The influenza A/M2 protein is a homotetrameric single-pass integral membrane protein encoded by the influenza A viral genome. Its transmembrane domain represents both a crucial drug target and a minimalistic model system for transmembrane proton transport and charge stabilization. Recent structural and functional studies of M2 have suggested that the proton transport mechanism involves sequential extraviral protonation and intraviral deprotonation of a highly conserved His37 side chain by the transported proton, consistent with a pH-activated proton shuttle mechanism. Multiple tautomeric forms of His can be formed, and it is not known whether they contribute to the mechanism of proton shuttling. Here we present the thermodynamic and functional characterization of an unnatural amino acid mutant at His37, where the imidazole side chain is substituted with a 4-thiazolyl group that is unable to undergo tautomerization and has a significantly lower solution pKa. The mutant construct has a similar stability to the wild-type protein at pH8 in bilayers and is virtually inactive at external pH7.4 in a semiquantitative liposome flux assay as expected from its lower sidechain pKa. However when the external buffer pH is lowered to 4.9 and 2.4, the mutant shows increasing amantadine sensitive flux of a similar magnitude to that of the wild type construct at pH7.4 and 4.9 respectively. These findings are in line with mechanistic hypotheses suggesting that proton flux through M2 is mediated by proton exchange from adjacent water molecules with the His37 sidechain, and that tautomerization is not required for proton translocation. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

The de novo design of membrane proteins remains difficult despite recent advances in understanding the factors that drive membrane protein folding and association. We have designed a membrane protein PRIME (PoRphyrins In MEmbrane) that positions two non-natural iron diphenylporphyrins (Fe(III)DPP's) sufficiently close to provide a multicentered pathway for transmembrane electron transfer. Computational methods previously used for the design of multiporphyrin water-soluble helical proteins were extended to this membrane target. Four helices were arranged in a D(2)-symmetrical bundle to bind two Fe(II/III) diphenylporphyrins in a bis-His geometry further stabilized by second-shell hydrogen bonds. UV-vis absorbance, CD spectroscopy, analytical ultracentrifugation, redox potentiometry, and EPR demonstrate that PRIME binds the cofactor with high affinity and specificity in the expected geometry.

The first computationally designed self-assembling oligomer consisting of exclusively β-amino acids (βAAs) is presented. The packing of a β-3(14) helix into coiled-coils of varying stoichiometries as a function of amino acid sequence is examined. β-Peptides with hVal repeating every third residue in the sequence appeared to have a strong propensity to pack into hexameric bundles. The designed sequence was synthesized and characterized with CD spectroscopy, NMR, and analytical ultracentrifugation, suggesting that the peptide adopts a well-folded hexameric structure.

The influenza A/M2 protein exhibits inwardly rectifying, pH-activated proton transport that saturates at low pH. A comparison of high-resolution structures of the transmembrane domain at high and low pH suggests that pH-dependent conformational changes may facilitate proton conduction by alternately changing the accessibility of the N-terminal and C-terminal regions of the channel as a proton transits through the transmembrane domain. Here, we show that M2 functionally reconstituted in liposomes populates at least three different conformational states over a physiologically relevant pH range, with transition midpoints that are consistent with previously reported His37 pK(a) values. We then develop and test two similar, quantitative mechanistic models of proton transport, where protonation shifts the equilibrium between structural states having different proton affinities and solvent accessibilities. The models account well for a collection of experimental data sets over a wide range of pH values and voltages and require only a small number of adjustable parameters to accurately describe the data. While the kinetic models do not require any specific conformation for the protein, they nevertheless are consistent with a large body of structural information based on high-resolution nuclear magnetic resonance and crystallographic structures, optical spectroscopy, and molecular dynamics calculations.

The influenza A virus M2 protein (A/M2) is a homotetrameric pH-activated proton transporter/channel that mediates acidification of the interior of endosomally encapsulated virus. This 97-residue protein has a single transmembrane (TM) helix, which associates to form homotetramers that bind the anti-influenza drug amantadine. However, the minimal fragment required for assembly and proton transport in cellular membranes has not been defined. Therefore, the conductance properties of truncation mutants expressed in Xenopus oocytes were examined. A short fragment spanning residues 21–61, M2(21-61), was inserted into the cytoplasmic membrane and had specific, amantadine-sensitive proton transport activity indistinguishable from that of full-length A/M2; an epitope-tagged version of an even shorter fragment, M2(21-51)-FLAG, had specific activity within a factor of 2 of the full-length protein. Furthermore, synthetic fragments including a peptide spanning residues 22–46 were found to transport protons into liposomes in an amantadine-sensitive manner. In addition, the functionally important His-37 residue pK a values are highly perturbed in the tetrameric form of the protein, a property conserved in the TM peptide and full-length A/M2 in both micelles and bilayers. These data demonstrate that the determinants for folding, drug binding, and proton translocation are packaged in a remarkably small peptide that can now be studied with confidence.

The forces that define the interactions of transmembrane helices have been evaluated using a model membrane-soluble peptide (MS1), whose packing is modeled on the two-stranded coiled-coil from GCN4. The thermodynamic stability of water-soluble coiled-coils depends on the side chain at the buried "a" position of the repeat, favoring large hydrophobic residues over small side chains. Here we show that just the opposite is true for the membrane-soluble peptide. Analytical ultracentrifugation and equilibrium disulfide interchange show that the stability of MS1 is greatest when Gly is at each "a" position of the heptad repeat (MS1-Gly), followed by Ala > Val > Ile. Moreover, MS1-Gly has a strong tendency to form antiparallel dimers, MS1-Ala forms a mixture of parallel and antiparallel dimers, while MS1-Val and MS1-Ile have a preference to form parallel dimers. Calculations based on exhaustive conformational searching and rotamer optimization were in excellent agreement with experiments, in terms of the overall stability of the structures and the preference for parallel vs antiparallel packing. The MS1-Gly helices are able to achieve more favorable and uniform packing in an antiparallel dimer, while MS1-Val and MS1-Ile have more favorable van der Waals interactions in a parallel dimer. Finally, the electrostatic component arising from the partial charges of the backbones become significant in the antiparallel MS1-Gly and MS1-Ala conformations, due to close packing of the helices. Thus, van der Waals interactions and electrostatic interactions contribute to the stability and orientational preferences of the dimers.

Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXIC321S is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXIC321S, FXIC321S,K331A, FXIC321S,E287A, FXIC321S,I290A, FXIC321S,Y329A, FXIC321S,L284A, FXIC321S,K331R, and FXIC321S,H343A were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXIC321S and FXIC321S,H343A existed in monomer/dimer equilibrium (K-d similar to 40 nM), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K-d = 3-38 mu M). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.

Glucocorticoid-induced TNF receptor ligand (GITRL) is a member of the TNF super family (TNFSF). GITRL plays an important role in controlling regulatory T cells. The crystal structure of the mouse GITRL (mGITRL) was determined to 1.8-Å resolution. Contrary to the current paradigm that all ligands in the TNFSF are trimeric, mGITRL associates as dimer through a unique C terminus tethering arm. Analytical ultracentrifuge studies revealed that in solution, the recombinant mGITRL exists as monomers at low concentrations and as dimers at high concentrations. Biochemical studies confirmed that the mGITRL dimer is biologically active. Removal of the three terminal residues in the C terminus resulted in enhanced receptor-mediated NF-κB activation than by the wild-type receptor complex. However, deletion of the tethering C-terminus arm led to reduced activity. Our studies suggest that the mGITRL may undergo a dynamic population shift among different oligomeric forms via C terminus-mediated conformational changes. We hypothesize that specific oligomeric forms of GITRL may be used as a means to differentially control GITR receptor signaling in diverse cells.

We explore the interplay between amino acid sequence, thermodynamic stability, and functional fitness in the M2 proton channel of influenza A virus. Electrophysiological measurements show that drug-resistant mutations have minimal effects on M2's specific activity, and suggest that resistance is achieved by altering a binding site within the pore rather than a less direct allosteric mechanism. In parallel, we measure the effects of these mutations on the free energy of assembling the homotetrameric transmembrane pore from monomeric helices in micelles and bilayers. Although there is no simple correlation between the evolutionary fitness of the mutants and their stability, all variants formed more stable tetramers in bilayers, and the least-fit mutants showed the smallest increase in stability upon moving from a micelle to a bilayer environment. We speculate that the folding landscape of a micelle, is rougher than that of a bilayer, and more accommodating of conformational variations in nonoptimized mutants.

BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE PAK1LC8 INTERACTION. Christine M. Lightcap Shangjin Sun, James D. Lear, Ulrich Rodeck, Tatyana Polenova, and John C. Williams Department of Biochemistry and Molecular Biology & Kimmel Cancer Center, Department of Dermatology, Thomas Jefferson University; Department of Chemistry and Biochemistry, University of Delaware; Department of Biochemistry and Biophysics, University of Pennsylvania.

We have developed a computational design strategy based on the alpha-helical coiled-coil to generate modular peptide motifs capable of assembling into metalloporphyrin arrays of varying lengths. The current study highlights the extension of a two-metalloporphyrin array to a four-metalloporphyrin array through the incorporation of a coiled-coil repeat unit. Molecular dynamics simulations demonstrate that the initial design evolves rapidly to a stable structure with a small rmsd compared to the original model. Biophysical characterization reveals elongated proteins of the desired length, correct cofactor stoichiometry, and cofactor specificity. The successful extension of the two-porphyrin array demonstrates how this methodology serves as a foundation to create linear assemblies of organized electrically and optically responsive cofactors.

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser(88) inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i. e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e. g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser88. Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. 2004; Jung et al. 2004; Rotondi and Gierasch 2005), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca(2+) or Mg(2+), has not been fully characterized. It has, however, been suggested that addition of Ca(2+) to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. 2004; Kopp et al. 2006), may also be important for Ca(2+) binding (Jung et al. 2004). In this work, we have tried: (1) to further pinpoint how Ca(2+) affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional (13)C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca(2+) and daptomycin. The (13)C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg(2+) are also presented. The implication of these results for the biological function of daptomycin is discussed.

A variety of methods exist for the design or selection of antibodies and other proteins that recognize the water-soluble regions of proteins; however, companion methods for targeting transmembrane (TM) regions are not available. Here, we describe a method for the computational design of peptides that target TM helices in a sequence-specific manner. To illustrate the method, peptides were designed that specifically recognize the TM helices of two closely related integrins (αIIbβ3 and αvβ3) in micelles, bacterial membranes, and mammalian cells. These data show that sequence-specific recognition of helices in TM proteins can be achieved through optimization of the geometric complementarity of the target-host complex.

Factor XI (FXI), a coagulation protein essential for normal hemostasis, is a homodimer consisting of two identical subunits of 80 KDa linked by a disulfide bond formed by Cys321 within the Apple 4 (A4) domain of each subunit. Prekallikrein (PK), in spite of its high homology with FXI both in amino acid sequence and domain structure, is a monomer. Cys321 in PK forms an intrachain disulfide bond with Cys326. However FXI/C321S (in which interchain disulfide bond formation is precluded) is a noncovalent dimer. Thus, there are interacting residues between the two subunits of FXI that are responsible for mediating its unique homodimeric structure. Examination of the crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J. Nature Structural & Molecular Biology . 2006; 13:557–8) shows salt bridges between Lys331 of one subunit with Glu287 of the other subunit as well as hydrophobic interactions at the interface of the A4 domains involving Ile290, Leu284 and Tyr329. FXI/C321S, FXI/C321S/K331A, FXI/C321S/E287A, FXI/C321S/I290A, FXI/C321S/Y329A, FXI/C321S/L284A and FXI/C321S/K331R were expressed in HEK293 cells and characterized using size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and functional assays. Whereas FXI/C321S existed in a monomer/dimer equilibrium (Kd ∼40 nM) all other mutants were predominantly monomers by SEC with impaired dimer formation by AUC (Kd 3.4–38 μM). All the monomeric mutants when converted to the active enzyme, FXIa, were able to hydrolyze the small chromogenic substrate S-2366 with normal values of Km and Vmax and cleaved the macromolecular substrate FIX at both its scissile bonds at rates similar to those observed with wtFXIa strongly suggesting that all mutant proteins were properly folded. However all the monomeric mutants displayed impaired clotting activity in an APTT assay and displayed markedly decreased rates of activation by FXIIa or thrombin and autoactivation in the presence or absence of dextran sulfate. We conclude that salt bridges formed between Lys331 of one subunit and Glu287 of the other together with hydrophobic interactions of residues Ile290 with Leu284 and Tyr329 with Tyr329 and are essential for normal homodimer formation, which is essential for normal proteolytic activation of FXI by FXIIa, thrombin and FXIa either in solution or on an anionic surface.

http://www.sciencemag.org/cgi/content/full/315/5820/1817 version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/cgi/content/full/315/5820/1817/DC1 can be found at: Supporting Online Material found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/cgi/content/full/315/5820/1817#related-content http://www.sciencemag.org/cgi/content/full/315/5820/1817#otherarticles , 19 of which can be accessed for free: cites 41 articles This article 8 article(s) on the ISI Web of Science. cited by This article has been http://www.sciencemag.org/cgi/content/full/315/5820/1817#otherarticles 4 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/biochem Biochemistry : subject collections This article appears in the following

We have designed a functional model membrane protein by engineering a bis-Histidine heme-binding site into a natural membrane protein, glycophorin A (GpA), structurally characterized by the dimerization of a single transmembrane helix. Out of the 32 residues comprising the transmembrane helix of GpA, five amino acids were mutated; the resulting protein, ME1, has been characterized in dodecyl phosphocholin (DPC) micelles by UV-vis, CD spectroscopy, gel electrophoresis, and analytical ultracentrifugation. ME1 binds heme with sub-micromolar affinity and maintains the highly helical secondary structure and dimeric oligomerization state of GpA. The ME1-Heme complex exhibits a redox potential of -128 +/- 2 mV vs SHE, indicating that the heme resides in a hydrophobic environment and is well shielded from the aqueous phase. Moreover, ME1 catalyzes the hydrogen peroxide dependent oxidation of organic substrates such as TMB (2,2',5,5'-tetramethyl-benzidine). This protein may provide a useful framework to investigate how the protein matrix tunes the cofactor properties in membrane proteins.

Anesthesia and surgery have been reported to produce long-term cognitive problems, and to accelerate neurodegenerative disorders in the elderly. In previous work, we found that inhaled anesthetics enhance fibril formation and cytotoxicity of amyloid beta peptide. In this work we show that the inhaled anesthetics halothane (2-bromo-2-chloro-1,1,1trifluoroethane) and isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) also favor intermediate oligomers of amyloid beta(1-40), and reduce solubility of amyloid beta(1-40) monomer. Size-exclusion chromatography, analytical ultracentrifugation and photo-induced cross-linking experiments indicate halothane enhancement of oligomeric species having molecular weight similar to 44-100 kDa. Bis-ANS fluorescence experiments revealed that halothane stabilizes a population of diffusible oligomers relative to the monomer or the mature fibril. These data show that inhaled anesthetics lower the amyloid beta(1-40) concentration necessary to initiate oligomer formation, probably by preferential binding to intermediate oligomers en route to fibril formation.