[18] Methods to study membrane protein structure in solution

Henry, Gillian D.; Sykes, Brian D.

doi:10.1016/s0076-6879(94)39020-7

Cited by 206 publications

(212 citation statements)

References 51 publications

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“…We compared three phospholipids that share the same zwitterionic phosphatidylcholine head group that predominates in mammalian membranes but differ in their acyl chains. In aqueous solutions these lipids form micelles that mimic the membrane environment and are well suited for NMR studies (36). The micelles formed by cyclohexylbutylphosphocholine (CYFOS-4) or DHPC are approximately three times smaller (6.3 kDa) and larger (62.6 kDa), respectively, than d 38 -DPC micelles (21.8 kDa) based on translational diffusion co-efficients measured by pulsed field gradient (33) NMR studies.…”

Section: Resultsmentioning

confidence: 99%

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Kutateladze

Capelluto

Ferguson

et al. 2004

Journal of Biological Chemistry

109

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Targeting of a wide variety of proteins to membranes involves specific recognition of phospholipid head groups and insertion into lipid bilayers. For example, proteins that contain FYVE domains are recruited to endosomes through interaction with phosphatidylinositol 3-phosphate (PtdIns(3)P). However, the structural mechanism of membrane docking and insertion by this domain remains unclear. Here, the depth and angle of micelle insertion and the lipid binding properties of the FYVE domain of early endosome antigen 1 are estimated by NMR spectroscopy. Spin label probes incorporated into micelles identify a hydrophobic protuberance that inserts into the micelle core and is surrounded by interfacially active polar residues. A novel proxyl PtdIns(3)P derivative is developed to map the position of the phosphoinositide acyl chains, which are found to align with the membrane insertion element. Dual engagement of the FYVE domain with PtdIns(3)P and dodecylphosphocholine micelles yields a 6-fold enhancement of affinity. The additional interaction of phosphatidylserine with a conserved basic site of the protein further amplifies the micelle binding affinity and dramatically alters the angle of insertion. Thus, the FYVE domain is targeted to endosomes through the synergistic action of stereospecific PtdIns(3)P head group ligation, hydrophobic insertion and electrostatic interactions with acidic phospholipids.Cellular processes including signal transduction, vesicular trafficking, and cytoskeletal rearrangement require selective recruitment of proteins to membrane surfaces. Well established mechanisms for localizing cytosolic proteins to membranes include electrostatic interactions through a basic peptide sequence, anchoring by covalently attached acyl chains, and association with the cytoplasmic domains of transmembrane proteins (reviewed in Refs. 1 and 2). (4), PDZ (5), and PTB (6) domains. Although the majority of these domains can interact with several PIs, the FYVE domain is remarkably selective for PtdIns(3)P (7-9). In addition to PI ligation, these domains often insert hydrophobic elements into the membrane bilayer, as has been demonstrated for the C2 (10), ENTH (11), FERM (12), FYVE (13), and PX (14) domains and the vinculin tail (15). Insertion into the membrane can be accompanied by interactions with multiple lipid head groups. For example, the PX domain of the p47 subunit of the NADPH oxidase binds cooperatively to PtdIns(3)P and phosphatidic acid (16), and the vinculin tail co-ligates phosphatidylinositol 4,5-bisphosphate and PtdSer (15). Although it is becoming evident that insertion of proteins into membranes is widespread, the three-dimensional orientations and quantitative binding properties remain challenging to characterize. The most common electron paramagnetic resonance and fluorescence approaches have provided important insights (17-20) but require covalent attachment of paramagnetic groups to various positions of the protein or mutations of residues. The inevitable effects of these modifications on lipi...

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Section: Resultsmentioning

confidence: 99%

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Kutateladze

Capelluto

Ferguson

et al. 2004

Journal of Biological Chemistry

109

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“…The myristoylated peptides were dissolved in membranemimic DPC micelles for NMR studies. DPC has the same zwitterionic phosphocholine head group as the dominant phospholipids of most eukaryotic membranes and provides a favorable balance between desirable NMR properties and biochemical compatibility with membrane-associated proteins (24)(25)(26). The myristoyl group has been shown to insert into the lipid bilayer or DPC micelles stabilizing peptide structures (21)(22)(23).…”

Section: Resultsmentioning

confidence: 99%

A structural basis for integrin activation by the cytoplasmic tail of the α _IIb -subunit

Vinogradova

Haas

Plow

et al. 2000

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

134

182

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A key step in the activation of heterodimeric integrin adhesion receptors is the transmission of an agonist-induced cellular signal from the short ␣-and͞or ␤-cytoplasmic tails to the extracellular domains of the receptor. The structural details of how the cytoplasmic tails mediate such an inside-out signaling process remain unclear. We report herein the NMR structures of a membraneanchored cytoplasmic tail of the ␣IIb-subunit and of a mutant ␣IIb-cytoplasmic tail that renders platelet integrin ␣IIb␤3 constitutively active. The structure of the wild-type ␣IIb-cytoplasmic tail reveals a ''closed'' conformation where the highly conserved N-terminal membrane-proximal region forms an ␣-helix followed by a turn, and the acidic C-terminal loop interacts with the Nterminal helix. The structure of the active mutant is significantly different, having an ''open'' conformation where the interactions between the N-terminal helix and C-terminal region are abolished. Consistent with these structural differences, the two peptides differ in function: the wild-type peptide suppressed ␣IIb␤3 activation, whereas the mutant peptide did not. These results provide an atomic explanation for extensive biochemical͞mutational data and support a conformation-based ''on͞off switch'' model for integrin activation.NMR ͉ cytoplasmic domain B y mediating numerous cell-cell and cell-matrix interactions, the integrin family of cell-surface receptors plays crucial roles in the development and health of all multicellular organisms (1-3). These noncovalent heterodimers (␣ and ␤) are expressed widely and regulate diverse biological processes such as matrix assembly, hemostasis, wound healing, inflammation, and tumor metastasis (1-3). Each subunit of an integrin consists of a single type I transmembrane domain, a large extracellular domain of several hundred amino acids, and typically, a short cytoplasmic tail of Ϸ20-70 residues (1, 2, 4). The extracellular domains interact with each other to form a complex binding site for a wide variety of ligands. Central to the function of the integrins is their capacity to undergo activation, a transition from a low to a high affinity͞avidity state for their ligands. Such activation is tightly regulated through a process termed ''insideout signaling'' (3, 4), i.e., agonist stimulation initiates intracellular changes that ultimately render the extracellular domain competent to bind ligands. The biological importance of such integrin activation is underscored by the major platelet integrin, ␣ IIb ␤ 3 , which cannot engage its abundant plasma protein ligands unless the cell has been stimulated by an agonist. This transition depends on the transmission of a cellular signal, typically initiated by occupancy of a G protein-coupled receptor, to the short cytoplasmic tails of the ␣ IIb -and͞or ␤ 3 -subunits, which is then propagated across the cell membrane to the extracellular ligandbinding domain. The structural basis of this inside-out signaling event remains poorly understood.The cytoplasmic tails of the ␣-subunits o...

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“…Of the three core regions defined by convergent backbone atoms, residues Asp 159 -Leu 163 are the most affected by these Although similar experimental resolution in NMR studies has been achieved using micelles in aqueous solution (46), such an environment would surround the TM IV peptide with hydrophobic detergent tails. In its natural setting, this segment of the NHE1 protein would contact lipid tail groups, surrounding polypeptide segments, and a pore region with a relatively high dielectric constant.…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

Slepkov¹,

Rainey

Li³

et al. 2005

Journal of Biological Chemistry

150

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The Na ؉ /H ؉ exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals. We characterized the structural and functional aspects of the critical transmembrane (TM) segment IV. Each residue was mutated to cysteine in cysteineless NHE1. TM IV was exquisitely sensitive to mutation with 10 of 23 mutations causing greatly reduced expression and/or activity. The Phe 161 3 Cys mutant was inhibited by treatment with the water-soluble sulfhydryl-reactive compounds [2-(trimethylammonium)ethyl]methanethiosulfonate and [2-sulfonatoethyl]methanethiosulfonate, suggesting it is a pore-lining residue. The structure of purified TM IV peptide was determined using high resolution NMR in a CD 3 OH:CDCl 3 :H 2 O mixture and in Me 2 SO. In CD 3 OH: CDCl 3 :H 2 O, TM IV was structured but not as a canonical ␣-helix. Residues Asp 159 -Leu 162 were a series of ␤-turns; residues Leu 165 -Pro 168 showed an extended structure, and residues Ile 169 -Phe 176 were helical in character. These three structured regions rotated quite freely with respect to the others. In Me 2 SO, the structure was much less defined. Our results demonstrate that TM IV is an unusually structured transmembrane segment that is exquisitely sensitive to mutagenesis and that Phe 161 is a pore-lining residue.

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[18] Methods to study membrane protein structure in solution

Cited by 206 publications

References 51 publications

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

A structural basis for integrin activation by the cytoplasmic tail of the α _IIb -subunit

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

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[18] Methods to study membrane protein structure in solution

Cited by 206 publications

References 51 publications

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

Multivalent Mechanism of Membrane Insertion by the FYVE Domain

A structural basis for integrin activation by the cytoplasmic tail of the α IIb -subunit

Structural and Functional Characterization of Transmembrane Segment IV of the NHE1 Isoform of the Na+/H+ Exchanger

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A structural basis for integrin activation by the cytoplasmic tail of the α _IIb -subunit