Dodecylphosphocholine Micelles Research Articles

C-terminal Elongation of Growth-blocking Peptide Enhances Its Biological Activity and Micelle Binding Affinity

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.

NMR structure of the transmembrane and cytoplasmic domains of human CD4 in micelles

The human cluster determinant 4 (CD4) is a type I transmembrane glycoprotein involved in T-cell signalling. It is expressed primarily on the surface of T helper cells but also on subsets of memory and regulatory T lymphocytes (CD4 + cells). It serves as a coreceptor in T-cell receptor recognition of MHC II antigen complexes. Besides its cellular functions, CD4 serves as the main receptor for human immunodeficiency virus type I (HIV-1). During T-cell infection, the CD4 extracellular domain is bound by HIV-1 gp120, the viral surface glycoprotein, which triggers a number of conformational changes ultimately resulting in virion entry of the cell. Subsequently, CD4 is downregulated in infected cells by multiple strategies that involve direct interactions of the HIV-1 proteins VpU and Nef with the cytoplasmic part of CD4. In the present work, we describe the NOE-based solution structure of the transmembrane and cytoplasmic domains of the cystein-free variant of CD4 (CD4mut) in dodecylphosphocholine (DPC) micelles. Furthermore, we have characterized micelle-inserted CD4mut by paramagentic relaxation enhancement (PRE) agents and 1H- 15N heteronuclear NOE data. CD4mut features a stable and well-defined transmembrane helix from M372 to V395 buried in the micellar core and a cytoplasmic helix ranging from A404 to L413. Experimental data suggest the amphipathic cytoplasmic helix to be in close contact with the micellar surface. The role of the amphipathic helix and its interaction with the micellar surface is discussed with respect to the biological function of the full-length CD4 protein.

Specific Interactions of Clausin, a New Lantibiotic, with Lipid Precursors of the Bacterial Cell Wall

We investigated the specificity of interaction of a new type A lantibiotic, clausin, isolated from Bacillus clausii, with lipid intermediates of bacterial envelope biosynthesis pathways. Isothermal calorimetry and steady-state fluorescence anisotropy (with dansylated derivatives) identified peptidoglycan lipids I and II, embedded in dodecylphosphocholine micelles, as potential targets. Complex formation with dissociation constants of ∼0.3 μM and stoichiometry of ∼2:1 peptides/lipid intermediate was observed. The interaction is enthalpy-driven. For the first time, to our knowledge, we evidenced the interaction between a lantibiotic and C 55-PP-GlcNAc, a lipid intermediate in the biosynthesis of other bacterial cell wall polymers, including teichoic acids. The pyrophosphate moiety of these lipid intermediates was crucial for the interaction because a strong binding with undecaprenyl pyrophosphate, accounting for 80% of the free energy of binding, was observed. No binding occurred with the undecaprenyl phosphate derivative. The pentapeptide and the N-acetylated sugar moieties strengthened the interaction, but their contributions were weaker than that of the pyrophosphate group. The lantibiotic decreased the mobility of the pentapeptide. Clausin did not interact with the water-soluble UDP-MurNAc- and pyrophosphoryl-MurNAc-pentapeptides, pointing out the importance of the hydrocarbon chain of the lipid target.

Prion Protein-Detergent Micelle Interactions Studied by NMR in Solution

Cellular prion proteins, PrP(C), carrying the amino acid substitutions P102L, P105L, or A117V, which confer increased susceptibility to human transmissible spongiform encephalopathies, are known to form structures that include transmembrane polypeptide segments. Herein, we investigated the interactions between dodecylphosphocholine micelles and the polypeptide fragments 90-231 of the recombinant mouse PrP variants carrying the amino acid replacements P102L, P105L, A117V, A113V/A115V/A118V, K110I/H111I, M129V, P105L/M129V, and A117V/M129V. Wild-type mPrP-(90-231) and mPrP[M129V]-(91-231) showed only weak interactions with dodecylphosphocholine micelles in aqueous solution at pH 7.0, whereas discrete interaction sites within the polypeptide segment 102-127 were identified for all other aforementioned mPrP variants by NMR chemical shift mapping. These model studies thus provide evidence that amino acid substitutions within the polypeptide segment 102-127 affect the interactions of PrP(C) with membranous structures, which might in turn modulate the physiological function of the protein in health and disease.

Micelle-bound structures and dynamics of the hinge deleted analog of melittin and its diastereomer: Implications in cell selective lysis by d-amino acid containing antimicrobial peptides

Melittin, the major component of the honey bee venom, is a 26-residue hemolytic and membrane active peptide. Structures of melittin determined either in lipid environments by NMR or by use of X-ray demonstrated two helical regions at the N- and C-termini connected by a hinge or a bend at the middle. Here, we show that deletion of the hinge residues along with two C-terminal terminal Gln residues (Q25 and Q26), yielding a peptide analog of 19-residue or Mel-H, did not affect antibacterial activity but resulted in a somewhat reduced hemolytic activity. A diastereomer of Mel-H or Mel- dH containing d-amino acids [ dV5, dV8, dL11 and dK16] showed further reduction in hemolytic activity without lowering antibacterial activity. We have carried out NMR structures, dynamics (H–D exchange and proton relaxation), membrane localization by spin labeled lipids, pulse-field-gradient (PFG) NMR and isothermal titration calorimetry (ITC) in dodecylphosphocholine (DPC) micelles, as a mimic to eukaryotic membrane, to gain insights into cell selectivity of these melittin analogs. PFG-NMR showed Mel-H and Mel- dH both were similarly partitioned into DPC micelles. ITC demonstrated that Mel-H and Mel- dH interact with DPC with similar affinity. The micelle-bound structure of Mel-H delineated a straight helical conformation, whereas Mel- dH showed multiple β-turns at the N-terminus and a short helix at the C-terminus. The backbone amide-proton exchange with solvent D 2O demonstrated a large difference in dynamics between Mel-H and Mel- dH, whereby almost all backbone protons of Mel- dH showed a much faster rate of exchange as compared to Mel-H. Proton T 1 relaxation had suggested a mobile backbone of Mel- dH peptide in DPC micelles. Resonance perturbation by paramagnetic lipids indicated that Mel-H inserted deeper into DPC micelles, whereas Mel- dH is largely located at the surface of the micelle. Taken together, results presented in this study demonstrated that the poor hemolytic activity of the d-amino acid containing analogs of antimicrobial peptides may be correlated with their flexible dynamics at the membrane surface.

Backbone dynamics of the antifungal Psd1 pea defensin and its correlation with membrane interaction by NMR spectroscopy

Plant defensins are cysteine-rich cationic peptides, components of the innate immune system. The antifungal sensitivity of certain exemplars was correlated to the level of complex glycosphingolipids in the membrane of fungi strains. Psd1 is a 46 amino acid residue defensin isolated from pea seeds which exhibit antifungal activity. Its structure is characterized by the so-called cysteine-stabilized α/β motif linked by three loops as determined by two-dimensional NMR. In the present work we explored the measurement of heteronuclear Nuclear Overhauser Effects, R1 and R2 15N relaxation ratios, and chemical shift to probe the backbone dynamics of Psd1 and its interaction with membrane mimetic systems with phosphatidylcholine (PC) or dodecylphosphocholine (DPC) with glucosylceramide (CMH) isolated from Fusarium solani. The calculated R2 values predicted a slow motion around the highly conserved among Gly12 residue and also in the region of the Turn3 His36–Trp38. The results showed that Psd1 interacts with vesicles of PC or PC:CMH in slightly different forms. The interaction was monitored by chemical shift perturbation and relaxation properties. Using this approach we could map the loops as the binding site of Psd1 with the membrane. The major binding epitope showed conformation exchange properties in the μs–ms timescale supporting the conformation selection as the binding mechanism. Moreover, the peptide corresponding to part of Loop1 (pepLoop1: Gly12 to Ser19) is also able to interact with DPC micelles acquiring a stable structure and in the presence of DPC:CMH the peptide changes to an extended conformation, exhibiting NOE mainly with the carbohydrate and ceramide parts of CMH.

Interaction of the Dengue Virus Fusion Peptide with Membranes Assessed by NMR: The Essential Role of the Envelope Protein Trp101 for Membrane Fusion

Dengue virus (DV) infection depends on a step of membrane fusion, which occurs in the acidic environment of the endosome. This process is mediated by virus surface envelope glycoprotein, in which the loop between residues D98–G112 is considered to be crucial, acting as a fusion peptide. Here, we have characterized functionally and structurally the interaction between the DV fusion peptide and different model membranes by fluorescence and NMR. Its interaction was strongest in dodecylphosphocholine (DPC) micelles and anionic phosphatidylcholine/phosphatidylglycerol vesicles, the only vesicle that was fused by DV fusion peptide. The three-dimensional structure of DV fusion peptide bound to DPC micelles was solved by solution homonuclear NMR with an r.m.s.d. of 0.98 Å. The most striking result obtained from the solution structure was the hydrophobic triad formed by residues W101, L107, and F108, pointing toward the same direction, keeping the segment between G102 and G106 in a loop conformation. The interaction of DV fusion peptide with phosphatidylcholine/phosphatidylglycerol vesicles was also mapped by transfer-nuclear Overhauser enhancement (NOE) experiments, in which the majority of the NOE cross-peaks were from the hydrophobic triad, corroborating the DPC-bound structure. Substitution of the residue W101 by an alanine residue completely abolished membrane binding and, thus, fusion by the peptide and its NOE cross-peaks. In conclusion, the 15-residue DV fusion peptide has intrinsic ability to promote membrane fusion, most likely due to the hydrophobic interaction among the residues W101, L107, and F108, which maintains its loop in the correct spatial conformation.

Correlating structure, dynamics, and function in transmembrane segment VII of the Na +/H + exchanger isoform 1

We place 15N nuclear magnetic resonance relaxation analysis and functional mutagenesis studies in the context of our previous structural and mutagenesis work to correlate structure, dynamics and function for the seventh transmembrane segment of the human Na +/H + exchanger isoform 1. Although G261–S263 was previously identified as an interruption point in the helical structure of this isolated transmembrane peptide in dodecylphosphocholine micelles, and rapid conformational exchange was implicated in the NOE measurements, the six 15N labelled residues examined in this study all have similar dynamics on the ps–ns time scale. A mathematical model incorporating chemical exchange is the best fit for residues G261, L264, and A268. This implies that a segment of residues from G261 to A268 samples different conformations on the μs–ms time scale. Chemical exchange on an intermediate time scale is consistent with an alternating-access cycle where E262 is bent away from the cytosol during proton translocation by the exchanger. The functional importance of chemical exchange at G261–A268 is corroborated by the abrogated activity of the full-length exchanger with the bulky and restricting Ile substitutions F260I, G261I, E262I, S263I, and A268I.

Headgroup-dependent membrane catalysis of apelin-receptor interactions is likely.

Apelin is the peptidic ligand for the G-protein-coupled receptor APJ. The apelin−APJ system is important in cardiovascular regulation, fluid homeostasis, and angiogenesis, among other roles. In this study, we investigate interactions between apelin and membrane-mimetic micelles of the detergents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG). Far-ultraviolet circular dichroism spectropolarimetry and diffusion-ordered spectroscopy indicate that apelin peptides bind to micelles of the anionic detergents SDS and LPPG much more favorably than to zwitterionic DPC micelles. Nuclear magnetic resonance spectroscopy allowed full characterization of the interactions of apelin-17 with SDS micelles. Titration with paramagnetic agents and structural determination of apelin-17 in SDS indicate that R6−K12 is highly structured, with R6−L9 directly interacting with headgroups of the micelle. Type I β-turns are initiated between R6 and L9, and a well-defined type IV β-turn is initiated at S10. Furthermore, binding of apelin-17 to SDS micelles causes structuring of M15-F17, with no evidence for direct binding of this region to the micelles. These results are placed into the context of the membrane catalysis hypothesis for peptide−receptor binding, and a hypothetical mechanism of APJ binding and activation by apelin is advanced.

Solution Structure of the HsapBK K+ Channel Voltage-Sensor Paddle Sequence,

Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

Refinement of Protein NMR Structure under Membrane-like Environments with an Implicit Solvent Model

Refinement of NMR structures by molecular dynamics (MD) simulations with a solvent model has improved the structural quality. In this study, we applied MD refinement with the generalized Born (GB) implicit solvent model to protein structure determined under membrane-like environments. Despite popularity of the GB model, its applications to the refinement of NMR structures of hydrophobic proteins, in which detergents or organic solvents enclose proteins, are limited, and there is little information on the use of another GB parameter for these cases. We carried out MD refinement of crambin NMR structure in dodecylphosphocholine (DPC) micelles (Ahn et al., J. Am. Chem. Soc. 2006, 128, 4398-4404) with GB/Surface area model and two different surface tension coefficients, one for aquatic and the other for hydrophobic conditions. Our data show that, of two structures by MD refinement with GB model, the one refined with the parameter to consider hydrophobic condition had the better qualities in terms of precision and solvent accessibility.

Three-dimensional structure of Phyllomedusin, a NK1 receptor agonist bound to dodecylphosphocholine micelles

Phyllomedusin, an amphibian tachykinin decapeptide, has been shown to be selective for Neurokinin 1 receptor. Because the micelle-associated structure may be relevant to the Phyllomedusin-receptor interaction, the three-dimensional structure of the Phyllomedusin in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Sequence specific resonance assignments of protons have been made from correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using DYANA. The CD and NMR results show that, while in water Phyllomedusin prefers to be in an extended chain conformation, whereas in the presence of dodecylphosphocholine micelles, a membrane model system, a partial helical conformation is induced. Analysis of NMR data indicates that the global fold of Phyllomedusin can be explained in terms of equilibrium between 3(10)-helix and alpha-helix from residue 4 to 10. An extended highly flexible N-terminus displays some degree of order and a possible turn structure. A comparison between the conformational features of Phyllomedusin and different Neurokinin 1 receptor agonist indicates several common features in the distribution of hydrophobic and hydrophilic residues. The conformational similarities suggest that the molecules interact with receptor in an analogous manner.

Structural and Functional Analysis of Transmembrane XI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na(+)/H(+) exchanger isoform 1 is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammals by extruding an intracellular H(+) in exchange for one extracellular Na(+). We characterized structural and functional aspects of the critical transmembrane (TM) segment XI (residues 449-470) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM XI was mutated to cysteine in the background of the cysteine-less protein and the sensitivity to water-soluble sulfhydryl reactive compounds MTSET ((2-(trimethylammonium) ethyl)methanethiosulfonate) and MTSES ((2-sulfonatoethyl) methanethiosulfonate) was determined for those residues with at least moderate activity remaining. Of the residues tested, only proteins with mutations L457C, I461C, and L465C were inhibited by MTSET. The activity of the L465C mutant was almost completely eliminated, whereas that of the L457C and I461C mutants was partially affected. The structure of a peptide representing TM XI (residues Lys(447)-Lys(472)) was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. The structure consisted of helical regions between Asp(447)-Tyr(454) and Phe(460)-Lys(471) at the N and C termini of the peptide, respectively, connected by a region with poorly defined, irregular structure consisting of residues Gly(455)-Gly(459). TM XI of NHE1 had a structural similarity to TM XI of the Escherichia coli Na(+)/H(+) exchanger NhaA. The results suggest that TM XI is a discontinuous helix, with residue Leu(465) contributing to the pore.

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.

Structure of the pore-helix of the hERG K+ channel

The hERG K(+) channel undergoes rapid inactivation that is mediated by 'collapse' of the selectivity filter, thereby preventing ion conduction. Previous studies have suggested that the pore-helix of hERG may be up to seven residues longer than that predicted by homology with channels with known crystal structures. In the present work, we determined structural features of a peptide from the pore loop region of hERG (residues 600-642) in both sodium dodecyl sulfate (SDS) and dodecyl phosphocholine (DPC) micelles using NMR spectroscopy. A complete structure calculation was done for the peptide in DPC, and the localization of residues inside the micelles were analysed by using a water-soluble paramagnetic reagent with both DPC and SDS micelles. The pore-helix in the hERG peptide was only two-four residues longer at the N-terminus, compared with the pore helices seen in the crystal structures of other K(+) channels, rather than the seven residues suggested from previous NMR studies. The helix in the peptide spanned the same residues in both micellar environments despite a difference in the localization inside the respective micelles. To determine if the extension of the length of the helix was affected by the hydrophobic environment in the two types of micelles, we compared NMR and X-ray crystallography results from a homologous peptide from the voltage gated potassium channel, KcsA.

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented 15N and 31P Solid-State NMR Spectroscopy

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.

Thermodynamics of partitioning of substance P in isotropic bicelles

The temperature dependence of the partition of a neuropeptide, substance P (SP), in isotropic (q = 0.5) bicelles was investigated by using pulsed field gradient NMR diffusion technique. The partition coefficient decreases as the temperature is increased from 295 to 325 K, indicating a favorable (negative) enthalpy change upon partitioning of the peptide. Thermodynamic analysis of the data shows that the partitioning of SP at 300 K is driven by the enthalpic term (DeltaH) with the value of - 4.03 kcal mol(-1), while it is opposed by the entropic term (-TDeltaS) by approximately 1.28 kcal mol(-1) with a small negative change in heat capacity (DeltaC(p)). The enthalpy-driven process for the partition of SP in bicelles is the same as in dodecylphosphocholine (DPC) micelles, however, the negative entropy change in bicelles of flat bilayer surface is in sharp contrast with the positive entropy change in DPC micelles of highly curved surface, indicating that the curvature of the membrane surface might play a significant role in the partitioning of peptides.

Interactions of SP-B Based Peptide with Lipid and Protein Components of Lung Surfactant

Lung surfactant (LS) is a mixture of lipids and proteins that reduces the surface tension at the alveolar air-water interface and thus prevents lung collapse and enables normal breathing. Surfactant Protein B (SP-B) is an essential component of LS and is absolutely necessary for survival. SP-B is thought to function by facilitating large-scale rearrangements of lipids and stabilizing the structures at various stages of the breathing cycle. However, neither the structural basis for this ability nor the physiological ramifications of lipid rearrangements are yet understood, in part because a high-resolution structure of SP-B is not yet available. Mini-B is a peptide fragment of SP-B that has been shown in in vitro and in vivo studies to retain similar activity to the full-length protein. Previously, we determined the structure of Mini-B, first in organic solvent hexafluoroisopropanol (HFIP) and then in detergent micelles composed of sodiumdodecylsulfate (SDS) using solution NMR. In our present work, we have studied the interactions of Mini-B with dodecylphosphocholine (DPC) and SDS micelles. DPC and SDS micelles provide an interfacial environment, with lipid headgroups corresponding to the headgroups of the most abundant lipids in LS, phosphatidylcholine (PC) and phosphatidylglycerol (PG) and thus solution NMR studies of interactions between Mini-B and these micelles can provide insight into LS protein-lipid interactions. We have also investigated the interactions of Mini-B with the most abundant surfactant protein SP-A under similar conditions. These studies further the understanding of the mechanisms of SP-B interactions with other surfactant components in native lung environment.

Backbone structure of a small helical integral membrane protein: A unique structural characterization

The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three-dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C-terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 A rmsd backbone structure with a transmembrane domain defined at 0.40 A. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long-range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins.

NMR Structure of a Disulfide-Crosslinked αIIbβ3 Cytoplasmic Domain Heterodimer

The platelet integrin αIIbβ3 is the prototypic example of regulated integrin function. Thus, αIIbβ3 is present in a resting conformation on unstimulated platelets, but switches to an active conformation following platelet stimulation. Recent experiments suggest that disrupting a heteromeric interaction between the αIIb and β3 transmembrane (TM) and cytoplasmic domains shifts αIIbβ3 from its resting to its active conformation. However, structural information about the heteromeric interaction is sparse. Thus far, the structure of the TM heterodimer has only been studied by molecular modeling. Interactions between soluble cytosolic tail peptides have been studied by NMR spectroscopy, but these studies may not reflect native contacts because they fail to account for constraining TM domain interactions. To obtain an NMR structure for the αIIbβ3 cytosolic tail heterodimer that reflects its native structure, we expressed 13C- and 15N-labeled peptides corresponding to αIIb residues 988–1008 and β3 residues 713–762 in E. coli. Residues 987 in αIIb and 712 in β3 were replaced with cysteines, based on modeling that predicts the resultant disulfide bond will fix the peptides in their native orientation. Crosslinked heterodimers were dissolved in dodecylphosphocholine micelles at pH 6.5 and analyzed at 37°C on a 750 MHz NMR spectrometer. Previously, we presented a preliminary analysis of this construct indicating that when constrained by the proximal disulfide bond, the αIIb and β3 cytoplasmic tails interact and the cytosolic tail of β3 consists of three helices. We have now solved the final structure which defines the β3 interface that interacts with the αIIb cytoplasmic tail. The αIIb-β3 heterodimer interface is dynamic, but can be localized to β3 residues 716 and 719 because they have different chemical shifts in the crosslinked heterodimer than they do in the component monomers. This positions β3 residue 723 at the αIIb-β3 interface, consistent with the putative Arg995-Asp723 salt bridge. Interestingly, the αIIb tail is natively unstructured so a static interface for αIIb could not be identified. Additionally, the completed structure defines the relative orientations of the three β3 helices. The β3 cytoplasmic tail contains a sharp kink at residue 724 that fixes the membrane embedded helix (residues 713–723) and the first cytoplasmic helix (residues 725–736) at a right angle. The kink was defined by multiple NMR parameters including NOE distance restraints between residues 721 and 727. The distal cytoplasmic helix (residues 746–757) is related to the rest of the molecule by a flexible loop (residues 737– 745). N15 NOESY-HSQC crosspeak intensities provide evidence that the flexible loop and distal helix undergo increased motion relative to the first two helices, and the final structure reflects this motion because there is no preferred orientation for the distal helix relative to the first two helices. Lastly, the distal helix and flexible loop are joined by β3's canonical NPXY motif which forms an N-terminal cap for the distal helix. In conclusion, we have solved the NMR structure of a disulfide-crosslinked αIIb/β3 cytoplasmic tail heterodimer. Our analysis indicates that, when constrained by a disulfide bond, the αIIb and β3 cytoplasmic tails interact, providing one mechanism for maintaining αIIbβ3 in a resting state.