. H. Oschkinat, FMP, Berlin). OmpG wurde von mir in Einschlusskörper exprimiert und durch die Nutzung von Detergenz mit einer Effizienz von mehr als 90% zur funktionell aktiven Form rückgefaltet. Die anschließende 2D Kristallisation von OmpG ergab eine Projektionskarte mit einer Auflösung von 8Å und einer dem nativen Protein ähnlichen Struktur. Außerdem wurden diese Kristalle zur Strukturbestimmung mittels Festkörper-NMR verwendet. Eines der ersten Spektren mit dem Schwerisotopen markiertem OmpG ermöglichte die Identifizierung von spezifischen Aminosäureresten einschließlich Threonin und Prolin. Weiterhin erhielt ich 3D Kristalle in Detergenz, welche bis zu 5,5Å beugten und nun verbessert werden

Archaeal Genome Guardians Give Insights into Eukaryotic DNA Replication and Damage Response Proteins

As the third domain of life, archaea, like the eukarya and bacteria, must have robust DNA replication and repair complexes to ensure genome fidelity. Archaea moreover display a breadth of unique habitats and characteristics, and structural biologists increasingly appreciate these features. As archaea include extremophiles that can withstand diverse environmental stresses, they provide fundamental systems for understanding enzymes and pathways critical to genome integrity and stress responses. Such archaeal extremophiles provide critical data on the periodic table for life as well as on the biochemical, geochemical, and physical limitations to adaptive strategies allowing organisms to thrive under environmental stress relevant to determining the boundaries for life as we know it. Specifically, archaeal enzyme structures have informed the architecture and mechanisms of key DNA repair proteins and complexes. With added abilities to temperature-trap flexible complexes and reveal core domains of transient and dynamic complexes, these structures provide insights into mechanisms of maintaining genome integrity despite extreme environmental stress. The DNA damage response protein structures noted in this review therefore inform the basis for genome integrity in the face of environmental stress, with implications for all domains of life as well as for biomanufacturing, astrobiology, and medicine

Characterisation of the MacA/MacB/TolC tripartite pump that confers resistance to macrolides in E. coli

Gram-negative bacteria possess tripartite pumps, composed of a membrane fusion protein (MFP), an inner membrane protein (IMP) and an outer membrane protein (OMP), to transport drugs across the inner and outer membranes. The plasmid encoding MacA, MacB and TolC confers resistance to the macrolide erythromycin in the host E. coli cell Kam3, indicating the three proteins are assembled and actively functional. MFPs are believed to have an important role in the stabilizing the pump complex; intriguingly, we found that the MFP MacA not only interacts directly with the IMP MacB and the OMP TolC, but regulates the function of MacB, apparently increasing its affinity for both ATP and erythromycin. As MacB hydrolyzes ATP there is a burst in phosphate production that is symptomatic of the reaction being rate- limited by product release; but the burst disappeared in the presence of MacA. Since MacA caused only a marginal increase in the k(_cat), but a significant decrease in the Km, for the steady-state ATPase activity, this suggests that the disappearance of the phosphate burst is due to a decrease in the rate of hydrolysis, rather than an increase in the rate of product release. This kinetic behaviour indicates that MacA promotes and stabilizes the ATP-binding form of the transporter. MacA regulates the activity of MacB via its ẞ-strand domain since S. aureus MacA, which lacks the coiled coil structure that is present in E. coli MacA and believed to be involved in the interaction with TolC, was able to abolish the Pi burst catalysed by MacB, in direct analogy with the effect of E. coli MacA on MacB. Analytical ultracentrifugation, mass spectrometry and atomic force microscopy indicated that MacB forms dimers, in analogy to ABC- transporters that require a pair of NBDs to bind ATP. Our data suggests a direct role for MacA in facilitating the delivery of drugs by MacB to TolC: by enhancing the binding of drugs to MacB and stabilizing the reorientation of MacB to the outward- facing conformation