Inhibited KdpFABC transitions into an E1 off-cycle state

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC state that we termed E1P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1P states to E2P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such states

Analyse der Struktur von RNA und DNA mittels ESR Spektroskopie

Natural occurring riboswitches, a class of RNA molecules, are able to control the implementation of genetic information through the regulation of gene expression. Both, natural and synthetic riboswitches, so-called aptamers, are complex folded structures, which bind specific ligands. Ligand binding in turn regulates gene expression. In the first section of this work continuous wave (cw) and pulse EPR spectroscopy in combination with site-directed spin labeling was performed to investigate the dynamics and conformational changes of the synthetic tetracycline (Tc) riboswitch. The results obtained herein indicate a thermodynamic equilibrium of two aptamer conformations in the absence of Tc, where one of these conformations is captured upon ligand binding. Aptamer structures have been modeled based on the two equilibrium conformations in the absence of the ligand, and on the captured aptamer conformation in the presence of Tc. All RNA models have been verified by MD simulations by comparing experimentally obtained interspin distances with simulated ones. The incorporation of DNA mutations such as nucleobase changes, double-strand breaks and mispairings can lead to structural and conformational changes of DNA domains which in turn are related to inheritable diseases, cancer and aging. Within this project, the copper(I)-catalyzed Huisgen-Sharpless-Meldal alkyne-azide cycloaddition (CuAAC) ‘click reaction’ was introduced as a powerful modification of existing spin labeling strategies. Interspin distances and exceptionally narrow distribution widths were determined by cw and pulse EPR experiments. The results of the MD simulations exhibit a very good agreement between simulated and experimentally obtained distances. Furthermore, the new spin labeling protocol was used to identify mismatch-induced conformational changes of spin labeled DNA. The application of cw and orientation selective pulse EPR measurements provided insights into the dynamics and structural alterations caused by mismatched base pairs. Interspin distances have been found to depend on the type and nearest neighbor environment of the mismatch. The changes are transferred to the base pairs carrying the spin labels

The synergetic effects of combining structural biology and epr spectroscopy on membrane proteins

Protein structures as provided by structural biology such as X-ray crystallography, cryo-electron microscopy and NMR spectroscopy are key elements to understand the function of a protein on the molecular level. Nonetheless, they might be error-prone due to crystallization artifacts or, in particular in case of membrane-imbedded proteins, a mostly artificial environment. In this review, we will introduce different EPR spectroscopy methods as powerful tools to complement and validate structural data gaining insights in the dynamics of proteins and protein complexes such that functional cycles can be derived. We will highlight the use of EPR spectroscopy on membrane-embedded proteins and protein complexes ranging from receptors to secondary active transporters as structural information is still limited in this field and the lipid environment is a particular challenge

Low affinity and slow Na+-binding precedes high affinity aspartate binding in GltPh

GltPh from Pyrococcus horikoshii is a homotrimeric Na+-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters (EAATs) that take up the neurotransmitter glutamate. Each protomer in GltPh consists of a trimerization domain involved in subunit interactions, and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na+ and aspartate binding to GltPh. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na+ binds with low affinity to the apo-protein (Kd 120 mM), with a particularly low kon value (5 M-1s-1). At least two Na+ ions bind prior to aspartate. The binding of Na+ requires very high activation energy (Ea 106.8 kJmol-1) and consequently has a large Q10 value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na+ concentration present. Binding of aspartate was not observed in the absence of Na+, whereas in the presence of high Na+ concentrations (above the Kd for Na+) the dissociation constants for aspartate were in the nanomolar range and the aspartate binding was fast (kon of 1.4*105 M-1s-1), with low Ea and Q10 values (42.6 KJmol-1 and 1.8, respectively). We conclude that Na+ binding is most likely the rate-limiting step for substrate binding

Regulation of lipid saturation without sensing membrane fluidity

Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. Here, we have reconstituted the core machinery for sensing and regulating lipid saturation in baker’s yeast to directly characterize its response to defined membrane environments. Using spectroscopic techniques and in vitro ubiquitylation, we uncover a unique sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains and provide unprecedented insight into the molecular rules of membrane adaptivity. Our data challenge the prevailing hypothesis that membrane viscosity serves as the measured variable for regulating lipid saturation. Rather, we show that the signaling output of Mga2 correlates with the size of a single sensor residue in the transmembrane helix, which senses the lateral pressure and/or compressibility profile in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such membrane properties in the future

Site-Directed Spin Labeling of DNA Reveals Mismatch-Induced Nanometer Distance Changes between Flanking Nucleotides

Multiple forms of DNA damages such as base modifications, double-strand breaks, and mispairings are related to inheritable diseases, cancer, and aging. Here, the structural changes of duplex DNA upon incorporation of mismatched base pairs are examined by EPR spectroscopy. Two ethynyl-7-deaza-2′-deoxyadenosine residues separated by two nucleotides were incorporated in DNA and functionalized with 4-azido-2,2,6,6-tetramethyl-piperidine-1-oxyl (4-azido TEMPO) by the click reaction. Mismatches such as dT·dT or dA·dA mispairs were positioned between these two spin labels in DNA duplexes. Pulse EPR experiments reveal that the mismatch-induced local conformational changes are transmitted to the flanking nucleotides and that the impact of this mismatch depends on the nearest neighbor environment

KtrB, a member of the superfamily of K+ transporters

KtrB is the K+-translocating subunit of the K+-uptake system KtrAB from bacteria. It is a member of the (s) under bar uperfamily of (K) under bar (+) (t) under bar ransporters (SKT proteins) with other sub-families occurring in archaea, bacteria, fungi, plants and trypanosomes. SKT proteins may have originated from small K+ channels by at least two gene duplication and two gene fusion events. They contain four covalently linked M1PM2 domains, in which M-1 and M-2 stand for transmembrane stretches, and P for a P-loop, which folds back from the external medium into the membrane. SKT proteins distinguish themselves in two important aspects from K+ channels: first, with just one conserved glycine residue in their P-loops they contain a much simpler K+-selectivity filter sequence than K+ channels with their conserved Thr-Val-Gly-Tyr-Gly sequence. Secondly, the middle part M-2C2 from the long transmembrane stretch M-2C of KtrB from the bacterium Vibrio alginolyticus forms a gate inside the membrane, which prevents K+ permeation to the cytoplasm. Beside the mechanism of K+ transport via KtrB and other SKT proteins existing hypotheses of how the KtrA protein regulates the K+-transport activity of KtrB are discussed. (C) 2011 Elsevier GmbH. All rights reserved

DNA with Parallel Strand Orientation: A Nanometer Distance Study with Spin Labels in the Watson–Crick and the Reverse Watson–Crick Double Helix

Parallel-stranded (ps) DNA characterized by its sugar–phosphate backbones pointing in the same direction represents an alternative pairing system to antiparallel-stranded (aps) DNA with the potential to inhibit transcription and translation. 25-mer oligonucleotides were selected containing only dA·dT base pairs to compare spin-labeled nucleobase distances over a range of 10 or 15 base pairs in ps DNA with those in aps DNA. By means of the copper­(I)-catalyzed Huisgen–Meldal–Sharpless alkyne–azide cycloaddition, the spin label 4-azido-2,2,6,6-tetramethylpiperidine-1-oxyl was clicked to 7-ethynyl-7-deaza-2′-deoxyadenosine or 5-ethynyl-2′-deoxyuridine to yield 25-mer oligonucleotides incorporating two spin labels. The interspin distances between spin labeled residues were determined by pulse EPR spectroscopy. The results reveal that in ps DNA these distances are between 5 and 10% longer than in aps DNA when the labeled DNA segment is located near the center of the double helix. The interspin distance in ps DNA becomes shorter compared with aps DNA when one of the spin labels occupies a position near the end of the double helix

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

RNA aptamers are in vitro–selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2′-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding