Published by De Gruyter

Volume 396 Issue 9-10

Issue of Biological Chemistry

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Contents
Journal Overview

Publicly Available August 6, 2015

Meeting Report

August 4, 2015

Membrane Transport and Communication in Frankfurt: Speakers’ Summary – Highlights

Robert Tampé

Page range: 949-954

HIGHLIGHT: MEMBRANE TRANSPORT AND COMMUNICATION

Publicly Available February 24, 2015

Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters

Michael Jaehme, Dirk Jan Slotboom

Page range: 955-966

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Abstract

Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications.

February 25, 2015

Team work at its best – TAPL and its two domains

Tina Zollmann, Christoph Bock, Philipp Graab, Rupert Abele

Page range: 967-974

Abstract

The transporter associated with antigen processing (TAPL, ABCB9) is a homodimeric ABC transporter, shuttling cytosolic polypeptides into the lumen of lysosomes energized by ATP hydrolysis. Here we give a short overview of the superfamily of ABC transporters and summarize the current state of knowledge on TAPL in detail. The architecture of TAPL and its substrate specificity are described and we discuss the function of an extra N-terminal transmembrane domain, called TMD0, in respect of subcellular targeting and interaction with proteins, contributing to long-term stability. As TAPL shows – besides a ubiquitous basal expression – an elevated expression in antigen presenting cells, we present models of TAPL function in adaptive immunity.

April 14, 2015

The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology

Tobias Stauber

Page range: 975-990

Abstract

Cellular volume regulation is fundamental for numerous physiological processes. The volume-regulated anion channel, VRAC, plays a crucial role in regulatory volume decrease. This channel, which is ubiquitously expressed in vertebrates, has been vastly characterized by electrophysiological means. It opens upon cell swelling and conducts chloride and arguably organic osmolytes. VRAC has been proposed to be critically involved in various cellular and organismal functions, including cell proliferation and migration, apoptosis, transepithelial transport, swelling-induced exocytosis and intercellular communication. It may also play a role in pathological states like cancer and ischemia. Despite many efforts, the molecular identity of VRAC had remained elusive for decades, until the recent discovery of heteromers of LRRC8A with other LRRC8 family members as an essential VRAC component. This identification marks a starting point for studies on the structure-function relation, for molecular biological investigations of its cell biology and for re-evaluating the physiological roles of VRAC. This review recapitulates the identification of LRRC8 heteromers as VRAC components, depicts the similarities between LRRC8 proteins and pannexins, and discussed whether VRAC conducts larger osmolytes. Furthermore, proposed physiological functions of VRAC and the present knowledge about the physiological significance of LRRC8 proteins are summarized and collated.

July 14, 2015

Extending native mass spectrometry approaches to integral membrane proteins

Albert Konijnenberg, Jeroen F. van Dyck, Lyn L. Kailing, Frank Sobott

Page range: 991-1002

Abstract

Recent developments in native mass spectrometry and ion mobility have made it possible to analyze the composition and structure of membrane protein complexes in the gas-phase. In this short review we discuss the experimental strategies that allow to elucidate aspects of the dynamic structure of these important drug targets, such as the structural effects of lipid binding or detection of co-populated conformational and assembly states during gating on an ion channel. As native mass spectrometry relies on nano-electrospray of natively reconstituted proteins, a number of commonly used lipid- and detergent-based reconstitution systems have been evaluated for their compatibility with this approach, and parameters for the release of intact, native-like folded membrane proteins studied in the gas-phase. The strategy thus developed can be employed for the investigation of the subunit composition and stoichiometry, oligomeric state, conformational changes, and lipid and drug binding of integral membrane proteins.

April 1, 2015

Functional diversity of the superfamily of K⁺ transporters to meet various requirements

Marina Diskowski, Vedrana Mikusevic, Charlott Stock, Inga Hänelt

Page range: 1003-1014

Abstract

The superfamily of K + transporters unites proteins from plants, fungi, bacteria, and archaea that translocate K + and/or Na + across membranes. These proteins are key components in osmotic regulation, pH homeostasis, and resistance to high salinity and dryness. The members of the superfamily are closely related to K + channels such as KcsA but also show several striking differences that are attributed to their altered functions. This review highlights these functional differences, focusing on the bacterial superfamily members KtrB, TrkH, and KdpA. The functional variations within the family and comparison to MPM-type K + channels are discussed in light of the recently solved structures of the Ktr and Trk systems.

July 4, 2015

The structure of Na⁺-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na⁺ transport

Julia Steuber, Georg Vohl, Valentin Muras, Charlotte Toulouse, Björn Claußen, Thomas Vorburger, Günter Fritz

Page range: 1015-1030

Abstract

The Na + -translocating NADH:ubiquinone oxidoreductase (Na + -NQR) of Vibrio cholerae is a respiratory complex that couples the exergonic oxidation of NADH to the transport of Na + across the cytoplasmic membrane. It is composed of six different subunits, NqrA, NqrB, NqrC, NqrD, NqrE, and NqrF, which harbor FAD, FMN, riboflavin, quinone, and two FeS centers as redox co-factors. We recently determined the X-ray structure of the entire Na + -NQR complex at 3.5-Å resolution and complemented the analysis by high-resolution structures of NqrA, NqrC, and NqrF. The position of flavin and FeS co-factors both at the cytoplasmic and the periplasmic side revealed an electron transfer pathway from cytoplasmic subunit NqrF across the membrane to the periplasmic NqrC, and via NqrB back to the quinone reduction site on cytoplasmic NqrA. A so far unknown Fe site located in the midst of membrane-embedded subunits NqrD and NqrE shuttles the electrons over the membrane. Some distances observed between redox centers appear to be too large for effective electron transfer and require conformational changes that are most likely involved in Na + transport. Based on the structure, we propose a mechanism where redox induced conformational changes critically couple electron transfer to Na + translocation from the cytoplasm to the periplasm through a channel in subunit NqrB.

April 1, 2015

Hybrid rotors in F₁F_o ATP synthases: subunit composition, distribution, and physiological significance

Karsten Brandt, Volker Müller

Page range: 1031-1042

Abstract

The c ring of the Na + F 1 F o ATP synthase from the anaerobic acetogenic bacterium Acetobacterium woodii is encoded by three different genes: atpE 1 , atpE 2 and atpE 3 . Subunit c 1 is similar to typical V-type c subunits and has four transmembrane helices with one ion binding site. Subunit c 2 and c 3 are identical at the amino acid level and are typical F-type c subunits with one ion binding site in two transmembrane helices. All three constitute a hybrid F o V o c ring, the first found in nature. To analyze whether other species may have similar hybrid rotors, we searched every genome sequence publicly available as of 23 February 2015 for F 1 F o ATPase operons that have more than one gene encoding the c subunit. This revealed no other species that has three different c subunit encoding genes but twelve species that encode one F o - and one V o -type c subunit in one operon. Their c subunits have the conserved binding motif for Na + . The organisms are all anaerobic. The advantage of hybrid c rings for the organisms in their environments is discussed.

April 3, 2015

Homeostatic control of biological membranes by dedicated lipid and membrane packing sensors

Kristina Puth, Harald F. Hofbauer, James P. Sáenz, Robert Ernst

Page range: 1043-1058

Abstract

Biological membranes are dynamic and complex assemblies of lipids and proteins. Eukaryotic lipidomes encompass hundreds of distinct lipid species and we have only begun to understand their role and function. This review focuses on recent advances in the field of lipid sensors and discusses methodical approaches to identify and characterize putative sensor domains. We elaborate on the role of integral and conditionally membrane-associated sensor proteins, their molecular mechanisms, and identify open questions in the emerging field of membrane homeostasis.

Publicly Available March 10, 2015

The transporter associated with antigen processing: a key player in adaptive immunity

Sabine Eggensperger, Robert Tampé

Page range: 1059-1072

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Abstract

The adaptive immune system co-evolved with sophisticated pathways of antigen processing for efficient clearance of viral infections and malignant transformation. Antigenic peptides are primarily generated by proteasomal degradation and translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, peptides are loaded onto major histocompatibility complex I (MHC I) molecules orchestrated by a multisubunit peptide-loading complex (PLC). Peptide-MHC I complexes are targeted to the cell surface for antigen presentation to cytotoxic T cells, which eventually leads to the elimination of virally infected or malignantly transformed cells. Here, we review MHC I mediated antigen processing with a primary focus on the function and structural organization of the heterodimeric ATP-binding cassette (ABC) transporter TAP1/2. We discuss recent data on the molecular transport mechanism of the antigen translocation complex with respect to structural and biochemical information of other ABC exporters. We further summarize how TAP provides a scaffold for the assembly of the macromolecular PLC, thereby coupling peptide translocation with MHC I loading. TAP inhibition by distinct viral evasins highlights the important role of TAP in adaptive immunity.

March 23, 2015

The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

Dijun Du, Jarrod Voss, Zhao Wang, Wah Chiu, Ben F. Luisi

Page range: 1073-1082

Abstract

Microorganisms encode several classes of transmembrane molecular pumps that can expel a wide range of chemically distinct toxic substances. These machines contribute to the capacity of the organisms to withstand harsh environments, and they help to confer resistance against clinical antimicrobial agents. In Gram-negative bacteria, some of the pumps comprise tripartite assemblies that actively transport drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the architecture and transport mechanism of the AcrA-AcrB-TolC pump of Escherichia coli . This multidrug efflux pump is powered by proton electrochemical gradients through the activity of AcrB, a member of the resistance/nodulation/cell division (RND) transporter family. Crystallographic data reveal how the small protein AcrZ binds to AcrB in a concave surface of the transmembrane domain, and we discuss how this interaction may affect the efflux activities of the transporter.

June 9, 2015

The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump

Reinke Tobias Müller, Klaas Martinus Pos

Page range: 1083-1089

Abstract

In Gram-negative bacteria, tripartite efflux pumps, like AcrAB-TolC from Escherichia coli , play a prominent role in the resistance against multiple antibiotics. Transport of the drugs across the outer membrane and its coupling to the electrochemical gradient is dependent on the presence of all three components. As the activity of the E. coli AcrAB-TolC efflux pump is dependent on both the concentration of substrates and the extent of the electrochemical gradient across the inner membrane, the dynamics of tripartite pump assembly and disassembly might be crucial for effective net transport of drugs towards the outside of the cell.

January 28, 2015

A universal mechanism for transport and regulation of CPA sodium proton exchangers

Octavian Călinescu, Klaus Fendler

Page range: 1091-1096

Abstract

Recent studies performed on a series of Na + /H + exchangers have led us to postulate a general mechanism for Na + /H + exchange in the monovalent cation/proton antiporter superfamily. This simple mechanism employs a single binding site for which both substrates compete. The developed kinetic model is self-regulatory, ensuring down-regulation of transport activity at extreme pH, and elegantly explains the pH-dependent activity of Na + /H + exchangers. The mechanism was experimentally verified and shown to describe both electrogenic and electroneutral exchangers. Using a small number of parameters, exchanger activity can be modeled under different conditions, providing insights into the physiological role of Na + /H + exchangers.

May 22, 2015

Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing

Helmut Merk, Ralf-Bernhardt Rues, Christine Gless, Kerstin Beyer, Fang Dong, Volker Dötsch, Michael Gerrits, Frank Bernhard

Page range: 1097-1107

Abstract

G protein-coupled receptors, like many other membrane proteins, are notoriously difficult to synthesize in conventional cellular systems. Although expression in insect cells is considered the preferred technique for structural characterizations in particular, inefficient membrane translocation, instability, toxic effects and low yields still pose clear limitations for their production in living cells. Recent studies started to explore alternative strategies for the in vitro production of problematic membrane proteins in cell-free lysates in combination with supplied membranes. We provide a detailed study on the production efficiencies and quality of G protein-coupled receptors, Fab fragments and other proteins synthesized in insect cell lysates containing endogenous microsomes. Effects of different reaction kinetics, redox conditions and sample preparations on the specific activities of synthesized proteins have been analyzed. The extent of glycosylation, membrane translocation and percentages of ligand binding active fractions of synthesized protein samples have been determined. We provide strong evidence that membrane insertion of integral membrane proteins can represent a prime limiting factor for their preparative scale in vitro production. Improved expression protocols resulting into higher production rates yielded more active protein in case of Fab fragments, but not in case of the human endothelin B receptor.

June 16, 2015

Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin

Elena Bühl, Markus Braun, Andrea Lakatos, Clemens Glaubitz, Josef Wachtveitl

Page range: 1109-1115

Abstract

The UV light absorbing species of proteorhodopsin with deprotonated Schiff base retinal was investigated using steady-state fluorescence and femtosecond pump-probe spectroscopy. Compared to the all- trans retinal with protonated Schiff base, the deprotonated chromophore absorbs at 365 nm and exhibits a blue-shifted fluorescence spectrum. The unusually long-lived excited state decays bi-exponentially with time constants of 8 ps and 130 ps to form a deprotonated 13- cis retinal as the primary photo-product.

July 4, 2015

Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum

Izabela Waclawska, Christine Ziegler

Page range: 1117-1126

Abstract

The trimeric transporter BetP counteracts hyperosmotic stress by a fast increase in transport rate in order to accumulate the compatible solute betaine. The positively charged α-helical C-terminal domain acts as an osmosensor perceiving the increase in the internal potassium (K + ) concentration. A second, still unidentified stimulus originates from stress-induced changes in the physical state of the membrane and depends on the amount of negatively charged lipids. BetP possesses a 60-amino acid (aa)-long negatively charged N-terminal domain, which is predicted to adopt a partly helical fold affecting osmoregulation by an unknown mechanism. It is assumed that the C-terminal domain, the N-terminal domain, and negatively charged lipids interact during stress sensing and regulation. Here, we have investigated the regulatory role of negatively charged clusters in the N-terminal domain. We identified one cluster, Glu24Glu25, to be crucial for osmoregulation. Cross-linking studies revealed an interaction between the C- and N-terminal domains of adjacent protomers modulating transport activation. A regulatory partner-switching mechanism emerges in which the C-terminal domain changes its interaction with the N-terminal domain of its own promoter and negatively charged lipids to an interaction with the N-terminal domain of an adjacent protomer and lipids bound to the central cavity of the BetP trimer.

March 23, 2015

The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex

Phong T. Nguyen, Qi Wen Li, Neena S. Kadaba, Jeffrey Y. Lai, Janet G. Yang, Douglas C. Rees

Page range: 1127-1134

Abstract

Despite the ubiquitous role of ATP-binding cassette (ABC) importers in nutrient uptake, only the Escherichia coli maltose and vitamin B 12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321 , 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 n m . Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ∼40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system.

April 3, 2015

The ABC exporter MsbA probed by solid state NMR – challenges and opportunities

Hundeep Kaur, Andrea Lakatos, Roberta Spadaccini, Ramona Vogel, Christian Hoffmann, Johanna Becker-Baldus, Olivier Ouari, Paul Tordo, Hassane Mchaourab, Clemens Glaubitz

Page range: 1135-1149

Abstract

ATP binding cassette (ABC) transporters form a superfamily of integral membrane proteins involved in translocation of substrates across the membrane driven by ATP hydrolysis. Despite available crystal structures and extensive biochemical data, many open questions regarding their transport mechanisms remain. Therefore, there is a need to explore spectroscopic techniques such as solid state NMR in order to bridge the gap between structural and mechanistic data. In this study, we investigate the feasibility of using Escherichia coli MsbA as a model ABC transporter for solid state NMR studies. We show that optimised solubilisation and reconstitution procedures enable preparing stable and homogenous protein samples. Depending on the duration of solubilisation, MsbA can be obtained in either an apo- or in a native lipid A bound form. Building onto these optimisations, the first promising MAS-NMR spectra with narrow lines have been recorded. However, further sensitivity improvements are required so that complex NMR experiments can be recorded within a reasonable amount of time. We therefore demonstrate the usability of paramagnetic doping for rapid data acquisition and explore dynamic nuclear polarisation as a method for general signal enhancement. Our results demonstrate that solid state NMR provides an opportunity to address important biological questions related to complex mechanisms of ABC transporters.

March 10, 2015

Functional properties of LptA and LptD in Anabaena sp. PCC 7120

Yi-Ching Hsueh, Eva-M. Brouwer, Julian Marzi, Oliver Mirus, Enrico Schleiff

Page range: 1151-1162

Abstract

Lipopolysaccharides (LPS) are central components of the outer membrane and consist of Lipid A, the core polysaccharide, and the O-antigen. The synthesis of LPS is initiated at the cytosolic face of the cytoplasmic membrane. The subsequent transport to and across the outer membrane involves multiple lipopolysaccharide transport (Lpt) proteins. Among those proteins, the periplasmic-localized LptA and the outer membrane-embedded LptD participate in the last steps of transfer and insertion of LPS into the outer membrane. While the process is described for proteobacterial model systems, not much is known about the machinery in cyanobacteria. We demonstrate that ana LptD (alr1278) of Anabaena sp. PCC 7120 is important for cell wall function and its pore domain shows a Lipid A sensitive cation-selective gating behavior. The N-terminal domain of ana LptD recognizes ana LptA (alr4067), but not ec LptA. Furthermore, ana LptA specifically interacts with the Lipid A from Anabaena sp. PCC 7120 only, while ana LptD binds to Lipid A isolated from Escherichia coli as well. Based on the comparative analysis of proteins from E. coli and Anabaena sp. we discuss the properties of the cyanobacterial Lpt system.

About this journal

Objective
Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences.

Topics

general biochemistry
pathobiochemistry
structural biology
molecular and cellular biology
genetics and epigenetics
molecular medicine
cancer research
virology
immunology
plant molecular biology and biochemistry
novel experimental methodologies

Article formats
Research Articles, Short Communications, Reviews and Minireviews
Reviews are published by invitation only, but suggestions to the Editor-in-Chief are welcome.

The journal is associated with the German Society for Biochemistry and Molecular Biology (GBM).

Volume 396 Issue 9-10

Issue of Biological Chemistry

Frontmatter

Membrane Transport and Communication in Frankfurt: Speakers’ Summary – Highlights

Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters

Abstract

Team work at its best – TAPL and its two domains

Abstract

The volume-regulated anion channel is formed by LRRC8 heteromers – molecular identification and roles in membrane transport and physiology

Abstract

Extending native mass spectrometry approaches to integral membrane proteins

Abstract

Functional diversity of the superfamily of K+ transporters to meet various requirements

Abstract

The structure of Na+-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na+ transport

Abstract

Hybrid rotors in F1Fo ATP synthases: subunit composition, distribution, and physiological significance

Abstract

Homeostatic control of biological membranes by dedicated lipid and membrane packing sensors

Abstract

The transporter associated with antigen processing: a key player in adaptive immunity

Abstract

The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump

Abstract

The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump

Abstract

A universal mechanism for transport and regulation of CPA sodium proton exchangers

Abstract

Biosynthesis of membrane dependent proteins in insect cell lysates: identification of limiting parameters for folding and processing

Abstract

Fluorescence and excited state dynamics of the deprotonated Schiff base retinal in proteorhodopsin

Abstract

Regulatory role of charged clusters in the N-terminal domain of BetP from Corynebacterium glutamicum

Abstract

The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter-substrate binding protein complex

Abstract

The ABC exporter MsbA probed by solid state NMR – challenges and opportunities

Abstract

Functional properties of LptA and LptD in Anabaena sp. PCC 7120

Abstract

Functional diversity of the superfamily of K⁺ transporters to meet various requirements

The structure of Na⁺-translocating of NADH:ubiquinone oxidoreductase of Vibrio cholerae: implications on coupling between electron transfer and Na⁺ transport

Hybrid rotors in F₁F_o ATP synthases: subunit composition, distribution, and physiological significance