Regulator of K+ conductance (RCK) domains control the activity of a variety of K+ channels and transporters, including the prokaryotic TrkA/H K+ transport complex and the eukaryotic BK channel, through binding of cytoplasmic ligands such as ATP, H+, and Ca2+. Thus RCK domains transduce ligand binding to gate transmembrane K+ flux in response to signaling events and cellular metabolism, in organisms ranging from bacteria to humans. In this work, I utilize the prokaryotic RCK domain containing K+ channel, MthK as a model system to provide insight toward the structural basis of ion channel gating by RCK domains. In MthK, binding of Ca2+ to an octameric ring of RCK domains (the gating ring) which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K+ conduction. Using electrophysiology and X-ray crystallography, I identify the presence of additional Ca2+ binding sites in the MthK RCK domain, showing that each RCK domain contributes to three different regulatory Ca2+ binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca2+ binding sites, resulting in a stoichiometry of 24 Ca2+ ions per channel, is consistent with the steep relation between [Ca2+] and MthK channel activity. Comparison of Ca2+ bound and unliganded RCK domains suggests a physical mechanism for Ca2+-dependent conformational changes that underlie gating in this class of channels. To gain insight toward mechanisms of RCK domain activation, I crystallized and solved the structure of the RCK domain of MthK bound with Ba2+. The Ba2+-bound RCK domain was assembled as an octomeric gating ring, as observed in structures of the full-length MthK channel, and shows Ba2+ bound at several positions, one of which overlaps with a known Ca2+ binding site. Functionally, I determined that Ba2+ could activate reconstituted MthK channels as observed in electrophysiological recordings. These results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating. In an effort to more accurately describe the Ca2+-dependent gating process in MthK, I crystallized and solved structures of mutant and wild-type RCK domains, and found that distinct Ca2+ activation sites near the N- and C-termini of the RCK domain (termed C1 and C3, respectively) are allosterically coupled to one another, to affect tuning of Ca2+ affinity and Ca2+-dependent channel activation. These results define a structural mechanism of allosteric modulation in a ligand-gated K+ channel, and provide a framework for understanding similar mechanisms in related RCK-containing channels and transporters. / Biochemistry

Diese Arbeit beschäftigt sich mit der cytoplasmatischen Untereinheit KtrA des K - Transportsystems KtrAB aus V. alginolyticus. KtrA gehört zur Familie der KTN/RCK Proteine. Ligandenbindung an entsprechende Proteine oder Proteindomänen ist direkt mit der Regulation entsprechender Transportsysteme verbunden. Im Verlauf dieser Untersuchung wurde KtrA mittels Affinitätschromatographie aufgereinigt und anstelle der erwarteten NAD(H)-Bindung, da die Aminosäuresequenz von KtrA den konservierten Rossman fold aufweist, eine ATP-Bindung festgestellt. Die Bindung von ATP führt zu einer Konformationsänderung des Proteins. Mittels Flowdialyse konnten Dissoziationskonstanten sowohl für ATP, als auch für andere Nukleotide ermittelt werden. Ebenfalls wurde gezeigt, dass ATP die Assemblierung des KtrAB-Komplexes unterstützt, sowohl unter denaturierenden, als auch unter nativen Bedingungen. Der Bedarf an ATP konnte letztlich durch Energiekopplungsexperimente in vivo bestätigt werden.

L’interaction avec les cellules hôtes est une étape primordiale du cycle infectieux des Salmonella. Elle implique entre autres des fimbriae permettant l’attachement des Salmonella aux cellules et des invasines permettant leur internalisation. Généralement, l’expression de ces facteurs est réprimée et ils ne sont exprimés qu’in vivo en des sites bien spécifiques. Notre objectif a été d’étudier les mécanismes de répression de l’expression des fimbriae Pef et de l’invasine Rck. Nos résultats montrent que les nucléoprotéines H-NS, Hha et YdgT régulent négativement l’expression de ces deux facteurs de virulence. Le mécanisme de répression de la transcription de l’opéron pef a été caractérisé et montre un rôle prépondérant d’H-NS. De plus, la régulation de cet opéron par Hha et YdgT semble être dépendante ou indépendante de H-NS en fonction des conditions de culture de la bactérie. La répression de l’opéron pefI-srgC, portant l’ORF rck, semble, quant à elle, plus complexe. A l’heure actuelle, seule la région cible de la répression par Hha et YdgT a pu être identifiée. Ces résultats renforcent l’hypothèse de l’existence de régulations par Hha-YdgT indépendantes de H-NS. / The interaction with host cells is an essential step of Salmonella infection cycle. Among other virulence factors, fimbriae allow attachment of Salmonella to eukaryotic cells and invasins enable cell internalisation. Generally, the expression of these factors is suppressed and they are only expressed in vivo at very specific locations. Our objective was to study the mechanisms of Pef fimbriae and Rck invasin repression. Our results show that the nucleoproteins H-NS, Hha and YdgT negatively regulate the expression of these two virulence factors. The mechanism of pef operon transcription repression was characterized and shows a predominant role of H-NS. Moreover, the regulation of this operon by Hha and YdgT appears to bedependent or independent of H-NS depending on bacterial culture conditions. Repression of the pefI-srgC operon, carrying rck ORF, shows a higher degree of complexity. Currently, only the Hha and YdgT targeted region for the repression has been identified. These results reinforce the hypothesis that Hha-YdgT can act independently of H-NS.