NMR solution structures of the MloK1 cyclic nucleotide-gated ion channel binding domain

Zyklische Nukleotid-gesteuerte Ionenkanäle spielen eine entscheidende Rolle in der neuronalen Erregbarkeit und in der Signaltransduktion primärer Sinneszellen. Die Kanäle werden durch die Bindung zyklischer Nukleotide an eine intrazelluläre zyklische Nukleotid-Bindedomäne (CNBD) aktiviert. Die direkte Bindung zyklischer Nukleotide an die CNBD begünstigt die Öffnung des Kanals, vermutlich werden fortlaufende Konformationsänderungen beginnend von der CNBD bis zur Kanalpore übertragen. Der grundlegende Mechanismus der zur Aktivierung des Kanals führt ist weitgehend unbekannt. Um den Mechanismus der Kanalaktivierung verstehen zu können, sind Informationen über die Raumstruktur der CNBD im zyklischen-Nukleotid gebundenen und im freien Zustand nötig. Der K$^{+}$-selektive MloK1 Kanal ist ein Mitglied der zyklischen Nukleotid-gesteuerten Ionenkanäle und wurde in dem Bakterium $\textit{Mesorhizobium loti}$ entdeckt. Der MloK1 Kanal setzt sich aus vier gleichen Untereinheiten zusammen, die zusammen ein Tetramer bilden. Jede Untereinheit enthält sechs Transmembransegmente, eine charakteristische Sequenz für die Selektivität von Kaliumionen und eine C-terminale, intrazellulär vorliegende CNBD. In der vorliegenden Arbeit wurde die Raumstruktur der CNBD des MloK1 Kanals im Komplex mit cAMP mittels mehrdimensionaler NMR-Spektroskopie bestimmt. Die Lösungsstruktur des cAMP-gebundenen Proteins wurde mit einer Präzision von 0.025 nm und 0.068 nm r.m.s.- Abweichung der Proteinrückgrat- und aller Schweratom-Positionen in einer Konformerenschar bestimmt. Ähnlich zu bereits strukturell charakterisierten Bindedomänen für zyklische Nukleotide wie der Proteinkinase A (PKA), dem Katabolit-Aktivatorprotein (CAP) und dem Guaninnukleotid-Austauschfaktor Protein (Epac) besteht die Raumstruktur aus einer $\beta$ Faltblattrolle und einer kurzen internen $\alpha$ Helix. Letztere ist auch bekannt als Phosphatbindekassette (PBC). Über der $\beta$ Faltblattrolle positioniert befinden sich vier zusätzliche $\alpha$ Helices. Ein Teilbereich der $\beta$ Faltblattrolle sowie die Phosphatbindekassette bilden die Bindestelle für zyklische Nukleotide. Dieser Bereich zeigt direkte Wechselwirkungen zum Phosphat und dem Ribosering des zyklischen Nukleotids. Die C-terminale Helix ist über der cAMP-Bindestelle positioniert und stabilisiert diesen Komplex indem die Seitenkette von R348 über der Purinbase liegt und mit dieser interagiert. Bisher war es nicht gelungen die Struktur der wildtyp cAMP-freien CNBD zu lösen. Nach der Proteinexpression liegt die CNBD weitgehend im cAMP-gebundenen Zustand vor und cAMP kann selbst durch intensive Dialyse nicht vom Protein entfernt werden. Im Rahmen dieser Arbeit wurde daher ein Protokoll entwickelt, um cAMP-freies CNBD Protein in ausreichender Menge für NMR-spektroskopische Untersuchungen herzustellen. Der wesentliche Bestandteil des Protokolls ist ein intensiver Waschschritt des Matrix-gebundenen GST-CNBD Fusions-[...

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels.

Cyclic nucleotide-binding domains (CNBDs) that are present in various channel proteins play crucial roles in signal amplification cascades. Although atomic resolution structures of some of those CNBDs are available, the detailed mechanism by which they confer cyclic nucleotide-binding to the ion channel pore remains poorly understood. In this review, we describe structural insights about cyclic nucleotide-binding-induced conformational changes in CNBDs and their potential coupling with channel gating

Analysis of the Ion Channel Gating Mechanism in Solution by Nuclear Magnetic Resonance (NMR) Spectroscopy

Ion channels activated by cyclic nucleotides play crucial roles in signal transduction pathways. Upon binding of cyclic nucleotides to the intracellular cyclic nucleotide-binding domain (CNBD) of HCN or CNG channels (hyperpolarization-activated and cyclic nucleotide-gated channels or cyclic nucleotide-gated channels) an opening of the membrane pore occurs.To analyze the underlying gating mechanism highly resolved structures of the cyclic nucleotide-binding domains are necessary. Until now, structures of CNBDs from eukaryotic HCN channels as well as prokaryotic CNG channels are known. However, CNBD crystal structures of the HCN channels reveal no significant differences between apo and holo state1,2. In contrast, CNBD structures of the prokaryotic Mesorhizobium loti K1 channel, solved by liquid state NMR spectroscopy, show substantial rearrangements upon binding of a cyclic nucleotide3,4.Further elucidation of the gating mechanism will be done by structural analysis of an eukaryotic CNBD using liquid state NMR spectroscopy

Recombinant Production of the Amino Terminal Cytoplasmic Region of Dengue Virus Non-Structural Protein 4A for Structural Studies

BACKGROUND:Dengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes.METHODOLOGY/PRINCIPAL FINDINGS:This report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded.CONCLUSIONS/SIGNIFICANCE:Small molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets

Structure and membrane interaction of the N-terminal region of Dengue virus NS4A protein

Dengue virus (DENV) infection presents a serious public health threat with more than one third of the world population at risk. DENV is a mosquito-transmitted virus that causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. There is no vaccine available against DENV and no specific treatment for dengue fever. DENV is believed to replicate its RNA genome in association with modified intracellular membranes. However, the details of the assembly of this replication complex are incompletely understood. We focused on the DENV non-structural protein 4A (NS4A) which has been implicated in the formation of the viral RNA replication complex. Sequence analysis identified conserved regions in the N-terminal 48 amino acids of NS4A that might form amphipathic helices (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization [1]. However, little is known about the three dimensional structure of NS4A(1-48). We used solution state nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to study the structure of wild type NS4A(1-48) and of the double mutant NS4A(1-48, L6E;M10E) in the presence and absence of model membranes. The hydrodynamic radius of liposomes and detergent micelles was determined by dynamic light scattering (DLS). Both peptides were recombinantly produced in E.coli and are basically unstructured in aqueous buffer [2]. Addition of liposomes made of POPC or POPC/DOPS mixtures induced formation of α-helical secondary structure in case of the wild type NS4A(1-48) but not for the mutant peptide. The degree of helicity of wt NS4A(1-48) is sensitive to the lipid composition and to the size of the liposomes. Formation of α-helical secondary structure was observed for both wt and mutant NS4A(1-48) upon addition of various membrane mimicking detergent micelles (SDS, DPC, DHPC, DM). The degree of helix formation depends on the type and concentration of the detergent and reaches a maximum at about 100 mM SDS or DPC. Solution state NMR spectroscopy provided a detailed picture of the structure and micelle interaction for both peptides in presence of 100 mM SDS. Backbone resonance assignment followed by analysis of secondary chemical shifts allowed us to identify two α-helical segments in each peptide which cover amino acid residues 5-10 and 15-29 in NS4A(1-48) and residues 4-9 and 15-29 in NS4A(1-48, L6E;M10E). Analysis of paramagnetic relaxation enhancement after addition of paramagnetic Mn2+ to the SDS micelle-containing buffer allowed us to distinguish buffer exposed from buried amino acid residues.[1] O. Stern et al. (2013) J. Virol. 87:4080-85[2] Y.F. Hung et al. (2014) PLoS One. 9: e8648

Solution structure of the Mesorhizobium loti K1 channel cyclic nucleotide-binding domain in complex with cAMP

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, are crucial in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels is not well understood. Here, we report the solution structure of the isolated CNBD of a cyclic nucleotide-sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide anti-parallel β-roll topped by a helical bundle comprising five α-helices and a short 310-helix. In contrast to the dimeric arrangement (‘dimer-of-dimers') in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other

Dengue virus NS4A cytoplasmic domain binding to liposomes is sensitive to membrane curvature

Dengue virus (DENV) infection is a growing public health threat with more than one-third of the world's population at risk. Non-structural protein 4A (NS4A), one of the least characterized viral proteins, is a highly hydrophobic transmembrane protein thought to induce the membrane alterations that harbor the viral replication complex. The NS4A N-terminal (amino acids 1–48), has been proposed to contain an amphipathic α-helix (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization. Nuclear magnetic resonance (NMR) spectroscopy was used to characterize the N-terminal cytoplasmic region (amino acids 1–48) of both wild type and mutant NS4A in the presence of SDS micelles. Binding of the two N-terminal NS4A peptides to liposomes was studied as a function of membrane curvature and lipid composition. The NS4A N-terminal was found to contain two AHs separated by a non-helical linker. The abovementioned mutations did not significantly affect the helical secondary structure of this domain. However, they reduced the affinity of the N-terminal NS4A domain for lipid membranes. Binding of wild type NS4A(1–48) to liposomes is highly dependent on membrane curvature