Helical structure of phospholamban in membrane bilayers.

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca(2+) pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-(13)C]Leu7 and [3-(13)C]Ala11 in the cytoplasmic region, between [1-(13)C]Pro21 and [3-(13)C]Ala24 in the juxtamembrane region and between [1-(13)C]Leu42 and [3-(13)C]Cys46 in the transmembrane domain of phospholamban are consistent with alpha-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long alpha-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca(2+) pump.

Structure of the transmembrane dimer interface of glycophorin A in membrane bilayers.

The hydrophobic transmembrane domain of glycophorin A contains a sequence motif that mediates dimerization in membrane environments. Long-range interhelical distance measurements using magic angle spinning NMR spectroscopy provide high-resolution structural constraints on the packing of the dimer interface in membrane bilayers. We show that direct packing contacts occur between glycine residues at positions 79 and 83 in the transmembrane sequence. Additional interhelical constraints between Ile76 and Gly79 and between Val80 and Gly83 restrict the rotational orientation and crossing angle of the interacting helices. These results refine our previously proposed structure of the glycophorin A dimer [Smith, S. O., and Bormann, B. J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 488-491] which revealed that the methyl groups of Val80 and Val84 are packed against Gly79 and Gly83, respectively.

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside.

Described herein is the synthesis of eight positional isomers of methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside. The compounds were generated simultaneously from methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography (HPLC). The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography (GLC) retention indices on three different capillary columns.

Internal packing of helical membrane proteins.

Helix packing is important in the folding, stability, and association of membrane proteins. Packing analysis of the helical portions of 7 integral membrane proteins and 37 soluble proteins show that the helices in membrane proteins have higher packing values (0.431) than in soluble proteins (0.405). The highest packing values in integral membrane proteins originate from small hydrophobic (G and A) and small hydroxyl-containing (S and T) amino acids, whereas in soluble proteins large hydrophobic and aromatic residues have the highest packing values. The highest packing values for membrane proteins are found in the transmembrane helix-helix interfaces. Glycine and alanine have the highest occurrence among the buried amino acids in membrane proteins, whereas leucine and alanine are the most common buried residue in soluble proteins. These observations are consistent with a shorter axial separation between helices in membrane proteins. The tight helix packing revealed in this analysis contributes to membrane protein stability and likely compensates for the lack of the hydrophobic effect as a driving force for helix-helix association in membranes.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5.

HIV-1 entry into CD4(+) cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Helix packing in polytopic membrane proteins: role of glycine in transmembrane helix association.

The nature and distribution of amino acids in the helix interfaces of four polytopic membrane proteins (cytochrome c oxidase, bacteriorhodopsin, the photosynthetic reaction center of Rhodobacter sphaeroides, and the potassium channel of Streptomyces lividans) are studied to address the role of glycine in transmembrane helix packing. In contrast to soluble proteins where glycine is a noted helix breaker, the backbone dihedral angles of glycine in transmembrane helices largely fall in the standard alpha-helical region of a Ramachandran plot. An analysis of helix packing reveals that glycine residues in the transmembrane region of these proteins are predominantly oriented toward helix-helix interfaces and have a high occurrence at helix crossing points. Moreover, packing voids are generally not formed at the position of glycine in folded protein structures. This suggests that transmembrane glycine residues mediate helix-helix interactions in polytopic membrane proteins in a fashion similar to that seen in oligomers of membrane proteins with single membrane-spanning helices. The picture that emerges is one where glycine residues serve as molecular notches for orienting multiple helices in a folded protein complex.

Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain.

The spleen focus forming virus (SFFV) gp55-P envelope glycoprotein specifically binds to and activates murine erythropoietin receptors (EpoRs) coexpressed in the same cell, triggering proliferation of erythroid progenitors and inducing erythroleukemia. Here we demonstrate specific interactions between the single transmembrane domains of the two proteins that are essential for receptor activation. The human EpoR is not activated by gp55-P but by mutation of a single amino acid, L238, in its transmembrane sequence to its murine counterpart serine, resulting in its ability to be activated. The converse mutation in the murine EpoR (S238L) abolishes activation by gp55-P. Computational searches of interactions between the membrane-spanning segments of murine EpoR and gp55-P provide a possible explanation: the face of the EpoR transmembrane domain containing S238 is predicted to interact specifically with gp55-P but not gp55-A, a variant which is much less effective in activating the murine EpoR. Mutational studies on gp55-P M390, which is predicted to interact with S238, provide additional support for this model. Mutation of M390 to isoleucine, the corresponding residue in gp55-A, abolishes activation, but the gp55-P M390L mutation is fully functional. gp55-P is thought to activate signaling by the EpoR by inducing receptor oligomerization through interactions involving specific transmembrane residues.

Magic angle spinning NMR of the protonated retinylidene Schiff base nitrogen in rhodopsin: expression of 15N-lysine- and 13C-glycine-labeled opsin in a stable cell line.

The apoprotein corresponding to the mammalian photoreceptor rhodopsin has been expressed by using suspension cultures of HEK293S cells in defined media that contained 6-15N-lysine and 2-13C-glycine. Typical yields were 1.5-1.8 mg/liter. Incorporation of 6-15N-lysine was quantitative, whereas that of 2-13C-glycine was about 60%. The rhodopsin pigment formed by binding of 11-cis retinal was spectrally indistinguishable from native bovine rhodopsin. Magic angle spinning (MAS) NMR spectra of labeled rhodopsin were obtained after its incorporation into liposomes. The 15N resonance corresponding to the protonated retinylidene Schiff base nitrogen was observed at 156.8 ppm in the MAS spectrum of 6-15N-lysine-labeled rhodopsin. This chemical shift corresponds to an effective Schiff base-counterion distance of greater than 4 A, consistent with structural water in the binding site hydrogen bonded with the Schiff base nitrogen and the Glu-113 counterion. The present study demonstrates that structural studies of rhodopsin and other G protein-coupled receptors by using MAS NMR are feasible.

Structural models of the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein is thought to be a type II integral membrane protein that exists as a disulfide-linked homodimer in transformed cells. Polarized-infrared measurements show that the E5 dimer in membrane bilayers is largely alpha-helical and has a transmembrane orientation. Computational searches of helix-helix conformations reveal two possible low-energy dimer structures. Correlation of these results with previous mutagenesis studies on the E5 protein suggests how the E5 dimer may serve as a molecular scaffold for dimerization and ligand-independent activation of the PDGF-beta receptor. We propose that on each face of the E5 dimer a PDGF-beta receptor molecule interacts directly with Gln17 from one E5 monomer and with Asp33 from the other E5 monomer. This model accounts for the requirement of Gln17 and Asp33 for complex formation and explains genetic results that dimerization of the E5 protein is essential for cell transformation.

Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation.

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.

Constitutive activation of opsin by mutation of methionine 257 on transmembrane helix 6.

Rhodopsin is a member of the large family of G protein-coupled receptors (GPCR's). Constitutive activity of GPCR's, defined as ligand-independent signaling, has been recognized as an important feature of receptor function and has also been implicated in the molecular pathophysiology of a number of human diseases. Rhodopsin has evolved a unique mechanism to minimize receptor basal activity. The chromophore 11-cis-retinal, which acts as an inverse agonist in rhodopsin, is covalently bound to the receptor to ensure extremely low receptor signaling in the dark. In this study, we replaced Met257 in TM helix 6 of opsin with each of the remaining 19 amino acids. Only mutant opsin M257R failed to be expressed in COS-cell membranes. Each of the remaining 18 mutant opsins, with the exception of M257L, was significantly constitutively active. Two mutants in particular, M257Y and M257N, displayed very high levels of constitutive activity. In addition, the double-site mutants with substitutions of both Met257 and Glu113 in TM helix 3 tended to be much more constitutively active than the sums of the activities of the individual single-site mutants. Based on existing structural models of rhodopsin, we conclude that Met257 may form an important and specific interhelical interaction with a highly conserved NPXXY motif in TM helix 7, which stabilizes the inactive receptor conformation by preventing TM helix 6 movement in the absence of all-trans-retinal. Furthermore, we are able to show that the pharmacological properties of the large number (approximately 50) of mutant opsins that we have characterized to date support the two-state model of GPCR function. These results suggest that rhodopsin and other GPCR's share a common mechanism of receptor activation that involves specific changes in helix-helix interactions.

Role of the C9 methyl group in rhodopsin activation: characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal.

Activation of the visual pigment rhodopsin involves both steric and electrostatic interactions between the chromophore and opsin within the retinal-binding site. Removal of the C9 methyl group of 11-cis-retinal inhibits light-dependent activation of the G protein, transducin, suggesting a direct steric contact. More recently, we have shown that steric interactions lead to receptor activation when Gly121 in the middle of transmembrane helix 3 is replaced by larger hydrophobic residues. In order to understand in more detail the role of the C9 methyl group of retinal in the structure and function of rhodopsin, we first studied the properties of recombinant 9-dm-Rho (opsin reconstituted with 11-cis-9-demethylretinal). The 9-dm-Rho pigment displayed a blue-shifted lambdamax, increased hydroxylamine reactivity, and decreased ability to activate transducin. These properties are consistent with the hypothesis that the C9 methyl group is a crucial structural anchor for the correct docking of the chromophore in its binding site. Next, we investigated the possible interaction between Gly121 of opsin and the C9 methyl group of retinal by characterizing recombinant pigments produced by combining mutant opsins (G121A, -V, -I, -L, and -W) with 11-cis-9-demethylretinal. Mutant opsins G121I, -L, and -W failed to bind the chromophore. However, the double mutant G121L/F261A bound 11-cis-9-demethylretinal to form a stable pigment with a lambdamax of 451 nm. When activity was assayed in membranes, the reduction in transducin activation by 9-dm-Rho caused by the lack of a C9 methyl group on the chromophore could be partially restored by replacing Gly121 with a bulky residue (leucine, isoleucine, or tryptophan). These results support a model of receptor activation that involves steric interaction between the C9 methyl group of the chromophore and the opsin in the vicinity of Gly121 on transmembrane helix 3.

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.

Partial agonist activity of 11-cis-retinal in rhodopsin mutants.

Rhodopsin, the photoreceptor molecule of the vertebrate rod cell, is a G protein-coupled receptor. Rhodopsin consists of the opsin apoprotein and its 11-cis-retinal chromophore, which is covalently bound to a specific lysine residue by a stable protonated Schiff base linkage. Rhodopsin activation occurs when light causes photoisomerization of the 11-cis chromophore to its all-trans form. The all-trans chromophore is the receptor agonist. The 11-cis-retinylidene chromophore is analogous pharmacologically to a potent inverse agonist of the receptor. We report here that replacement of a highly conserved glycine residue (Gly121) causes 11-cis-retinal to become a pharmacologic partial agonist. Although the mutant apoproteins do not display constitutive activity, they are active in the dark when bound to an 11-cis-retinylidene chromophore, or to a "locked" chromophore analogue, Ret-7. The degree of partial agonism is directly related to the size of the amino acid replacement at position 121, and it can be reversed by a specific second-site replacement of Phe261. Thus, mutation of Gly121 in rhodopsin causes 11-cis-retinal to act as a partial agonist rather than an inverse agonist, allowing the mutant pigment to activate transducin in the dark.

The steric trigger in rhodopsin activation.

Rhodopsin is the seven transmembrane helix receptor responsible for dim light vision in vertebrate rod cells. The protein has structural homology with the other G protein-coupled receptors, which suggests that the tertiary structures and activation mechanisms are likely to be similar. However, rhodopsin is unique in several respects. The most striking is the fact that the receptor "ligand", 11-cis retinal, is covalently bound to the protein and is converted from an "antagonist" to an "agonist" upon absorption of light. NMR studies of rhodopsin and its primary photoproduct, bathorhodopsin, have generated structural constraints that enabled docking of the 11-cis and all-trans retinal chromophores into a low-resolution model of the protein proposed by Baldwin. These studies also suggest a mechanism for how retinal isomerization leads to rhodopsin activation. More recently, mutagenesis studies have extended these results by showing how the selectivity of the retinal-binding site can be modified to favor the all-trans over the 11-cis isomer. The structural constraints produced from these studies, when placed in the context of a high-resolution model of the protein, provide a coherent picture of the activation mechanism, which we show involves a direct steric interaction between the retinal chromophore and transmembrane helix 3 in the region of Gly121.

Structure of the transmembrane cysteine residues in phospholamban.

Phospholamban, a 52-residue membrane protein, associates to form a pentameric complex of five long alpha-helices traversing the sarcoplasmic reticulum membrane of cardiac muscle cells. The transmembrane domain of the protein is largely hydrophobic, with only three cysteine residues having polar side chains, yet it functions as a Ca2+-selective ion channel. In this report, infrared spectroscopy is used to probe the conformation of the three cysteine side chains and to establish whether the free S-H groups form intrahelical hydrogen bonds in the pentameric complex. Vibrational spectra of a transmembrane peptide were obtained which corresponded to the transmembrane domain of wild-type phospholamban and three peptides each containing a cysteine --> alanine substitution. The observed S-H frequencies argue that each of the sulfhydryl groups is hydrogen-bonded to an i-4 backbone carbonyl oxygen. Electrostatic calculations on a model of phospholamban based on molecular dynamics and mutagenesis studies, show that the sulfhydryl groups may significantly contribute to the electrostatic potential field of the protein.

Structural perspectives of phospholamban, a helical transmembrane pentamer.

Phospholamban is a 52-amino-acid protein that assembles into a pentamer in sarcoplasmic reticulum membranes. The protein has a role in the regulation of the resident calcium ATPase through an inhibitory association that can be reversed by phosphorylation. The phosphorylation of phospholamban is initiated by beta-adrenergic stimulation, identifying phospholamban as an important component in the stimulation of cardiac activity by beta-agonists. In this role of phospholamban that has motivated studies in recent decades. There is evidence that phospholamban may also function as a Ca(2+)-selective ion channel. The structural properties of phospholamban have been studied by mutagenesis, modeling, and spectroscopy, resulting in a new view of the organization of this key molecule in membranes.

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin.

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor-ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor-ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly121 was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark-i.e., without chromophore isomerization-was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly121 and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.

Channels formed by the transmembrane helix of phospholamban: a simulation study.

Phospholamban is a small membrane protein which can form cation selective ion channels in lipid bilayers. Each subunit contains a single, largely hydrophobic transmembrane helix. The helices are thought to assemble as a pentameric and approximately parallel bundle surrounding a central pore. A model of this assembly (PDB code IPSL) has been used as the starting point for molecular dynamics (MD) simulations of a system consisting of the pentameric helix bundle, plus 217 water molecules located within and at either mouth of the pore. Interhelix distance restraints were employed to maintain the integrity of the helix bundle during a 500 ps MD simulation. Water molecules within the pore exhibited reduced diffusional and rotational mobility. Interactions between the alpha-helix dipoles and the water dipoles, the latter aligned anti-parallel to the former, contribute to the stability of the system. Analysis of the potential energy of interaction of a K+ ion as it was moved through the pore suggested that unfavourable interactions of the cation with the aligned helix dipoles at the N-terminal mouth were overcome by favourable ion-water interactions. Comparable analysis for a Cl ion revealed that the ion-(pore + water) interactions were unfavourable along the whole of the pore, increasingly so from the N- to the C-terminal mouth. Overall, the interaction energy profiles were consistent with a pore selective for cations over anions. Pore radius profiles were used to predict a channel conductance of 50 to 70 ps in 0.2 M KCl, which compares well with an experimental value of 100 ps.