Structural and functional implications of a proline residue in the antimicrobial peptide gaegurin.

Although it is commonly known as a helix breaker, proline residues have been found in the alpha-helical regions of many peptides and proteins. The antimicrobial peptide gaegurin displays alpha-helical structure and has a central proline residue (P14). The structure and activity of gaegurin and its alanine derivative (P14A) were determined by various spectroscopic methods, restrained molecular dynamics, and biological assays. Both P14 and P14A exhibited cooperative helix formation in solution, but the helical stability of P14 was reduced substantially when compared to that of P14A. Chemical-shift analysis indicated that both of the peptides formed curved helices and that P14 showed diminished stability in the region around the central proline. However, hydrogen-exchange data revealed remarkable differences in the location of stable amide protons. P14 showed a stable region in the concave side of the curved helix, while P14A exhibited a stable region in the central turn of the helix. The model structure of P14 exhibited a pronounced kink, in contrast to the uniform helix of P14A. Both peptides showed comparable binding affinities for negatively charged lipids, while P14 had a considerably reduced affinity for a neutral lipid. With its destabilized alpha-helix, P14 exhibited greater antibacterial activity than did P14A. Hence, electrostatic interaction between helical peptides and lipid membranes is believed to be the dominant factor for antibacterial activity. Moreover, helical stability can modulate peptide binding to membranes that is driven by electrostatic interactions. The observation that P14 is a more potent antibacterial agent than P14A implies that the helical kink of P14 plays an important role in the disruption of bacterial membranes.     

Structural and functional consequences of the mutation of a conserved arginine residue in alphaA and alphaB crystallins.

A point mutation of a highly conserved arginine residue in alphaA and alphaB crystallins was shown to cause autosomal dominant congenital cataract and desmin-related myopathy, respectively, in humans. To study the structural and functional consequences of this mutation, human alphaA and alphaB crystallin genes were cloned and the conserved arginine residue (Arg-116 in alphaA crystallin and Arg-120 in alphaB crystallin) mutated to Cys and Gly, respectively, by site-directed mutagenesis. The recombinant wild-type and mutant proteins were expressed in Escherichia coli and purified. The mutant and wild-type proteins were characterized by SDS-polyacrylamide gel electrophoresis, Western immunoblotting, gel permeation chromatography, fluorescence, and circular dichroism spectroscopy. Biophysical studies reveal significant differences between the wild-type and mutant proteins. The chaperone-like activity was studied by analyzing the ability of the recombinant proteins to prevent dithiothreitol-induced aggregation of insulin. The mutations R116C in alphaA crystallin and R120G in alphaB crystallin reduce the chaperone-like activity of these proteins significantly. Near UV circular dichroism and intrinsic fluorescence spectra indicate a change in tertiary structure of the mutants. Far UV circular dichroism spectra suggest altered packing of the secondary structural elements. Gel permeation chromatography reveals polydispersity for both of the mutant proteins. An appreciable increase in the molecular mass of the mutant alphaA crystallin is also observed. However, the change in oligomer size of the alphaB mutant is less significant. These results suggest that the conserved arginine of the alpha-crystallin domain of the small heat shock proteins is essential for their structural integrity and subsequent in vivo function.     

Structural and functional consequences of mutating a proteobacteria-specific surface residue in the catalytic domain of Escherichia coli GluRS

Nucleotides whose mutations seriously affect glutamylation efficiency are experimentally known for Escherichia coli tRNA(Glu). However, not much is known about functional hotspots on the complementary enzyme, glutamyl-tRNA synthetase (GluRS). From structural and functional studies on an Arg266Leu mutant of E. coli GluRS, we demonstrate that Arg266 is essential for efficient glutamylation of tRNA(Glu). Consistent with this result, we found that Arg266 is a conserved signature of proteobacterial GluRS. In contrast, most non-proteobacterial GluRS contain Leu, and never Arg, at this position. Our results imply a unique strategy of glutamylation of tRNA(Glu) in proteobacteria under phylum-specific evolutionary compulsions.     

Structural and functional consequences of peptide-carbohydrate mimicry. Crystal structure of a carbohydrate-mimicking peptide bound to concanavalin A

The functional consequences of peptide-carbohydrate mimicry were analyzed on the basis of the crystal structure of concanavalin A (ConA) in complex with a carbohydrate-mimicking peptide, DVFYPYPYASGS. The peptide binds to the non-crystallographically related monomers of two independent dimers of ConA in two different modes, in slightly different conformations, demonstrating structural adaptability in ConA-peptide recognition. In one mode, the peptide has maximum interactions with ConA, and in the other, it shows relatively fewer contacts within this site but significant contacts with the symmetry-related subunit. Neither of the peptide binding sites overlaps with the structurally characterized mannose and trimannose binding sites on ConA. Despite this, the functional mimicry between the peptide and carbohydrate ligands was evident. The peptide-inhibited ConA induced T cell proliferation in a dose-dependent manner. The effect of the designed analogs of the peptide on ConA-induced T cell proliferation and their recognition by the antibody response against alpha-d-mannopyranoside indicate a role for aromatic residues in functional mimicry. Although the functional mimicry was observed between the peptide and carbohydrate moieties, the crystal structure of the ConA-peptide complex revealed that the two peptide binding sites are independent of the methyl alpha-d-mannopyranoside binding site.     

Structural and functional insights into a peptide bond-forming bidomain from a nonribosomal peptide synthetase

The crystal structure of the bidomain PCP-C from modules 5 and 6 of the nonribosomal tyrocidine synthetase TycC was determined at 1.8 A resolution. The bidomain structure reveals a V-shaped condensation domain, the canyon-like active site groove of which is associated with the preceding peptidyl carrier protein (PCP) domain at its donor side. The relative arrangement of the PCP and the peptide bond-forming condensation (C) domain places the active sites approximately 50 A apart. Accordingly, this PCP-C structure represents a conformational state prior to peptide transfer from the donor-PCP to the acceptor-PCP domain, implying the existence of additional states of PCP-C domain interaction during catalysis. Additionally, PCP-C exerts a mode of cyclization activity that mimics peptide bond formation catalyzed by C domains. Based on mutational data and pK value analysis of active site residues, it is suggested that nonribosomal peptide bond formation depends on electrostatic interactions rather than on general acid/base catalysis.     

Structural and functional consequences of methionine oxidation in thrombomodulin

Thrombomodulin (TM) is an endothelial cell surface glycoprotein that is responsible for switching the catalytic activity of thrombin away from fibrinogen cleavage (pro-coagulant) and towards protein C cleavage (anticoagulant). Although TM is a large protein, only the fourth and fifth epidermal growth factor-like (EGF-like) domains are required for anticoagulant function. These two domains must work together, and the linker between the two domains contains a single methionine residue, Met 388. Oxidation of Met 388 is deleterious for TM activity. Structural studies, both X-ray and NMR, of wild type and variants at position 388 show that Met 388 provides a key linkage between the two domains. Oxidation of the methionine has consequences for the structure of the fifth domain, which binds to thrombin. Oxidation also appears to disrupt the interdomain contacts resulting in structural and dynamic changes. The functional consequences of oxidation of Met 388 include decreased anticoagulant activity. Oxidative stress from several causes is reflected in lower serum levels of activated protein C and a higher thrombotic tendency, and this is thought to be linked to the oxidation of Met 388 in TM. Thus, TM structure and function are altered in a subtle but functionally critical way upon oxidation of Met 388.     

C-terminal anchoring of a peptide to class II MHC via the P10 residue is compatible with a peptide bulge.

The binding of antigenic peptide to class II MHC is mediated by hydrogen bonds between the MHC and the peptide, by salt bridges, and by hydrophobic interactions. The latter are confined to a number of deeper pockets within the peptide binding groove, and peptide side chains that interact with these pockets are referred to as anchor residues. T cell recognition involves solvent-accessible peptide residues along with minor changes in MHC helical pitch induced by the anchor residues. In class I MHC there is an added level of epitope complexity that results from binding of longer peptides that bulge out into the solvent-accessible, T cell contact area. Unlike class I MHC, class II MHC does not bind peptides of discrete length, and the possibility of peptide bulging has not been clearly addressed. A peptide derived from position 24-37 of integrin beta(3) can either bind or not bind to the class II MHC molecule HLA DRB3*0101 based on a polymorphism at the P9 anchor. We show that the loss of binding can be compensated by changes at the P10 position. We propose that this could be an example of a class II peptide bulge. Although not as efficient as P9 anchoring, the use of P10 as an anchor adds another possible mechanism by which T cell epitopes can be generated in the class II presentation system.     

Structural transition of a 15 amino acid residue peptide induced by GM1

The ganglioside GM1-binding peptide, p3, with a sequence of VWRLLAPPFSNRLLP, displayed a clear structural alteration depending on the presence or absence of GM1 micelles. The three-dimensional structures of the p3 peptide in the free and GM1 bound states were analyzed using two-dimensional NMR spectroscopic experiments with distance-restrained simulated annealing calculations. The NMR experiments for the p3 peptide alone indicated that the peptide has two conformers derived from the exchange of cis and trans forms at Pro(7)-Pro(8). Further study with theoretical modeling revealed that the p3 peptide has a curb conformation without regular secondary structure. On the other hand, the NMR studies for the p3 peptide with the GM1 micelles elucidated a trans conformer and gave a structure stabilized by hydrophobic interactions of beta- and helical turns. Based on these structural investigations, tryptophan, a core residue of the hydrophobic cluster, might be an essential residue for the recognition of the GM1 saccharides. The dynamic transition of the p3 peptide may play an important role in the function of GM1 as a multiple receptor as in the traditional pathway of the infection by cholera toxin.     

Structural and dynamic consequences of increasing repeats in a MUC1 peptide tumor antigen

MUC1 mucin is a large transmembrane glycoprotein whose extracelluler domain is composed of repeating units of a 20 amino acid sequence. In the cancer associated state, this protein expression becomes upregulated and underglycosylated. Previous studies, which show an enhanced binding of a 5-repeat over a 1-repeat MUC1 peptide to a panel of anti-MUC1 antibodies, have led us to investigate the structural and dynamic consequences of increasing repeat number. Two MUC1 peptides were studied: a 16mer corresponding to slightly less than one full repeat of the MUC1 tandem repeat sequence (GVTSAPDTRPAPGSTA) and a 40mer corresponding to two full repeats of the MUC1 sequence (VTSAPDTRPAPGSTAPPAHG)2. Isotopically labeled versions of these MUC1 peptides were cloned, expressed, purified, and evaluated structurally and dynamically using 15N- and 13C-edited NMR approaches. The data show that MUC1 structure, dynamics, and antibody binding affinity are invariant with increasing repeat number. In light of these results, we conclude that the enhanced antibody affinity of the 5-repeat over the 1-repeat MUC1 peptide is due to multivalency effects, and not due to the development of higher order structure in the longer length peptides. The implications of these results are discussed within the context of a multiple repeat MUC1 breast cancer vaccine design.     

Structural and functional characterization of a mutant of Pseudocerastes persicus natriuretic peptide

We hereby report on a mutational analysis of a novel natriuretic peptide (PNP), recently isolated by us from the Iranian snake venom. The PNP variant (mutPNP) with four substitutions (G16T, K18S, R21S, G23R) and a disulfide bonded ring shortened by 3 residues. mutPNP peptide was expressed in pET32 and purified by affinity separation on nickel resin followed by RP-HPLC chromatography. The conformation of mutPNP was characterized in solution by 1H nuclear magnetic resonance spectroscopy, where it was found that the 14-residue disulfide bonded ring, like the 17-residue ring in PNP, retains a high degree of conformational flexibility. The conformation of mutPNP bound to NPR-C receptor was predicted by homology protein structure modeling. When injected intravenously into rats, mutPNP, in contrast to PNP had no physiological effect on blood pressure or on diuresis. The loss of physiological activity is explained in terms of the modeled bound conformation and the ensemble of solution conformations obtained using the NMR constraints.     

Structural and functional consequences of galactolipids on thylakoid membrane organization

Photosynthetic membranes of higher plant chloroplasts are composed primarily of polar, but uncharged, galactolipids unlike most mammalian membranes which contain large amounts of phosphatidylcholine. It is unclear what role(s) the galactolipids play in maintaining the differentiated thylakoid membranes, or in stabilizing the photosynthetically active enzyme complexes. Some of the membrane complexes show no lipid selectivity for maintaining structural or functional integrity. Others are poisoned or dissociated in the presence of high concentrations of a trace lipid class. The efficiency of energy transfer and the reconstitution of protein complexes into liposomes are dependent on the lipid class employed. The lipids are asymmetrically arranged along and across the thylakoid membranes but not as distinctly as the proteins.Abbreviations DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SQDG sulfoquinovosyldiglyceride - PG phosphatidylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - PSI photosystem I - PSII photosystem II - LHC chlorophylla/b lightharvesting complex - cytb ₆ f cytochromeb ₆ f complex - CF₀/CF₁ coupling factor ATPase - DCIP 2,6-dichlorophenolindophenol - LRa galactolipase fromRhizopus arrhis     

Cutting edge: a single MHC anchor residue alters the conformation of a peptide-MHC complex inducing T cells that survive negative selection

We generated transgenic mice that expressed hen egg-white lysozyme (HEL) under a class II MHC promoter. The A7 line expressed HEL with a point mutation in the Asp(52) residue, the main anchor amino acid responsible for the selection of the chemically dominant family of peptides (52-60) by I-A(k) molecules. Mice expressing HEL with Ala(52) were completely unresponsive when immunized with the same protein, i.e., HEL A52. However, the same mice immunized with wild-type HEL elicited T cells that recognized a conformation of the 52-61 core sequence uniquely different between Asp(52) and Ala(52) containing peptides. Importantly, some T cells also recognized the HEL A52 peptide given exogenously but not the same peptide processed from HEL A52 protein. Thus, a core MHC anchor residue influences markedly the specificity of the T cells. We discuss the relevance of these findings to autoimmunity and vaccination with altered peptides.     

Structural and functional effects of mutations altering the subunit interface of mitochondrial malate dehydrogenase

Among highly conserved residues in eucaryotic mitochondrial malate dehydrogenases are those with roles in maintaining the interactions between identical monomeric subunits that form the dimeric enzymes. The contributions of two of these residues, Asp-43 and His-46, to structural stability and catalytic function were investigated by construction of mutant enzymes containing Asn-43 and Leu-46 substitutions using in vitro mutagenesis of the Saccharomyces cerevisiae gene (MDH1) encoding mitochondrial malate dehydrogenase. The mutant enzymes were expressed in and purified from a yeast strain containing a disruption of the chromosomal MDH1 locus. The enzyme containing the H46L substitution, as compared to the wild type enzyme, exhibits a dramatic shift in the pH profile for catalysis toward an optimum at low pH values. This shift corresponds with an increased stability of the dimeric form of the mutant enzyme, suggesting that His-46 may be the residue responsible for the previously described pH-dependent dissociation of mitochondrial malate dehydrogenase. The D43N substitution results in a mutant enzyme that is essentially inactive in in vitro assays and that tends to aggregate at pH 7.5, the optimal pH for catalysis for the dimeric wild type enzyme.     

MHC allele-specific binding of a malaria peptide makes it become promiscuous on fitting a glycine residue into pocket 6

Peptide 1585 (EVLYLKPLAGVYRSLKKQLE) has a highly conserved amino-acid sequence located in the Plasmodium falciparum main merozoite surface protein (MSP-1) C-terminal region, required for merozoite entry into human erythrocytes and therefore represents a vaccine candidate for P. falciparum malaria. Original sequence-specific binding to five HLA DRB1* alleles (0101, 0102, 0401, 0701, and 1101) revealed this peptide's specific HLA DRB1*0102 allele binding. This peptide's allele-specific binding to HLA DRB1*0102 took on broader specificity for the DRB1*0101, -0401, and -1101 alleles when lysine was replaced by glycine in position 17 (peptide 5198: EVLYLKPLAGVYRSLKG(17)QLE). Binding of the identified G(10)VYRSLKGQLE(20) C-terminal register to these alleles suggests that peptide promiscuous binding relied on fitting Y(12), L(15), and G(17) into P-1, P-4, and P-6, respectively. The implications of the findings and the future of this synthetic vaccine candidate are discussed.     

Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 A. In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.     

Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme.

To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102----Lys (M102K) and Leu 133----Asp (L133D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pKa of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with 13C incorporated at the C epsilon position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.     

Structural and functional dissection of an MHC class I antigen-binding adenovirus glycoprotein.

The early transmembrane glycoprotein E19 of adenovirus-2 binds to class I antigens of the major histocompatibility complex (MHC). The association is initiated in the endoplasmic reticulum of infected cells and abrogates the intracellular transport of the class I molecules. To examine which parts of the E19 molecule are responsible for the association with the class I antigens and which parts confine the protein to the endoplasmic reticulum we have constructed a series of mutated E19 genes, which have been expressed in an improved mammalian expression vector. By various manipulations the membrane anchoring and the cytoplasmic domains were removed from the protein. The biosynthesis of the mutant protein was examined. All mutant proteins were secreted from the cells suggesting that the transmembrane and/or cytoplasmic portions of the E19 molecule are responsible for its confinement to the endoplasmic reticulum. The ability to associate with class I antigens was retained by the lumenal domain of the E19 protein.     

Structural and functional characterization of AtPTR3, a stress-induced peptide transporter of Arabidopsis

A T-DNA tagged mutant line of Arabidopsis thaliana, produced with a promoter trap vector carrying a promoterless gus (uidA) as a reporter gene, showed GUS induction in response to mechanical wounding. Cloning of the chromosomal DNA flanking the T-DNA revealed that the insert had caused a knockout mutation in a PTR-type peptide transporter gene named At5g46050 in GenBank, here renamed AtPTR3. The gene and the deduced protein were characterized by molecular modelling and bioinformatics. Molecular modelling of the protein with fold recognition identified 12 transmembrane spanning regions and a large loop between the sixth and seventh helices. The structure of AtPTR3 resembled the other PTR-type transporters of plants and transporters in the major facilitator superfamily. Computer analysis of the AtPTR3 promoter suggested its expression in roots, leaves and seeds, complex hormonal regulation and induction by abiotic and biotic stresses. The computer-based hypotheses were tested experimentally by exposing the mutant plants to amino acids and several stress treatments. The AtPTR3 gene was induced by the amino acids histidine, leucine and phenylalanine in cotyledons and lower leaves, whereas a strong induction was obtained in the whole plant upon exposure to salt. Furthermore, the germination frequency of the mutant line was reduced on salt-containing media, suggesting that the AtPTR3 protein is involved in stress tolerance in seeds during germination.Figure a Induction of AtPTR3 gene by amino acids. GUS staining of line 9 plants eight hours after induction with amino acids. Control indicates plant treated with water. His, Leu and Phe indicate plants treated with 10 mM amino acids histidine, leucine or phenylalanine, respectively. b Induction of AtPTR3 gene by salt. GUS staining of line 9 plants grown on MS medium on different salt concentrations: Control indicates plant grown on MS medium and 100 mM, 120 mM and 140 mM indicate plants grown on MS medium supplemented with the indicated NaCl concentrations. Size of the plants grown on salt medium has been magnified. c Germination frequency of Atptr3 knockout mutant line is reduced on salt medium. Atptr3 knockout mutant (9) and wild type C24 (WT) sown on MS medium (Control) and MS medium supplemented with salt (140 mM NaCl).