A novel haem-binding interface in the 22 kDa haem-binding protein p22HBP

The 22 kDa haem-binding protein, p22HBP, is highly expressed in erythropoietic tissues and binds to a range of metallo- and non-metalloporphyrin molecules with similar affinities, suggesting a role in haem regulation or synthesis. We have determined the three-dimensional solution structure of p22HBP and mapped the porphyrin-binding site, which comprises a number of loops and a alpha-helix all located on a single face of the molecule. The structure of p22HBP is related to the bacterial multi-drug resistance protein BmrR, and is the first protein with this fold to be identified in eukaryotes. Strikingly, the porphyrin-binding site in p22HBP is located in a similar position to the drug-binding site of BmrR. These similarities suggest that the broad ligand specificity observed for both BmrR and p22HBP may result from a conserved ligand interaction mechanism. Taken together, these data suggest that the both the fold and its associated function, that of binding to a broad range of small hydrophobic molecules, are ancient, and have been adapted throughout evolution for a variety of purposes.     

Human immunodeficiency virus type 1 envelope glycoprotein oligomerization requires the gp41 amphipathic alpha-helical/leucine zipper-like sequence.

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) oligomerization was investigated by coexpressing wild-type and truncated envelope glycoproteins to determine the minimum sequence required for mutant-wild-type hetero-oligomerization. The gp41 putative amphipathic alpha-helix, Leu-550 to Leu-582, was essential for hetero-oligomer formation. Alanine substitution of 9 of the 10 residues composing the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence was required to inhibit mutant-wild-type hetero-oligomerization and to render the envelope glycoprotein precursor, gp160, monomeric. This indicates that multiple hydrophobic contacts contribute to the stable envelope glycoprotein oligomeric structure. Single alanine substitutions within the hydrophobic repeat sequence did not affect gp160 oligomeric structure but abolished syncytium-forming function. Some mutations also diminished gp160 processing efficiency and the association between gp120 and gp41 in a position-dependent manner. These results indicate that the gp41 amphipathic alpha-helix 4-3 hydrophobic repeat sequence plays a central role in HIV-1 envelope glycoprotein oligomerization and fusion function.     

Influence of lipids on membrane assembly and stability of the potassium channel KcsA

A novel antibacterial peptide, moricin, isolated from the silkworm Bombyx mori, consists of 42 amino acids. It is highly basic and the amino acid sequence has no significant similarity to those of other antibacterial peptides. The 20 structures of moricin in methanol have been determined from two-dimensional 1H-nuclear magnetic resonance spectroscopic data. The solution structure reveals an unique structure comprising of a long alpha-helix containing eight turns along nearly the full length of the peptide except for four N-terminal residues and six C-terminal residues. The electrostatic surface map shows that the N-terminal segment of the alpha-helix, residues 5-22, is an amphipathic alpha-helix with a clear separation of hydrophobic and hydrophilic faces, and that the C-terminal segment of the alpha-helix, residues 23-36, is a hydrophobic alpha-helix except for the negatively charged surface at the position of Asp30. The results suggest that the amphipathic N-terminal segment of the alpha-helix is mainly responsible for the increase in permeability of the membrane to kill the bacteria.     

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.     

NMR solution structure of the mitochondrial F1beta presequence from Nicotiana plumbaginifolia

We have isolated, characterized and determined the three-dimensional NMR solution structure of the presequence of ATPsynthase F1beta subunit from Nicotiana plumbaginifolia. A general method for purification of presequences is presented. The method is based on overexpression of a mutant precursor containing a methionine residue introduced at the processing site, followed by CNBr-cleavage and purification of the presequence on a cation-exchange column. The F1beta presequence, 53 amino acid residues long, retained its native properties as evidenced by inhibition of in vitro mitochondrial import and processing at micromolar concentrations. CD spectroscopy revealed that the F1beta presequence formed an alpha-helical structure in membrane mimetic environments such as SDS and DPC micelles (approximately 50% alpha-helix), and in acidic phospholipid bicelles (approximately 60% alpha-helix). The NMR solution structure of the F1beta presequence in SDS micelles was determined on the basis of 518 distance and 21 torsion angle constraints. The structure was found to contain two helices, an N-terminal amphipathic alpha-helix (residues 4-15) and a C-terminal alpha-helix (residues 43-53), separated by a largely unstructured 27 residue long internal domain. The N-terminal amphipathic alpha-helix forms the putative Tom20 receptor binding site, whereas the C-terminal alpha-helix is located upstream of the mitochondrial processing peptidase cleavage site.     

The structure of the lactose permease derived from Raman spectroscopy and prediction methods.

The secondary structure of the lactose permease of Escherichia coli reconstituted in lipid membranes was determined by Raman spectroscopy. The alpha-helix content is approximately 70%, the beta-strand content below 10% and beta-turns contribute 15%. About 1/3 of the residues in alpha-helices and most other residues are exposed to water. Employing a method for structural prediction which accounts for amphipathic helices, 10 membrane-spanning helices are predicted which are either hydrophobic or amphipathic. They are expected to form an outer ring of helices in the membrane. The interior of the ring would be made of residues which are predominantly hydrophilic and, evoking the analogy to sugar-binding proteins, suited to provide the sugar binding site.     

Identification of a common protein association region in the neuronal Cdk5 activator

Cyclin-dependent protein kinase 5 (Cdk5) depends on the association with neuronal Cdk5 activator (Nck5a) for kinase activity. A variety of cellular proteins have been shown to undergo high affinity association with Nck5a, including three novel proteins, C42, C48, and C53 found by a yeast two-hybrid screen (Ching, Y. P., Qi, Z., and Wang, J. H. (2000) Gene 242, 285-294). The three proteins show competitive binding to Nck5a suggesting that they bind at a common site. The binding site has been mapped to a region of 26 amino acid residues (residues 145 to 170) at the N-terminal boundary of the kinase activation domain of Nck5a. This region of Nck5a contains an amphipathic alpha-helix whose hydrophobic face is involved in Cdk5 activation (Chin, K. T., Ohki, S, Tang, D., Cheng, H. C., Wang, J. H. , and Zhang, M. (1999) J. Biol. Chem. 274, 7120-7127). Several lines of evidence suggest that Nck5a interacts with the binding proteins at the hydrophilic face of the amphipathic alpha-helix. First, the Nck5a-(145-170) peptide can bind Cdk5 and Nck5a-binding proteins simultaneously. Second, the association of Nck5a-(145-170) to C48 can be markedly reduced by high ionic strength whereas the interaction between Nck5a and Cdk5 is not affected. Third, substitution of Glu(157) by glutamine in Nck5a-(145-170) abolishes the peptide's ability to bind to the three Nck5a-binding proteins without diminishing its Cdk5 binding activity.     

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane α-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (≤ 1 μM) in SDS-EmrE is monomeric, but upon increasing EmrE concentration, a variety of multimeric states can be observed by SDS-Tricine polyacrylamide gel electrophoresis (PAGE). Addition of the (QCC), tetraphenyl phosphonium (TPP), to SDS-EmrE samples enhanced EmrE multimer formation using SDS-Tricine PAGE. The relative shapes of EmrE multimers in SDS with or without TPP addition were determined by small angle neutron scattering (SANS) analysis and revealed that EmrE dimers altered in conformation depending on the SDS concentration. SANS analysis also revealed that relative shapes of larger EmrE multimers (≥ 100 nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.     

1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin and RACK1

The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.     

High-level expression and mutagenesis of recombinant human phosphatidylcholine transfer protein using a synthetic gene: evidence for a C-terminal membrane binding domain

Phosphatidylcholine transfer protein (PC-TP) is a 214-amino acid cytosolic protein that promotes intermembrane transfer of phosphatidylcholines, but no other phospholipid class. To probe mechanisms for membrane interactions and phosphatidylcholine binding, we expressed recombinant human PC-TP in Escherichia coli using a synthetic gene. Optimization of codon usage for bacterial protein translation increased expression of PC-TP from trace levels to >10% of the E. coli cytosolic protein mass. On the basis of secondary structure predictions of an amphipathic alpha-helix (residues 198-212) in proximity to a hydrophobic alpha-helix (residues 184-193), we explored whether the C-terminus might interact with membranes and promote binding of phosphatidylcholines. Consistent with this possibility, truncation of five residues from the C-terminus shortened the predicted amphipathic alpha-helix and decreased PC-TP activity by 50%, whereas removal of 10 residues eliminated the alpha-helix, abolished activity, and markedly decreased the level of membrane binding. Circular dichroic spectra of synthetic peptides containing one ((196-214)PC-TP) or both ((183-214)PC-TP) predicted C-terminal alpha-helices in aqueous buffer were most consistent with random coil structures. However, both peptides adopted alpha-helical configurations in the presence of trifluoroethanol or phosphatidylcholine/phosphatidylserine small unilamellar vesicles. The helical content of (196-214)PC-TP increased in proportion to vesicle phosphatidylserine content, consistent with stabilization of the alpha-helix at the membrane surface. In contrast, the helical content of (183-214)PC-TP was not influenced by vesicle composition, implying that the more hydrophobic of the alpha-helices penetrated into the membrane bilayer. These studies suggest that tandem alpha-helices located near the C-terminus of PC-TP facilitate membrane binding and extraction of phosphatidylcholines.     

Structural analysis of the heparin-binding site of the NC1 domain of collagen XIV by CD and NMR.

Type XIV collagen, a fibril-associated collagen with interrupted triple helices (FACIT), interacts with the surrounding extracellular matrix and/or with cells via its binding to glycosaminoglycans (GAGs). To further characterize such interactions in the NC1 domain of chicken collagen XIV, we identified amino acids essential for heparin binding by affinity chromatography analysis after proteolytic digestion of the synthetic peptide NC1(84-116). The 3D structure of this peptide was then obtained using circular dichroism and NMR. The NC1(84-116) peptide appeared poorly structured in water, but the stabilization of its conformation by the interaction with hydrophobic surfaces or by using cosolvents (TFE, SDS) revealed a high propensity to adopt an alpha-helical folding. A 3D structure model of NC1(84-116), calculated from NMR data recorded in a TFE/water mixture, showed that the NC1-heparin binding site forms a amphipathic alpha-helix exhibiting a twisted basic groove. It is structurally similar to the consensus spatial alpha-helix model of heparin-binding [Margalit et al. (1993) J. Biol. Chem. 268, 19228-19231], except that the GAG binding domain of NC1 may be extended over 18 residues, that is, the NC1(94-111) segment. In addition, the formation of a hydrophobic groove upon helix formation suggests the contribution of additional sequences to ensure the stability of the GAG-binding domain. Overall the NC1(84-116) model exhibits a nativelike conformation which presents suitably oriented residues for the interaction with a specific GAG.     

Conformational changes in the multidrug transporter EmrE associated with substrate binding

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP(+)) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP(+) binding; the data showed that one TPP(+) molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP(+) produced well ordered two-dimensional crystals. Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7A resolution. An average native EmrE projection structure was calculated from the c222 and p222(1) crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP(+) bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPP(+) bound at the monomer-monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane alpha-helix.     

A role of the C-terminal region adjacent to the zinc-fingers in the DNA binding ability of Rme1p, a regulator of meiosis in S. cerevisiae.

Rme1p is a zinc-finger protein and has a pivotal role in control of meiosis in Saccharomyces cerevisiae. The DNA binding domain of Rme1p consists of three zinc-finger segments and the C-terminal 16 amino acid residues (called C-TR). To examine the role of C-TR, a series of mutant Rme1p fused with maltose binding protein (MBP) were constructed, purified, and characterized, in terms of the DNA binding ability. The basic amino acid residues R287 and K290, and the hydrophobic residues F288, L292, 1295, and L296 play an important role for DNA binding, suggesting that the C-TR forms an amphipathic alpha-helix. Also, it was shown that the mutations in the basic amino acid residues abolish the repression and inhibition of spore formation by Rme1p in vivo. Hence, the C-TR is important for in vivo function of Rme1p.     

Shared and unique determinants of the erythropoietin (EPO) receptor are important for binding EPO and EPO mimetic peptide.

We have shown previously that Phe93 in the extracellular domain of the erythropoietin (EPO) receptor (EPOR) is crucial for binding EPO. Substitution of Phe93 with alanine resulted in a dramatic decrease in EPO binding to the Escherichia coli-expressed extracellular domain of the EPOR (EPO-binding protein or EBP) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the EBP bound to an EPO-mimetic peptide (EMP1), suggesting that Phe93 is also important for EMP1 binding. We used alanine substitution of EBP residues that contact EMP1 in the crystal structure to investigate the function of these residues in both EMP1 and EPO binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both EPO and EMP1 binding, Met150 is not important for EPO binding but is critical for EMP1 binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both EPO and EMP1, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the EPOR for productive ligand binding.     

Molecular mapping of a histocompatibility-restricted immunodominant T cell epitope with synthetic and natural peptides: implications for T cell antigenic structure

We have prepared synthetic and natural peptides that have allowed delineation of a major antigenic site of myoglobin recognized by histocompatibility (I-Ed)-restricted T cell clones. The smallest peptide capable of stimulating T cell proliferation consisted of residues 136-146. Residues Glu 136, Lys 140, and Lys 145 were essential for antigenicity, whereas Lys 133 and Tyr 146 added potency but were not required for antigenicity. The periodicity of these residues suggests that folding the peptide into its native alpha-helical structure may be essential for antigenicity either by forming a hydrophilic binding site for the T cell receptor or by participating in antigen presentation. The same folding could also produce a hydrophobic site on the opposite side of the alpha-helix that could participate in hydrophobic interactions. The extrapolation of these findings to other known peptide antigens suggests that this tendency to form an amphipathic alpha-helix may be a general property of antigenic sites recognized by T cells, perhaps due to a different functional role of each type of site in eliciting T cell responses.