NMR Solution Structure of the Integral Membrane Enzyme DsbB: Functional Insights into DsbB-Catalyzed Disulfide Bond Formation

Zhou, Yunpeng; Cierpicki, Tomasz; Jiménez, Ricardo H. Flores; Lukasik, Stephen M.; Ellena, Jeffrey F.; Cafiso, David S.; Kadokura, Hiroshi; Beckwith, Jon; Bushweller, John H.

doi:10.1016/j.molcel.2008.08.028

Cited by 177 publications

(234 citation statements)

References 28 publications

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“…In contrast, solid-state NMR using oriented membrane protein samples can give molecular details regarding both backbone structure and topology. Because a number of membrane protein structures recently determined show similar folds in micelles, lipid bilayers, and crystals (34,(37)(38)(39)(40)(41), we propose to combine the restraints from these techniques into a unique protocol with the goal of obtaining the highresolution structure (backbone and side chains) and topology within the lipid bilayer. In the literature, there are several examples of backbone structure determination of membrane protein using solid-state NMR data alone (42,43) and a few examples reported for the combined use of solution and solid-state NMR information in a qualitative fashion (39, 44, 45).…”

Section: Discussionmentioning

confidence: 99%

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Traaseth

Shi²,

Verardi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

158

194

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Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1-16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23-30) and transmembrane domain II (residues 31-52) traverses the bilayer with a tilt angle of Ϸ24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.hybrid method ͉ membrane proteins ͉ oriented solid-state NMR ͉ molecular modeling ͉ PISEMA S tructure and topology are central to membrane protein function (1). Recently determined high-resolution structures reveal the compact folds for several membrane proteins, such as electron and proton-conducting proteins involved in photosynthesis and respiration (http://blanco.biomol.uci.edu/ Membrane Proteins xtal.html). However, a significant population of membrane proteins does not possess a compact tertiary fold, but has its fold space defined through interactions of secondary structure elements (helices, turns, and loops) with the lipid membrane, i.e., the topology (1). This is the case for phospholamban (PLN), a mammalian protein that is essential in the regulation of cardiac muscle contractility (2), and that has recently become a major target for gene therapy to ameliorate cardiomyopathies (3, 4). PLN is located in the sarco(endo)plasmic reticulum (SR) of cardiac myocytes, inhibiting the SR Ca 2ϩ -ATPase (SERCA) by shifting its relative Ca 2ϩ affinity (5). In vitro and in vivo experiments have shown PLN to exist as a homopentamer that deoligomerizes into active monomers that bind SERCA in a 1:1 molar ratio (6). The monomeric form of PLN exists in equilibrium between a dynamically disordered R state and a more restricted T state (7,8) and has 3 ...

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Section: Discussionmentioning

confidence: 99%

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Traaseth

Shi²,

Verardi

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

158

194

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“…DsbB also has its pairs of redox-active cysteines located in or close to the periplasm (15,16,30). The redox-active cysteines in both Mtb VKOR and DsbB are in or close to the two periplasmic domains formed by the first through the fourth TM segments.…”

Section: Discussionmentioning

confidence: 99%

Membrane Topology and Mutational Analysis ofMycobacterium tuberculosisVKOR, a Protein Involved in Disulfide Bond Formation and a Homologue of Human Vitamin K Epoxide Reductase

Wang

Dutton

Beckwith

et al. 2011

Antioxidants & Redox Signaling

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We have presented evidence that a homologue of vertebrate membrane protein vitamin K epoxide reductase (VKOR) is an important component of the protein disulfide bond-forming pathway in many bacteria. Bacterial VKOR appears to take the place of the nonhomologous DsbB found in Escherichia coli. We also determined the structure of a VKOR from a Cyanobacterium and showed that two or four conserved cysteines are required, according to different reductants for activity in an in vitro assay. Here we present evidence for the topologic arrangement in the cytoplasmic membrane of the VKOR from Mycobacterium tuberculosis (Mtb). The results show that Mtb VKOR is a membrane protein that spans the membrane 5 times with its N-terminus in the cytoplasm, C-terminus in the periplasm, and all four cysteines facing the periplasm. The essentiality of the four conserved cysteine residues has also been demonstrated in promoting disulfide bond formation in vivo and a mixed disulfide between a cysteine of DsbA of E. coli, and one of the cysteines (Cys 57 ) of the VKOR homologue has been identified to be a likely intermediate in the disulfide bond-forming pathway. These studies may inform future resolution of issues surrounding the functioning of human VKOR.

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“…Therefore, it is generally considered a mild detergent that largely preserves native structures of membrane proteins. Indeed, DPC is a preferred lipid for determining structures of membrane proteins by solution NMR (21)(22)(23)(24)(25). However, and despite the earlier NMR successes with DPC, caution is still advised when extrapolating some aspects of our synaptobrevin structure to situations in lipid bilayers.…”

Section: Discussionmentioning

confidence: 99%

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation

Ellena

Liang

Wiktor

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

107

130

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The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most Nterminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.structure ͉ dynamics ͉ membrane fusion ͉ SNARE proteins ͉ NMR S ynaptic release of neurotransmitter requires the docking and fusion of synaptic vesicles at the presynaptic membrane. The fusion reaction is thought to be mediated by the formation of the SNARE complex from its components syntaxin-1a and SNAP-25 in the target membrane and synaptobrevin-2 (Syb) in the vesicle membrane (1). These three proteins together form a complex that consists of a thermally very stable coiled-coil four-helix bundle, as revealed by the crystal structure of the soluble SNARE core complex (2). SNAP-25 contributes two helices to this complex and syntaxin and synaptobrevin each contribute one. More recently, the structure of the postfusion cis-SNARE complex with its C-terminal transmembrane (TM) domain extensions was solved at 3.4 Å resolution (3). Most interestingly, the helices continue through the juxtamembrane linker region and into the membrane, suggesting that force could be transmitted through this region into the membrane leading to membrane bending and eventually membrane fusion. This interesting finding immediately raises the question: how exactly is the assembly and folding of the four-helix bundle coupled into the membrane and how does the energy derived from this reaction ultimately fuse two different membranes into one? A common notion is that an initial trans-SNARE complex forms by pairing vesicle and target membrane SNAREs from their Nterminal ends and progressively folds in a zipper-like fashion toward the C-terminal TM domains. This reaction is thought to pull the two membranes into closer contact until, at some stage they merge into a single membrane. However, structural data on a trans-SNARE complex do not yet exist. It is also not yet known whether zipperfolding of trans-SNARE complexes progresses smoothly and continuously into the membrane or whether this reaction is discontinuous and segmented in some fashion.To understand how the SNARE complex is formed and to find possible reaction intermediates, several structural studies of SNARE proteins in isolation or in binary comp...

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NMR Solution Structure of the Integral Membrane Enzyme DsbB: Functional Insights into DsbB-Catalyzed Disulfide Bond Formation

Cited by 177 publications

References 28 publications

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Membrane Topology and Mutational Analysis ofMycobacterium tuberculosisVKOR, a Protein Involved in Disulfide Bond Formation and a Homologue of Human Vitamin K Epoxide Reductase

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation

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