The architecture of parallel beta-helices and related folds.

Three-dimensional structures have been determined of a large number of proteins characterized by a repetitive fold where each of the repeats (coils) supplies a strand to one or more parallel beta-sheets. Some of these proteins form superfamilies of proteins, which have probably arisen by divergent evolution from a common ancestor. The classical example is the family including four families of pectinases without obviously related primary sequences, the phage P22 tailspike endorhamnosidase, chrondroitinase B and possibly pertactin from Bordetella pertusis. These show extensive stacking of similar residues to give aliphatic, aromatic and polar stacks such as the asparagine ladder. This suggests that coils can be added or removed by duplication or deletion of the DNA corresponding to one or more coils and explains how homologous proteins can have different numbers of coils.This process can also account for the evolution of other families of proteins such as the beta-rolls, the leucine-rich repeat proteins, the hexapeptide repeat family, two separate families of beta-helical antifreeze proteins and the spiral folds. These families need not be related to each other but will share features such as relative untwisted beta-sheets, stacking of similar residues and turns between beta-strands of approximately 90 degrees often stabilized by hydrogen bonding along the direction of the parallel beta-helix.Repetitive folds present special problems in the comparison of structures but offer attractive targets for structure prediction. The stacking of similar residues on a flat parallel beta-sheet may account for the formation of amyloid with beta-strands at right-angles to the fibril axis from many unrelated peptides.     

Twist and shear in beta-sheets and beta-ribbons

The structures of the beta-sheets and the beta-ribbons have been analysed using high-resolution protein structure data. Systematic asymmetries measured in both parallel and antiparallel beta-structures include the sheet twist and the strand shear. In order to determine the origin of these asymmetries, numerous interactions and correlations were examined. The strongest correlations are observed for residues in antiparallel beta-sheets and beta-ribbons that form non-H-bonded pairs. For these residues, the sheet twist is correlated to the backbone phi angle but not to the psi angle. Our analysis supports the existence of an inter-strand C(alpha)H(alpha)...O weak H-bond, which, together with the CO...HN H-bond, constitutes a bifurcated H-bond that links neighbouring beta-strands. Residues of beta-sheets and beta-ribbons in high-resolution protein structures form a distinct region of the Ramachandran plot, which is determined by the formation of the bifurcated H-bond, the formation of an intra-strand O...H(alpha) non-bonded polar interaction, and an intra-strand O...C(beta) steric clash. Using beta-strands parameterised by phi-psi values from the allowed beta-sheet region of the Ramachandran plot, the shear and the right-hand twist can be reproduced in a simple model of the antiparallel and parallel beta-ribbon that models the bifurcated H-bonds specifically. The conformations of interior residues of beta-sheets are shown to be subsets of the conformations of residues of beta-ribbons.     

Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution.

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 A resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 A resolution with residual 15.5%. The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure. The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.     

Sequence specificity, statistical potentials, and three-dimensional structure prediction with self-correcting distance geometry calculations of beta-sheet formation in proteins

A statistical analysis of a representative data set of 169 known protein structures was used to analyze the specificity of residue interactions between spatial neighboring strands in beta-sheets. Pairwise potentials were derived from the frequency of residue pairs in nearest contact, second nearest and third nearest contacts across neighboring beta-strands compared to the expected frequency of residue pairs in a random model. A pseudo-energy function based on these statistical pairwise potentials recognized native beta-sheets among possible alternative pairings. The native pairing was found within the three lowest energies in 73% of the cases in the training data set and in 63% of beta-sheets in a test data set of 67 proteins, which were not part of the training set. The energy function was also used to detect tripeptides, which occur frequently in beta-sheets of native proteins. The majority of native partners of tripeptides were distributed in a low energy range. Self-correcting distance geometry (SECODG) calculations using distance constraints sets derived from possible low energy pairing of beta-strands uniquely identified the native pairing of the beta-sheet in pancreatic trypsin inhibitor (BPTI). These results will be useful for predicting the structure of proteins from their amino acid sequence as well as for the design of proteins containing beta-sheets.     

Structural principles for the propeller assembly of beta-sheets: the preference for seven-fold symmetry.

Twisted beta-sheets, packed face to face, may be arranged in circular formation like blades of a propeller or turbine. This beta-propeller fold has been found in three proteins: that in neuraminidase consists of six beta-sheets while those in methylamine dehydrogenase and galactose oxidase are composed of seven beta-sheets. A model for multisheet packing in the beta-propeller fold is proposed. This model gives both geometrical parameters of the beta-propellers composed of different numbers of sheets and patterns of residue packing at their sheet-to-sheet interfaces. All the known beta-propeller structures have been analyzed, and the observed geometries and residue packing are found to be in good agreement with those predicted by models. It is shown that unusual seven-fold symmetry is preferable to six- or eight-fold symmetry for propeller-like multi-sheet assembly. According to the model, a six-beta-sheet propeller has to have predominantly small residues in the beta-strands closed to its six-fold axis, but no strong sequence constraints are necessary for a seven-fold beta-propeller.     

Analysis and prediction of inter-strand packing distances between beta-sheets of globular proteins

Any two beta-strands belonging to two different beta-sheets in a protein structure are considered to pack interactively if each beta-strand has at least one residue that undergoes a loss of one tenth or more of its solvent contact surface area upon packing. A data set of protein 3-D structures (determined at 2.5 A resolution or better), corresponding to 428 protein chains, contains 1986 non-identical pairs of beta-strands involved in interactive packing. The inter-axial distance between these is significantly correlated to the weighted sum of the volumes of the interacting residues at the packing interface. This correlation can be used to predict the changes in the inter-sheet distances in equivalent beta-sheets in homologous proteins and, therefore, is of value in comparative modelling of proteins.     

On the significance of alternating patterns of polar and non-polar residues in beta-strands

A common assumption about protein sequences in beta-strands is that they have alternating patterns of polar and non-polar residues. It is thought that such patterns reflect the interior/exterior geometry of amino acid residue side-chains on a beta-sheet. Here we study the prevalence of simple hydrophobicity patterns in parallel and antiparallel beta-sheets in proteins of known structure and in the sequences of amyloidogenic proteins. The occurrence of 32 possible pentapeptide binary patterns (polar (P)/non-polar (N)) is computed in 1911 non-homologous protein structures. Despite their tendency to aggregate in experimentally designed proteins, the purely alternating hydrophobic/polar patterns (PNPNP and NPNPN) are most frequent in beta-sheets, typically occurring in antiparallel strands. The overall distribution of the pentapeptide binary patterns is significantly different in strands within parallel and antiparallel sheets. In both types of sheets, complementary patterns (where the hydrophobic and polar residues pair with one another) associate preferentially. We do not find alternating patterns to be common in amyloidogenic proteins or in short fragments involved directly in amyloid formation. However, we do note some similarities between patterns present in amyloidogenic sequences and those in parallel strands.     

Secondary-structure analysis of denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

To elucidate the structure of denatured proteins, we measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra from 260 to 172 nm of three proteins (metmyoglobin, staphylococcal nuclease, and thioredoxin) in the native and the acid-, cold-, and heat-denatured states, using a synchrotron-radiation VUVCD spectrophotometer. The circular dichroism spectra of proteins fully unfolded by guanidine hydrochloride (GdnHCl) were also measured down to 197 nm for comparison. These denatured proteins exhibited characteristic VUVCD spectra that reflected a considerable amount of residual secondary structures. The contents of alpha-helices, beta-strands, turns, poly-L-proline type II (PPII), and unordered structures were estimated for each denatured state of the three proteins using the SELCON3 program with Protein Data Bank data and the VUVCD spectra of 31 reference proteins reported in our previous study. Based on these contents, the characteristics of the four types of denaturation were discussed for each protein. In all types of denaturation, a decrease in alpha-helices was accompanied by increases in beta-strands, PPII, and unordered structures. About 20% beta-strands were present even in the proteins fully unfolded by GdnHCl in which beta-sheets should be broken. From these results, we propose that denatured proteins constitute an ensemble of residual alpha-helices and beta-sheets, partly unfolded (or distorted) alpha-helices and beta-strands, PPII, and unordered structures.     

A new protein folding algorithm based on hydrophobic compactness: Rigid Unconnected Secondary Structure Iterative Assembly (RUSSIA). II: Applications

The RUSSIA procedure (Rigid Unconnected Secondary Structure Iterative Assembly) produces structural models of cores of small- and medium-sized proteins. Loops are omitted from this treatment and regular secondary structures are reduced to points, the centers of their hydrophobic faces. This methodology relies on the maximum compactness of the hydrophobic residues, as described in detail in Part I. Starting data are the sequence and the predicted limits and natures of regular secondary structures (alpha or beta). Helices are treated as rigid cylinders, whereas beta-strands are collectively taken into account within beta-sheets modeled by helicoid surfaces. Strands are allowed to shift along their mean axis to allow some flexibility and the alpha-helices can be placed on either side of beta-sheets. Numerous initial conformations are produced by discrete rotations of the helices and sheets around the direction going from the center of their hydrophobic face to the global center of the protein. Selection of proposed models is based upon a criterion lying on the minimization of distances separating hydrophobic residues belonging to different regular secondary structures. The procedure is rapid and appears to be robust relative to the quality of starting data (nature and length of regular secondary structures). This dependence of the quality of the model on secondary structure prediction and in particular the beta-sheet topology, is one of the limits of the present algorithm. We present here some results for a set of 12 proteins (alpha, beta and alpha/beta classes) of lengths 40-166 amino acids. The r.m.s. deviations for core models with respect to the native proteins are in the range 1.4-3.7 A.     

A general model of the P2 protein of peripheral nervous system myelin based on secondary structure predictions, tertiary folding principles, and experimental observations

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable alpha-helix, beta-structure, beta-turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high beta-sheet content and virtual absence of alpha-helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the beta-structure over the alpha-helical conformation. In order to define those beta-sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted beta-strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential beta-strands in the predicted secondary structure were then ordered into beta-sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a "Greek key" beta-barrel, consisting of eight antiparallel beta-strands with a two-stranded ribbon of antiparallel beta-structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative beta-strand orderings yield three specific models having different interstrand connections across the barrel ends.     

Prediction of prolyl residues in cis-conformation in protein structures on the basis of the amino acid sequence

In proteins most peptide bonds are in trans-conformation: the torsion angle omega = 180 degrees. Only few show cis-conformation in known protein structures (omega = 0 degrees). Most of them are prolyl residues. About 6% of about 4000 prolyl residues are in cis-conformation. Between trans- and cis-prolyl residues significant differences are observed in the surrounding sequences. E.g. there are large amounts of aromatic residues N-terminally in case of cis-prolyl residues, but in the case of trans-prolyl residues more aromatic amino acids occur C-terminally. But in all cases there are only complex patterns which are indicative of cis- and trans-conformation, respectively. Considering the neighbours (+/- 6 residues) of prolyl residues and their physicochemical properties we find 6 different patterns which allow one to assign correctly about 75% of known cis-structured prolyl residues, whereby no false positive one is predicted.     

Effect of mutations in the beta5-beta7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11)

The human genome encodes ten different small heat shock proteins, each of which contains the so-called alpha-crystallin domain consisting of 80-100 residues and located in the C-terminal part of the molecule. The alpha-crystallin domain consists of six or seven beta-strands connected by different size loops and combined in two beta-sheets. Mutations in the loop connecting the beta5 and beta7 strands and conservative residues of beta7 in alphaA-, alphaB-crystallin and HSP27 correlate with the development of different congenital diseases. To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants. These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis-ANS bound to HSP22. Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis. Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking. The wild-type HSP22 possessed higher chaperone-like activity than their mutants when insulin or rhodanase were used as the model substrates. Because conservative Lys residues located in the beta5-beta7 loop and in the beta7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases.     

Kinetic rationale for selectivity toward N- and C-terminal oxygen-dependent degradation domain substrates mediated by a loop region of hypoxia-inducible factor prolyl hydroxylases

Hydroxylation of two conserved prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of the alpha-subunit of hypoxia-inducible factor (HIF) signals for its degradation via the ubiquitin-proteasome pathway. In human cells, three prolyl hydroxylases (PHDs 1-3) belonging to the Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenase family catalyze prolyl hydroxylation with differing selectivity for CODD and NODD. Sequence analysis of the catalytic domains of the PHDs in the light of crystal structures for PHD2, and results for other 2OG oxygenases, suggested that either the C-terminal region or a loop linking two beta-strands (beta2 and beta3 in human PHD2) are important in determining substrate selectivity. Mutation analyses on PHD2 revealed that the beta2beta3 loop is a major determinant in conferring selectivity for CODD over NODD peptides. A chimeric PHD in which the beta2beta3 loop of PHD2 was replaced with that of PHD3 displayed an almost complete selectivity for CODD (in competition experiments), as observed for wild-type PHD3. CODD was observed to bind much more tightly to this chimeric protein than the wild type PHD2 catalytic domain.     

Electrostatic screening and backbone preferences of amino acid residues in urea-denatured ubiquitin

Local structures in denatured proteins may be important in guiding a polypeptide chain during the folding and misfolding processes. Existence of local structures in chemically denatured proteins is a highly controversial issue. NMR parameters [coupling constants (3) J(H(alpha),H(N)) and chemical shifts] of chemically denatured proteins in general deviate little from their values in small peptides. These peptides were presumed to be completely unstructured; therefore, it was considered that chemically denatured proteins are random coils. But recent experimental studies show that small peptides adopt relatively stable structures in aqueous solutions. Small deviations of the NMR parameters from their values in small peptides may thus actually indicate the existence of local structures in chemically denatured proteins. Using NMR data and theoretical predictions we show here that fluctuating beta-strands exist in urea-denatured ubiquitin (8 M urea at pH 2). Residues in such beta-strands populate more frequently the left side of the broad beta region of -psi space. Urea-denatured ubiquitin contains no detectable beta-sheet secondary structures; nevertheless, the fluctuating beta-strands in urea-denatured ubiquitin coincide to the beta-strands in the native state. Formation of beta-strands is in accord with the electrostatic screening model of unfolded proteins. The free energy of a residue in an unfolded protein is in this model determined by the local backbone electrostatics and its screening by backbone solvation. These energy terms introduce strong electrostatic coupling between neighboring residues, which causes cooperative formation of beta-strands in denatured proteins. We propose that fluctuating beta-strands in denatured proteins may serve as initiation sites to form fibrils.     

Dependence of alpha-helical and beta-sheet amino acid propensities on the overall protein fold type

ABSTRACT: BACKGROUND: A large number of studies have been carried out to obtain amino acid propensities for alpha- helices and beta-sheets. The obtained propensities for alpha-helices are consistent with each other, and the pair-wise correlation coefficient is frequently high. On the other hand, the beta-sheet propensities obtained by several studies differed significantly, indicating that the context significantly affects beta-sheet propensity. RESULTS: We calculated amino acid propensities for alpha-helices and beta-sheets for 39 and 24 protein folds, respectively, and addressed whether they correlate with the fold. The propensities were also calculated for exposed and buried sites, respectively. Results showed that alpha-helix propensities do not differ significantly by fold, but beta-sheet propensities are diverse and depend on the fold. The propensities calculated for exposed sites and buried sites are similar for alpha-helix, but such is not the case for the beta-sheet propensities. We also found some fold dependence on amino acid frequency in beta-strands. Folds with a high Ser, Thr and Asn content at exposed sites in beta-strands tend to have a low Leu, Ile, Glu, Lys and Arg content (correlation coefficient = 0.90) and to have flat beta-sheets. At buried sites in beta-strands, the content of Tyr, Trp, Gln and Ser correlates negatively with the content of Val, Ile and Leu (correlation coefficient = 0.93). "All-beta" proteins tend to have a higher content of Tyr, Trp, Gln and Ser, whereas alpha/beta proteins tend to have a higher content of Val, Ile and Leu. CONCLUSIONS: The alpha-helix propensities are similar for all folds and for exposed and buried residues. However, beta-sheet propensities calculated for exposed residues differ from those for buried residues, indicating that the exposed-residue fraction is one of the major factors governing amino acid composition in beta-strands. Furthermore, the correlations we detected suggest that amino acid composition is related to folding properties such as the twist of a beta-strand or association between two beta sheets.     

Three-dimensional solution structure of apo-neocarzinostatin.

Two and three-dimensional solution nuclear magnetic resonance studies of the 11K apoprotein from natural antitumor agent neocarzinostatin (NCS) were extended to elucidation of the high-resolution structure by the use of restrained molecular dynamics computations. The refined structures attained convergency upon three steps of iterative calculations, in which more distance restraints were extracted from experimental data, and the existing distance bounds were optimized on the basis of computed structures. The solution structures of apo-NCS contain seven antiparallel beta-strands, which form two closely located beta-sheets and a short beta-segment. This protein lacks any alpha-helical component. The alignment of the seven beta-strands gives rise to a beta-barrel with an elongated diameter in one direction. The global structure of apo-NCS resembles that of the Ig-fold domain found in immunoglobulins and other structurally related beta-proteins. Residues responsible for side-chain packing and the possible salt-bridge formation important for protein folding were identified. Neocarzinostatin and the analogous proteins are known to exert their biological activity through the interaction of DNA with a chromophoric molecule, which is non-covalently bound to the apo-proteins. This molecular chromophore-binding site in apo-NCS is made of a cavity consisting of residues from the four-beta-stranded sheet and the short beta-segment. Although the solution structures of apo-NCS are similar to that of the analogous apoauromomycin in the crystalline state, difference in the shape of the binding cavities between the two was found. This study provides a structural basis for characterization of the specific recognition and molecular mechanism of the antitumor NCS chromophore binding to its host protein.     

Beta-breakers: an aperiodic secondary structure

We have studied the architecture of parallel beta-sheets in proteins and focused on the residues that initiate and terminate the beta-strands. These beta-breaker residues are at the origin of the kink between the beta-strand and the turn that precedes or follows it. beta-Breakers can be located automatically using a consensus approach based on algorithmic secondary structure assignment, solvent accessibility and backbone dihedral angles. These beta-breakers are conformationally homogeneous with respect to side-chain solvent accessibility and backbone dihedral angle profile. A sequence-structure correlation is noted: a restricted subset of amino acids is observed at these positions. Analysis of homologous protein sequences shows that these residues are more highly conserved than other residues in the loop. We conclude that beta-breakers are the structural analogs of the N and C-terminal caps of alpha-helices. The identification of this aperiodic substructure suggests a strategy for improving secondary structure prediction and may guide site-directed mutagenesis experiments.     

Generation of efficient fingerprint for GFP-like fold and computational identification of potential GFP-like homologs

There is a considerable interest in the detection of GFP-like proteins due to their structural stability and functional usefulness. GFP-like proteins share highly conserved beta-barrel fold with 11 beta-strands. However, their low sequence identity hampers efficient identification of their homologous proteins from database. In this study, an attempt was made to generate a fingerprint for efficient detection of GFP-like proteins. Overlapped conserved residues (OCR)-based approach has been used to design a protein fingerprint based on sequentially and structurally conserved residues in secondary structures to detect homologous proteins very efficiently. Therefore, a fingerprint for GFP-like fold was designed using the OCR approach. However, its specificity was too low to be used for the identification of novel proteins. The conserved residues of loop regions were added and optimized to improve its specificity without losing its high sensitivity. The optimized fingerprint was employed to scan NR database. A total of 20 hypothetical proteins were detected, among which nine were validated as potential GFP-like homologs.     

Correlation between the 1.6 A crystal structure and mutational analysis of keratinocyte growth factor

A comprehensive deletion, mutational, and structural analysis of the native recombinant keratinocyte growth factor (KGF) polypeptide has resulted in the identification of the amino acids responsible for its biological activity. One of these KGF mutants (delta23KGF-R144Q) has biological activity comparable to the native protein, and its crystal structure was determined by the multiple isomorphous replacement plus anomalous scattering method (MIRAS). The structure of KGF reveals that it folds into a beta-trefoil motif similar to other members of fibroblast growth factor (FGF) family whose structures have been resolved. This fold consists of 12 anti-parallel beta-strands in which three pairs of the strands form a six-stranded beta-barrel structure and the other three pairs of beta-strands cap the barrel with hairpin triplets forming a triangular array. KGF has 10 well-defined beta strands, which form five double-stranded anti-parallel beta-sheets. A sixth poorly defined beta-strand pair is in the loop between residues 133 and 144, and is defined by only a single hydrogen bond between the two strands. The KGF mutant has 10 additional ordered amino terminus residues (24-33) compared to the other FGF structures, which are important for biological activity. Based on mutagenesis, thermal stability, and structural data we postulate that residues TRP125, THR126, and His127 predominantly confer receptor binding specificity to KGF. Additionally, residues GLN152, GLN138, and THR42 are implicated in heparin binding. The increased thermal stability of delta23KGF-R144Q can structurally be explained by the additional formation of hydrogen bonds between the GLN side chain and a main-chain carbonyl on an adjoining loop. The correlation of the structure and biochemistry of KGF provides a framework for a rational design of this potentially important human therapeutic.