Amino acid pairing preferences in parallel beta-sheets in proteins.

Statistical approaches have been applied to examine amino acid pairing preferences within parallel beta-sheets. The main chain hydrogen bonding pattern in parallel beta-sheets means that, for each residue pair, only one of the residues is involved in main chain hydrogen bonding with the strand containing the partner residue. We call this the hydrogen bonded (HB) residue and the partner residue the non-hydrogen bonded (nHB) residue, and differentiate between the favorability of a pair and that of its reverse pair, e.g. Asn(HB)-Thr(nHB)versus Thr(HB)-Asn(nHB). Significantly (p < or = 0.000001) favoured pairings were rationalised using stereochemical arguments. For instance, Asn(HB)-Thr(nHB) and Arg(HB)-Thr(nHB) were favoured pairs, where the residues adopted favoured chi1 rotamer positions that allowed side-chain interactions to occur. In contrast, Thr(HB)-Asn(nHB) and Thr(HB)-Arg(nHB) were not significantly favoured, and could only form side-chain interactions if the residues involved adopted less favourable chi1 conformations. The favourability of hydrophobic pairs e.g. Ile(HB)-Ile(nHB), Val(HB)-Val(nHB) and Leu(HB)-Ile(nHB) was explained by the residues adopting their most preferred chi1 and chi2 conformations, which enabled them to form nested arrangements. Cysteine-cysteine pairs are significantly favoured, although these do not form intrasheet disulphide bridges. Interactions between positively and negatively charged residues were asymmetrically preferred: those with the negatively charged residue at the HB position were more favoured. This trend was accounted for by the presence of general electrostatic interactions, which, based on analysis of distances between charged atoms, were likely to be stronger when the negatively charged residue is the HB partner. The Arg(HB)-Asp(nHB) interaction was an exception to this trend and its favorability was rationalised by the formation of specific side-chain interactions. This research provides rules that could be applied to protein structure prediction, comparative modelling and protein engineering and design. The methods used to analyse the pairing preferences are automated and detailed results are available (http://www.rubic.rdg.ac.uk/betapairprefsparallel/).

Characterization of a serine protease that cleaves pro-gamma-melanotropin at the adrenal to stimulate growth.

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-gamma-melanotropin (pro-gamma-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-gamma-MSH, at a specific bond.

Barrel structures in proteins: automatic identification and classification including a sequence analysis of TIM barrels.

Automated methods for identifying and characterizing regular beta-barrels from coordinate data have been developed to analyze and classify various kinds of barrel structures based on geometric parameters such as the barrel strand number (n) and shear number (S). In total, we find 1,316 barrels in the January 1998 release of Protein Data Bank. Of 1,316 barrels, 1,277 barrels had an even shear number, corresponding to 50 nonhomologous families. The (beta alpha)8 triose phosphate isomerase (TIM) barrel (n = 8, S = 8) fold has the largest number of apparently nonhomologous entries, 16, although the trypsin like antiparallel (n = 6, S = 8) barrels (representing only three families) are the most common with 527 barrels. Of all the protein families that exhibit barrel structures, 68% are found to be various kinds of enzymes, the remainder being binding proteins and transport membrane proteins. In addition, the layers of side chains, which form the cores of barrels with S = n and S = 2n, are also analyzed. More sophisticated methods were developed for detecting TIM barrels specifically, including consideration of the amino acid propensities for the side chains that form the layers. We found that the residues on the outside of the eight stranded parallel beta-barrel, buried by the alpha-helices, are much more hydrophobic than the residues inside the barrel.

Correlation of observed fold frequency with the occurrence of local structural motifs.

It is well known that some protein folds (superfolds) occur very frequently. We show that compared to other folds, most superfold structures have a higher proportion of their alpha-helical or beta-strand residues in one of three basic units of supersecondary structure (alpha-hairpin, beta-hairpin or betaalphabeta-unit). Furthermore, by taking into consideration two more complex motifs, the four-stranded Greek-key (beta4) and the betaalpha-Greek key (betaalphabetabeta), we demonstrate that the remaining superfold structures contain many of these higher order units of three-dimensional packing. The implications of these results for folding are discussed.

Determinants of strand register in antiparallel beta-sheets of proteins.

Antiparallel beta-sheets present two distinct environments to inter-strand residue pairs: beta(A,HB) sites have two backbone hydrogen bonds; whereas at beta(A,NHB) positions backbone hydrogen bonding is precluded. We used statistical methods to compare the frequencies of amino acid pairs at each site. Only approximately 10% of the 210 possible pairs showed occupancies that differed significantly between the two sites. Trends were clear in the preferred pairs, and these could be explained using stereochemical arguments. Cys-Cys, Aromatic-Pro, Thr-Thr, and Val-Val pairs all preferred the beta(A,NHB) site. In each case, the residues usually adopted sterically favored chi1 conformations, which facilitated intra-pair interactions: Cys-Cys pairs formed disulfide bonds; Thr-Thr pairs made hydrogen bonds; Aromatic-Pro and Val-Val pairs formed close van der Waals contacts. In contrast, to make intimate interactions at a beta(A,HB) site, one or both residues had to adopt less favored chi1 geometries. Nonetheless, pairs containing glycine and/or aromatic residues were favored at this site. Where glycine and aromatic side chains combined, the aromatic residue usually adopted the gauche conformation, which promoted novel aromatic ring-peptide interactions. This work provides rules that link protein sequence and tertiary structure, which will be useful in protein modeling, redesign, and de novo design. Our findings are discussed in light of previous analyses and experimental studies.

Protein folds and functions.

BACKGROUND: The recent rapid increase in the number of available three-dimensional protein structures has further highlighted the necessity to understand the relationship between biological function and structure. Using structural classification schemes such as SCOP, CATH and DALI, it is now possible to explore global relationships between protein fold and function, something which was previously impractical. RESULTS: Using a relational database of CATH data we have generated fold distributions for arbitrary selections of proteins automatically. These distributions have been examined in the light of protein function and bound ligand. Different enzyme classes are not clearly reflected in distributions of protein class and architecture, whereas the type of bound ligand has a much more dramatic effect. CONCLUSIONS: The availability of structural classification data has enabled this novel overview analysis. We conclude that function at the top level of the EC number enzyme classification is not related to fold, as only a very few specific residues are actually responsible for enzyme activity. Conversely, the fold is much more closely related to ligand type.

Contemporary approaches to protein structure classification.

In a similar manner to sequence database searching, it is also possible to compare three-dimensional protein structure. Such methods can be extremely useful because a structural similarity may represent a distant evolutionary relationship that is undetectable by sequence analysis. In this review, we summarise the most popular structure comparison methods, show how they can be used for database searching, and then describe some of the most advanced attempts to develop comprehensive protein structure classifications. With such data, it is possible to identify distant evolutionary relationships, provide libraries of unique folds for structure prediction, estimate the total number of folds that exist, and investigate the preference for certain types of structures over others.

PROMOTIF--a program to identify and analyze structural motifs in proteins.

We describe a suite of programs, PROMOTIF, that analyzes a protein coordinate file and provides details about the structural motifs in the protein. The program currently analyzes the following structural features: secondary structure; beta-and gamma-turns; helical geometry and interactions; beta-strands and beta-sheet topology; beta-bulges; beta-hairpins; beta-alpha-beta units and psi-loops; disulphide bridges; and main-chain hydrogen bonding patterns. PROMOTIF creates postscript files showing the examples of each type of motif in the protein, and a summary page. The program can also be used to compare motifs in a group of related structures, such as an ensemble of NMR structures.

A revised set of potentials for beta-turn formation in proteins.

Three thousand eight hundred ninety-nine beta-turns have been identified and classified using a nonhomologous data set of 205 protein chains. These were used to derive beta-turn positional potentials for turn types I' and II' for the first time and to provide updated potentials for formation of the more common types I, II, and VIII. Many of the sequence preferences for each of the 4 positions in turns can be rationalized in terms of the formation of stabilizing hydrogen bonds, preferences for amino acids to adopt a particular conformation in phi, psi space, and the involvement of turn types I' and II' in beta-hairpins. Only 1,632 (42%) of the turns occur in isolation; the remainder have at least 1 residue in common with another turn and have hence been classified as multiple turns. Several types of multiple turn have been identified and analyzed.

Identification, classification, and analysis of beta-bulges in proteins.

A beta-bulge is a region of irregularity in a beta-sheet involving two beta-strands. It usually involves two or more residues in the bulged strand opposite to a single residue on the adjacent strand. These irregularities in beta-sheets were identified and classified automatically, extending the definition of beta-bulges given by Richardson et al. (Richardson, J.S., Getzoff, E.D., & Richardson, D.C., 1978, Proc. Natl. Acad. Sci. USA 75, 2574-2578). A set of 182 protein chains (170 proteins) was used, and a total of 362 bulges were extracted. Five types of beta-bulges were found: classic, G1, wide, bent, and special. Their characteristic amino acid preferences were found for most classes of bulges. Basically, bulges occur frequently in proteins; on average there are more than two bulges per protein. In general, beta-bulges produce two main changes in the structure of a beta-sheet: (1) disrupt the normal alternation of side-chain direction; (2) accentuate the twist of the sheet, altering the direction of the surrounding strands.

Topological and stereochemical restrictions in beta-sandwich protein structures.

Chain topology in beta-structured protein domains and handedness associated with it are discussed. Previously, other workers have shown that by considering just two restrictions--structures that are left-handed and/or have loops that cross can be disregarded--the number of topologies associated with such structures is expected to be severely limited. By way of example, we determine the number of topologies compatible with a six-stranded antiparallel beta-sandwich. Without restriction on the type of strand-strand connection allowed but with elimination of symmetry related structures 360 topologies are possible. If connections between parallel strands are disqualified the number is reduced, 10-fold, to 36. The figure is cut to 24 when structures with loop crossings are eliminated. Handedness in these structures is examined in detail and from this a rationale for the observed predominance of right-handed forms of beta-structures is presented. The 24 structures can be considered as a set of right- and left-handed pairs of 12 topologies. All but two of these pairs can be assigned hands on the basis of existing rules. Six of the structures are found to occur in the Brookhaven Protein Databank and all are right-handed. This study provides a basis for protein design projects which might, for example, attempt the synthesis of unobserved protein topologies. Of the 24 structures in the final set eight are examples of the classic Greek key fold. Thus, the predominance of this motif among all-beta proteins can be attributed in part to these topological constraints. The possible physicochemical origins of the structural selection rules and additional factors which might contribute to the particular favourability of certain structures are also explored.

The Greek key motif: extraction, classification and analysis.

The Greek key is a very common structural motif in proteins. It has been traditionally defined as four beta-strands with '+3,-1,-1' topology. This definition encompasses motifs with several different three-dimensional structures. We have classified Greek keys, based on their hydrogen bonding patterns, into three groups with similar three-dimensional structures. All examples of Greek keys in each of these classes have been automatically extracted using a set of programs. Analysis of these examples shows the variability of secondary structure segment length and sequences of Greek keys even within one class. This variability suggests that no single folding pathway is likely to fit all Greek key structures.

Depicting topology and handedness in jellyroll structures.

The jellyroll structure is a special case of the Greek key topology and, to date, has only been observed in complete form in one of its four possible arrangements. Like other elements of super-secondary structure involving the beta-strand (e.g. the beta alpha beta unit) the known structure forms a right-handed superhelix. The possibility of losing such tertiary information and other problems associated with representing these structures by two-dimensional topology diagrams are discussed. A series of rules are presented which allow this three-dimensional information to be represented in two-dimensional topology diagrams from which the handedness of a jellyroll structure can be determined.

Stereochemical quality of protein structure coordinates.

Methods have been developed to assess the stereochemical quality of any protein structure both globally and locally using various criteria. Several parameters can be derived from the coordinates of a given structure. Global parameters include the distribution of phi, psi and chi 1 torsion angles, and hydrogen bond energies. There are clear correlations between these parameters and resolution; as the resolution improves, the distribution of the parameters becomes more clustered. These features show a broad distribution about ideal values derived from high-resolution structures. Some structures have tightly clustered distributions even at relatively low resolutions, while others show abnormal scatter though the data go to high resolution. Additional indicators of local irregularity include proline phi angles, peptide bond planarities, disulfide bond lengths, and their chi 3 torsion angles. These stereochemical parameters have been used to generate measures of stereochemical quality which provide a simple guide as to the reliability of a structure, in addition to the most important measures, resolution and R-factor. The parameters used in this evaluation are not novel, and are easily calculated from structure coordinates. A program suite is currently being developed which will quickly check a given structure, highlighting unusual stereochemistry and possible errors.

HERA--a program to draw schematic diagrams of protein secondary structures.

A program is described which generates hydrogen bonding diagrams of protein structures and optionally helical wheels and helical nets. The program can also beta-strands beta-strands and to automatically extract simple structural motifs such as hairpins or Greek keys. The program greatly reduces the effort required to produce these diagrams and offers considerable flexibility in the information which can be represented. The usefulness of the program is illustrated by several examples including comparing homologous families, correlating protein structure with attributes of individual residues, and extracting all examples of the psi-loop motif from the Brookhaven Data Bank.

Identification and electron microscopic analysis of a chaperonin oligomer from Neurospora crassa mitochondria.

A 7-fold symmetric particle has been identified in Neurospora crassa which is most probably the mitochondrial chaperonin. The particle, about 12 nm in diameter, appears in preparations of cytochrome reductase, and is shown to contain a 60 kd protein which cross-reacts with anti-GroEL antibodies. Results of STEM mass measurement suggest that the particle is composed of 14 subunits. A preliminary interpretation of the structure of the particle based on electron microscopy is given. Its quaternary structure and molecular weight are similar to those of the recently discovered family of particles called chaperonins, found in bacteria, chloroplasts and mitochondria.