Plectin-vimentin interaction

Das Cytolinkerprotein Plectin spielt bei der Aufrechterhaltung der Integrität des Zytoskeletts eine entscheidende Rolle, indem es Intermediärfilamente (IFs) mit anderen zytoskelettären Netzwerksystemen verbindet und diese an der Plasmamembran verankert. Weiters dient es als strukturelles Grundgerüst für Signalkaskaden und dürfte ebenfalls eine Funktion in der Netzwerkanordnung und –dynamik haben. Das Plectinmolekül mit seinem hohen Molekulargewicht (>500,000), besitzt eine Drei-Domänen-Organisation, wobei eine zentrale α-helikale Stabdomäne von zwei terminalen globulären Domänen begrenzt wird. Indem es eine einzige Phosphorylierungsstelle für die Proteinkinase Cdk1 and Bindungsstellen für verschiedene IF-Proteine und eine Vielfalt an Proteinen, die im „Signaling“ involviert sind, besitzt, ist die C-terminale Domäne von Plectin (bestehend aus den sechs strukturellen Wiederholungen - R1-6) von strategisch wichtiger Bedeutung. Abhängig von der Spezies, gibt es zumindest 13 Cysteine in der C-terminalen Domäne von Plectin, vier davon befinden sich in der Repeatdomäne R5. Cysteine könnten einen wichtigen Beitrag zur Stabilisierung der Konformation von Proteinen leisten. Der erste Teil der Arbeit ist auf die Aufreinigung und Kristallisation von Pectinfragmenten, in denen die IF-Bindungsstelle lokalisiert ist, fokussiert. Alle rekombinanten Proteine wurden durch mindestens einen säulenchromatographischen Schritt gereinigt und die Homogenität der Proben durch Größenausschluss-Chromatographie bestätigt. Umfangreiches Kristallisationsscreening zeigte, dass nur die cysteinfreie Version des Plectin R5 und ein Fragment, das die Repeat-Domäne R4-5 enthielt, in der Lage waren, Kristalle zu bilden. Aber auch in diesen Fällen traten nur kleine stabförmige oder sternförmig angeordnete nadelige Kristalle auf. Weder die Optimierung der Kristallisationsbedingungen noch Großansätze (Macroseeding) führten zu Kristallen mit Dimensionen, die für die Röntgenbeugungsanalysen geeignet waren. Im zweiten Teil der Arbeit untersuchte ich strukturelle und biologische Funktionen der in R5 enthaltenen Cysteine und die Auswirkungen ihrer Nitrosylierung auf Plectins Vimentinbindung und den Kollaps von IFs. Mit Hilfe von Cystein-Mutagenese (zu Serinen) zeigte ich, dass vier der in der R5 Repeat-Domäne enthaltenen Cysteine intra- und intermolekulare Disulfidbrücken bilden konnten. Darüber hinaus, konnte gezeigt werden, dass das einzige in Vimentin enthaltene Cystein mit den R5-Cysteinen Disulfidbindungen eingehen können. Dennoch war die Vimentinbindung signifikant effektiver wenn das R5-Fragment in reduzierter Form vorlag. Von den vier Cysteinen in R5 wurde nur eines (Cys4) gefunden, das besonders reaktiv hinsichtlich Disulfidbrückenbildung war und auch in vitro nitrosiliert werden konnte. Unter Verwendung immortalisierter Endothelzellen konnte ich zeigen, dass Plectin auch in vivo S-nitrosiliert wird, und außerdem zeigte sich, dass Stickoxid (NO)-Donor-induzierter IF-Netzwerkzerfall in Plectin-defizienten Zellen dramatisch schneller ablief als in Wildtyp-Zellen. Zusätzlich beobachtete ich, dass sich Aktin-Stressfasern durch NO-Donor-Behandlung im Zentrum der Zellen ansammelten. Die Messung des von Endothelzellen nach endothelialer Stickoxidsynthase (eNOS)-Stimulation freigesetzte NO ergab, dass die NO-Produktion in Plectin-defizienten im Vergleich zu Wildtyp-Zellen drastisch reduziert war. Die NO-Freisetzung korrelierte mit der Menge und dem Aktivierungsstatus von eNOS. Auch die Verteilung der eNOS entsprach ihrem Aktivierungzustand, wobei sie in Plectin-defizienten Zellen an der Zellperipherie lokalisiert (inaktive Form) war, während in Wildtyp-Zellen eine diffuse Verteilung (aktive Form) zu finden war. Im dritten Teil meiner Arbeit untersuchte ich die Auswirkungen der Phosphorylierung von Plectin auf dessen Vimentinbindung, IF-Netzwerkbildung und Filamentdynamik. In Bindungsstudien fand ich heraus, dass die Phosphorylierung von Vimentin und Plectin durch die Mitose-spezifische Kinase Cdk1 deren Interaktion beeinflusste. Im speziellen führte phosphoryliertesVimentin, in Gegenwart der phosphorylierten Bindungsdomäne Plectin R4-5, zur Bildung globulärer Strukturen unterschiedlicher Größe. Sehr ähnliche Strukturen, vorwiegend in Form von Granula und kurzen Filamenten, wurden in postmitotischen Fibroblasten und in Zellen unmittelbar nach der Trypsinierung beobachtet. Ich konnte zeigen, dass die Bildung dieser Vimentin-Filament-Zwischenstufen von Plectin abhängig war, da sie nur in Plectin-positiven Zellen zu beobachten war. Zusätzlich beobachtete ich in mitotischen Zellen multipolare Spindeln in Plectin-defizienten, jedoch nicht in Wildtyp-Zellen. Während der Großteil der Wildtyp-Zellen nach der Cytokinese eine ungleiche Verteilung des Vimentin-Netzwerks auf die Tochterzellen zeigte, wurde eine wesentlich gleichmäßigere Verteilung in Plectin-knockout Zellen beobachtet. Darüber hinaus zeigte sich, dass die Mitose in Plectin-knockout Fibroblasten schneller als in Wildtyp-Zellen ablief. Die in meiner Arbeit präsentierten Ergebnisse deuten darauf hin, dass Plectin nicht nur ein bedeutendes Organisationselement der IF-Netzwerk-Cytoarchitektur darstellt, sondern auch eine wichtige Rolle bei dynamischen Prozessen des IF-Netzwerks spielt.The cytolinker protein plectin plays a crucial role in maintaining the integrity of the cytoskeleton by interlinking intermediate filaments (IFs) with other cytoskeletal network systems, and anchoring them to the plasma membrane. It also serves as a scaffolding platform for signaling cascades, and may well have also a function in IF network assembly and dynamics. The plectin molecule, with its high molecular weight (>500,000), has a three-domain organization, where a central α-helical rod domain is flanked by two terminal globular domains. Harboring a unique phosphorylation site for protein kinase Cdk1 and binding sites for different IF proteins and for a variety of proteins involved in signaling, plectin’s C-terminal domain consisting of six structural repeats (R1-6) is of strategic functional importance. Depending on the species, there are at least 13 cysteines in plectin's C-terminal domain, 4 of which reside in the repeat domain 5 (R5). Cysteines may play an important role in stabilizing the protein structure and conformation. The first part of the thesis was focused on the purification and crystallization of the plectin fragments harboring the IF-binding site. All recombinant proteins were purified by at least one purification step and homogeneity of the proteins used for crystallization was confirmed by size exclusion chromatography. Extensive crystallization screening revealed that only a cysteine-free version of plectin R5 and a fragment corresponding to repeat domains R4-5 were able to form crystals. However, only small rod-shaped or clustered needle-shaped crystals occurred. Neither optimizing crystallization conditions nor macroseeding led to crystals with dimensions appropriate for collecting X-ray diffraction data. In the second part, I investigated the structural and biological functions of R5 cysteines and the effects of plectin nitrosylation on vimentin-binding and involvement in IF collapse using biochemical and functional analyses. Performing cysteine to serine mutagenesis and biochemical analyses I showed that the four cysteines of R5 can form intra- and intermolecular disulfide bridges. In addition it could be shown that the single cysteine in vimentin as well as the cysteines in R5 formed disulfide bonds between each other. However, vimentin-binding was significantly more efficient when R5 was in its reduced form, probably reflecting distinct conformations of the reduced and the nonreduced forms. Out of the four cysteines in R5 only one (Cys4) was found to be particularly reactive with respect to disulfide bridges formation ability and serving as a target for nitrosylation in vitro. Using immortalized endothelial cells, I could show that endogenous plectin is the target of S-nitrosylation in vivo and I found that NO donor-induced IF collapse proceeded dramatically faster in plectin-deficient compared to wild-type cells. Additionally, I observed that actin stress fibers accumulated in the center of the cells upon NO donor treatment. By measuring the amount of NO released from endothelial cells upon eNOS stimulation, I found that NO production was dramatically reduced in plectin-deficient compared to wild-type cells. NO release correlated with the expression level of eNOS and its activation status. Also the distribution of eNOS corresponded with its activation in both cell types, as it was localized at the cell periphery in plectin-deficient cells (inactive form) and diffusely distributed in wild-type cells (active form). In a third part of my thesis I studied the effects of plectin phosphorylation on vimentin-binding and on IF network formation and dynamics. Using an in vitro binding assay I found that vimentin and plectin phosphorylation by Cdk1 (a typical mitotic event) influenced the interaction of both proteins. In particular, phosphorylated vimentin in the presence of phosphorylated plectin R5-6 led to the formation of globule-like structures of various sizes. Very similar structures, mainly in the form of granules and squiggles were observed in newly spreading postmitotic fibroblasts and in cells after trypsinization/replating. I could show that the formation of these vimentin intermediates were plectin dependent, as they showed association with plectin and were not observed in the absence of plectin. In addition in mitotic cells I observed multipolar spindles in plectin-deficient contrary to wild-type cells. Moreover, while the majority of wild-type cells undergoing cytokinesis showed an uneven distribution of the vimentin network to their daughter cells, a much more even distribution was observed in plectin knockout cells. Also I found that mitosis progressed faster in plectin knockout compared to wild-type fibroblasts. The data presented in my thesis suggest that plectin is not only a major organizing element of the IF network cytoarchitecture, but also has an important function in IF network assembly and dynamics

Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABAA/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels

Family of prokaryote cyclic nucleotide-modulated ion channels

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.status: publishe

Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site

Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site

Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.status: publishe

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8-β9 loop in the extracellular ligand-binding domain. The β8-β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8-β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.status: publishe