Folding simulations of a three-stranded antiparallel β-sheet peptide

Protein folding is a grand challenge of the postgenomic era. In this paper, 58 folding events sampled during 47 molecular dynamics trajectories for a total simulation time of more than 4 μs provide an atomic detail picture of the folding of a 20-residue synthetic peptide with a stable three-stranded antiparallel β-sheet fold. The simulations successfully reproduce the NMR solution conformation, irrespective of the starting structure. The sampling of the conformational space is sufficient to determine the free energy surface and localize the minima and transition states. The statistically predominant folding pathway involves the formation of contacts between strands 2 and 3, starting with the side chains close to the turn, followed by association of the N-terminal strand onto the preformed 2–3 β-hairpin. The folding mechanism presented here, formation of a β-hairpin followed by consolidation, is in agreement with a computational study of the free energy surface of another synthetic three-stranded antiparallel β-sheet by Bursulaya and Brooks [(1999) J. Am. Chem. Soc. 121, 9947–9951]. Hence, it might hold in general for antiparallel β-sheets with short turns.

An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated β-sheet structure for amyloid

X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-β x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, β-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-Å resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded β-sheets. The unusually high density of the crystals shows that the packed β-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed β-sheets. This dry β-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.

Peptide hybrids containing α- and β-amino acids: Structure of a decapeptide β-hairpin with two facing β-phenylalanine residues

A β-hairpin conformation has been characterized in crystals of the decapeptide t-butoxycarbonyl-Leu-Val-βPhe-Val-DPro-Gly-Leu-βPhe-Val-Val-methyl ester [βPhe; (S)-β3 homophenylalanine] by x-ray diffraction. The polypeptide chain reversal is nucleated by the centrally positioned DPro-Gly segment, which adopts a type-I′ β-turn conformation. Four intramolecular cross-strand hydrogen bonds stabilize the peptide fold. The βPhe(3) and βPhe(8) residues occupy facing positions on the hairpin, with the side chains projecting on opposite faces of the β-sheet. At the site of insertion of β-residues, the polarity of the peptide units along each strand reverses, as compared with the α-peptide segments. In this analog, a small segment of a polar sheet is observed, where adjacent CO and NH groups line up in opposite directions in each strand. In the crystal, an extended β-sheet is formed by hydrogen bonding between strands of antiparallel pairs of β-hairpins. The crystallographic parameters for C65H102N10O13⋅ 3H2O are: space group P212121; a = 19.059(8) Å, b = 19.470(2) Å, c = 21.077(2) Å; Z = 4; agreement factor R1 = 9.12% for 3,984 data observed >4σ(F) and a resolution of 0.90 Å.

A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.

The structure of bovine F1-ATPase complexed with the peptide antibiotic efrapeptin.

In the previously determined structure of mitochondrial F1-ATPase determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. AMP-PNP and ADP are bound to subunits beta TP and beta DP, respectively, and the third beta-subunit (beta E) has no bound nucleotide. The efrapeptins are a closely related family of modified linear peptides containing 15 amino acids that inhibit both ATP synthesis and hydrolysis by binding to the F1 catalytic domain of F1F0-ATP synthase. In crystals of F1-ATPase grown in the presence of both nucleotides and inhibitor, efrapeptin is bound to a unique site in the central cavity of the enzyme. Its binding is associated with small structural changes in side chains of F1-ATPase around the binding pocket. Efrapeptin makes hydrophobic contacts with the alpha-helical structure in the gamma-subunit, which traverses the cavity, and with subunit beta E and the two adjacent alpha-subunits. Two intermolecular hydrogen bonds could also form. Intramolecular hydrogen bonds probably help to stabilize efrapeptin's two domains (residues 1-6 and 9-15, respectively), which are connected by a flexible region (beta Ala-7 and Gly-8). Efrapeptin appears to inhibit F1-ATPase by blocking the conversion of subunit beta E to a nucleotide binding conformation, as would be required by an enzyme mechanism involving cyclic interconversion of catalytic sites.

Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides.

Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.

Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution.

The measurement of dipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported. The splittings appear as small field-dependent perturbations of normal scalar couplings. Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein. Implications as an additional source of information for protein structure determination in solution are discussed.

Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.

Structure of the gene for porcine peptide antibiotic PR-39, a cathelin gene family member: comparative mapping of the locus for the human peptide antibiotic FALL-39.

PR-39 is a porcine 39-aa peptide antibiotic composed of 49% proline and 24% arginine, with an activity against Gram-negative bacteria comparable to that of tetracycline. In Escherichia coli, it inhibits DNA and protein synthesis. PR-39 was originally isolated from pig small intestine, but subsequent cDNA cloning showed that the gene is expressed in the bone marrow. The open reading frame of the clone showed that PR-39 is made as 173-aa precursor whose proregion belongs to the cathelin family. The PR39 gene, which is rather compact and spans only 1784 bp has now been sequenced. The coding information is split into four exons. The first exon contains the signal sequence of 29 residues and the first 37 residues of the cathelin propart. Exons 2 and 3 contain only cathelin information, while exon 4 codes for the four C-terminal cathelin residues and the mature PR-39 peptide extended by three residues. The sequenced upstream region (1183 bp) contains four potential recognition sites for NF-IL6 and three for APRF, transcription factors known to regulate genes for both cytokines and acute phase response factors. Genomic hybridizations revealed a fairly high level of restriction fragment length polymorphism and indicated that there are at least two copies of the PR39 gene in the pig genome. PR39 was mapped to pig chromosome 13 by linkage and in situ hybridization mapping. The gene for the human peptide antibiotic FALL-39 (also a member of the cathelin family) was mapped to human chromosome 3, which is homologous to pig chromosome 13.

The crystal structure of an N-terminal two-domain fragment of vascular cell adhesion molecule 1 (VCAM-1): a cyclic peptide based on the domain 1 C-D loop can inhibit VCAM-1-alpha 4 integrin interaction.

Vascular cell adhesion molecule 1 (VCAM-1) represents a structurally and functionally distinct class of immunoglobulin superfamily molecules that bind leukocyte integrins and are involved in inflammatory and immune functions. X-ray crystallography defines the three-dimensional structure of the N-terminal two-domain fragment that participates in ligand binding. Residues in domain 1 important for ligand binding reside in the C-D loop, which projects markedly from one face of the molecule near the contact between domains 1 and 2. A cyclic peptide that mimics this loop inhibits binding of alpha 4 beta 1 integrin-bearing cells to VCAM-1. These data demonstrate how crystallographic structural information can be used to design a small molecule inhibitor of biological function.

Computer determination of peptide conformations in water: different roads to structure.

Fragments of proteins (short peptides) that "fold" suggest a mechanism of how complete conformational search in protein folding is avoided. We used a computational method to determine structures of two foldable peptides in explicit water: RVEW and CSVTC. The optimization starts from random structures and no experimental constraints are used. In agreement with NMR data, the simulations find a hydrophobic pair (Val/Trp) in REVW. The structure of CSVTC is induced by a surface water that bridges two amide hydrogens, a drive to structure hypothesized by Ben-Naim [Ben-Naim, A. (1990) J. Chem. Phys. 93, 8196-8210] that is largely ignored in studies of folding. Tendency to structure in short peptide chains suggests a mechanism for the formation of short-range nucleation sites in protein folding.

Crystal structure of an H-2Kb-ovalbumin peptide complex reveals the interplay of primary and secondary anchor positions in the major histocompatibility complex binding groove.

Sequence analysis of peptides naturally presented by major histocompatibility complex (MHC) class I molecules has revealed allele-specific motifs in which the peptide length and the residues observed at certain positions are restricted. Nevertheless, peptides containing the standard motif often fail to bind with high affinity or form physiologically stable complexes. Here we present the crystal structure of a well-characterized antigenic peptide from ovalbumin [OVA-8, ovalbumin-(257-264), SIINFEKL] in complex with the murine MHC class I H-2Kb molecule at 2.5-A resolution. Hydrophobic peptide residues Ile-P2 and Phe-P5 are packed closely together into binding pockets B and C, suggesting that the interplay of peptide anchor (P5) and secondary anchor (P2) residues can couple the preferred sequences at these positions. Comparison with the crystal structures of H-2Kb in complex with peptides VSV-8 (RGYVYQGL) and SEV-9 (FAPGNYPAL), where a Tyr residue is used as the C pocket anchor, reveals that the conserved water molecule that binds into the B pocket and mediates hydrogen bonding from the buried anchor hydroxyl group could not be likewise positioned if the P2 side chain were of significant size. Based on this structural evidence, H-2Kb has at least two submotifs: one with Tyr at P5 (or P6 for nonamer peptides) and a small residue at P2 (i.e., Ala or Gly) and another with Phe at P5 and a medium-sized hydrophobic residue at P2 (i.e., Ile). Deciphering of these secondary submotifs from both crystallographic and immunological studies of MHC peptide binding should increase the accuracy of T-cell epitope prediction.

Characterization of the Structure and Dynamics of Biomimetic Peptides by Solid-State NMR

Thesis (Ph.D.)--University of Washington, 2016-05