The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.     

Conformational dynamics of human α-fetoprotein-derived heptapeptide LDSYQCT analogs

Conformational dynamics of a biologically active fragment of alpha-fetoprotein, the heptapeptide LDSYQCT, and its analogs obtained by site-directed substitutions of amino acid residues were studied. The conformational dynamics of the peptide were conservative under the substitutions Y17F, Y17S, and D15E. Substitutions C19A and S16V resulted only in local changes in the dynamic behavior of the peptide. Chemical modification of cysteine (C19) or dimerization of the peptide by producing a disulfide bond between cysteine residues of two parallel peptide chains, as well as the substitutions C19G, C19S, Q18E, and D15N changed a set of possible conformations and dynamic behavior of all amino acid residues. The most significant changes were caused by substitution of uncharged amino acid residues by charged ones, and vice versa.     

Conformational similarity among amino acid residues: 1. Analysis of protein crystal structure data

The probability distribution in the (?,ψ)-plane obtained for each amino acid residue from cyrstal structure data of globular proteins is compared. This has shown amino acid residues. Pro and Gly to be conformationally unique. Conformational similarity in the (?,ψ)-plane of amino acid reced does not necessarily mean that they will have the same chemical or biochemical properties or similar secondary structures. A set of amino acid residues are given which can adopt the conformations of other amino acid residues without much difficulty either in the whole (?,ψ)-plane or in regions, where the observed conformations are maximum.     

The interrelationships of side-chain and main-chain conformations in proteins

The accurate determination of a large number of protein structures by X-ray crystallography makes it possible to conduct a reliable statistical analysis of the distribution of the main-chain and side-chain conformational angles, how these are dependent on residue type, adjacent residue in the sequence, secondary structure, residue-residue interactions and location at the polypeptide chain termini. The interrelationship between the main-chain (phi, psi) and side-chain (chi 1) torsion angles leads to a classification of amino acid residues that simplify the folding alphabet considerably and can be a guide to the design of new proteins or mutational studies. Analyses of residues occurring with disallowed main-chain conformation or with multiple conformations shed some light on why some residues are less favoured in thermophiles.     

Comparison of NMR and MD N–H bond order parameters: example of HIV-1 protease

It has been suggested that the cause of disagreements between molecular dynamics (MD) and NMR N–H bond order parameters is the fact that the NMR order parameter is determined for different amino acid residues at different time intervals, while the MD one is derived for all residues from the same MD trajectory of the same time interval. Therefore, it has been proposed for correct comparison with NMR data to calculate the MD order parameter for different amino acid residues separately for trajectory ranges close to NMR correlation time. The MD simulation of the human immunodeficiency virus type-1 protease (HIV-1 PR) with monoprotonated active centre was performed for verification of the proposition. It has been shown that the protease in aqueous solution adopts a set of conformations, which are intermediate between semiopen and closed ones. The calculated MD N–H bond order parameters are in agreement with literature NMR data in confidence interval limits.     

Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat Ends

A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian sarcoma virus (SIV), and avian sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ' process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val(72), Ser(153), Lys(160)-Ile(161), Gly(163)-Val(165), and His(171)-Leu(172). Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser(153) and Val(72) in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance.     

Energy minimization method using automata network for sequence and side-chain conformation prediction from given backbone geometry

Globular proteins have high packing densities as a result of residue side chains in the core achieving a tight, complementary packing. The internal packing is considered the main determinant of native protein structure. From that point of view, we present here a method of energy minimization using an automata network to predict a set of amino acid sequences and their side-chain conformations from a desired backbone geometry for de novo design of proteins. Using discrete side-chain conformations, that is, rotamers, the sequence generation problem from a given backbone geometry becomes one of combinatorial problems. We focused on the residues composing the interior core region and predicted a set of amino acid Sequences and their side-chain conformations only from a given backbone geometry. The kinds of residues were restricted to six hydrophobic amino acids (Ala, Ile, Met, Leu, Phe, and Val) because the core regions are almost always composed of hydrophobic residues. The obtained sequences were well packed as was the native sequence. The method can be used for automated sequence generation in the de novo design of proteins. © 1994 Wiley-Liss, Inc.     

Effects of glycosylation on the conformation and dynamics of O-linked glycoproteins: carbon-13 NMR studies of ovine submaxillary mucin

Carbon-13 NMR spectroscopic studies of native and sequentially deglycosylated ovine submaxillary mucin (OSM) have been performed to examine the effects of glycosylation on the conformation and dynamics of the peptide core of O-linked glycoproteins. OSM is a large nonglobular glycoprotein in which nearly one-third of the amino acid residues are Ser and Thr which are glycosylated by the alpha-Neu-NAc(2-6)alpha-GalNAc- disaccharide. The beta-carbon resonances of glycosylated Ser and Thr residues in intact and asialo mucin display considerable chemical shift heterogeneity which, upon the complete removal of carbohydrate, coalesces to single sharp resonances. This chemical shift heterogeneity is due to peptide sequence variability and is proposed to reflect the presence of sequence-dependent conformations of the peptide core. These different conformations are thought to be determined by steric interactions of the GalNAc residue with adjacent peptide residues. The absence of chemical shift heterogeneity in apo mucin is taken to indicate a loss in the peptide-carbohydrate steric interactions, consistent with a more relaxed random coiled structure. On the basis of the 13C relaxation behavior (T1 and NOE) the dynamics of the alpha-carbons appear to be unique to each amino acid type and glycosylation state, with alpha-carbon mobilities decreasing in the order Gly greater than Ala = Ser greater than Thr much greater than monoglycosylated Ser/Thr approximately greater than disaccharide linked Ser/Thr.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid composition of protein termini are biased in different manners

An exhaustive statistical analysis of the amino acid sequences at the carboxyl (C) and amino (N) termini of proteins and of coding nucleic acid sequences at the 5' side of the stop codons was undertaken. At the N ends, Met and Ala residues are over-represented at the first (+1) position whereas at positions 2 and 5 Thr is preferred. These peculiarities at N-termini are most probably related to the mechanism of initiation of translation (for Met) and to the mechanisms governing the life-span of proteins via regulation of their degradation (for Ala and Thr). We assume that the C-terminal bias facilitates fixation of the C ends on the protein globule by a preference for charged and Cys residues. The terminal biases, a novel feature of protein structure, have to be taken into account when molecular evolution, three-dimensional structure, initiation and termination of translation, protein folding and life-span are concerned. In addition, the bias of protein termini composition is an important feature which should be considered in protein engineering experiments.     

Protein truncation is required for the activation of the c-myb proto-oncogene.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.     

Asymmetric amino acid compositions of transmembrane beta-strands

In contrast to water-soluble proteins, membrane proteins reside in a heterogeneous environment, and their surfaces must interact with both polar and apolar membrane regions. As a consequence, the composition of membrane proteins' residues varies substantially between the membrane core and the interfacial regions. The amino acid compositions of helical membrane proteins are also known to be different on the cytoplasmic and extracellular sides of the membrane. Here we report that in the 16 transmembrane beta-barrel structures, the amino acid compositions of lipid-facing residues are different near the N and C termini of the individual strands. Polar amino acids are more prevalent near the C termini than near the N termini, and hydrophobic amino acids show the opposite trend. We suggest that this difference arises because it is easier for polar atoms to escape from the apolar regions of the bilayer at the C terminus of a beta-strand. This new characteristic of beta-barrel membrane proteins enhances our understanding of how a sequence encodes a membrane protein structure and should prove useful in identifying and predicting the structures of trans-membrane beta-barrels.     

A molecular dynamics simulation study of human alpha-fetoprotein-derived peptide and its analogues. Influence of dielectric constant

A comparative study of the conformation dynamics of a series of heptapeptides: the human alpha-fetoprotein fragment LDSYQCT and its seven analogues has been conducted. The effect of the dielectric constant of medium on the dynamics of heptapeptide conformation is considered. It is shown that electrostatic interactions have a marked effect on several accessible conformations and the dynamics of the behavior of amino acid residues.     

Distance-constraint approach to protein folding. II. Prediction of three-dimensional structure of bovine pancreatic trypsin inhibitor

The possibility of predicting the three-dimensional structure of bovine pancreatic trypsin inhibitor (BPTI) is examined using a distance-constraint approach. The mean distances between the amino acid residues in globular proteins, calculated in a previous paper, are utilized as distance constraints. In this study, as in previous work of others, root-mean-square deviations of the predicted conformations from the native one of less than 4 Å could not be obtained if the only input information consisted of the mean distances between amino acid residues (which involve information about the hydrophobicity and hydrophilicity of each amino acid residue) and the locations of disuifide bonds, -helices, and -structures. An examination is made of the kinds of structural features of BPTI that appear in the conformations predicted without explicit inclusion of information about such structural features, and of the kinds of information required in a given set of distance constraints for successful folding of BPTI. For example, structures that resemble incipient-forming -helices, bends, and -structures are observed in the conformations predicted when only the mean distances between the amino acid residues and the locations of the disulfide bonds (without information about the locations of -helices, bends, and -structures) are used. One type of additional required information is knowledge of spatially distant pairs involving the active site and the terminal residues. Furthermore, examination of the missing information indicates an important role for the strong nonbonded interaction between sulfur atoms and the side chains of aromatic amino acid residues in BPTI. When such information is introduced into the set of distance constraints, in terms of the exact distances of five pairs, (which implicitly include sulfur/aromatic interactions), the root-mean-square deviations of the predicted conformations decrease to 2.2–3.2 Å. Several methods for comparing conformations are also discussed; in particular, comparisons between conformations of short segments are carried out by a differential-geometry procedure.     

Approaching a complete classification of protein secondary structure

A complete classification of types of the protein secondary structure is developed on the basis of computer analysis of the crystallographic structural data deposited in the protein Data Bank. The majority of amino acid residues fall into five conformation types. A conclusion is drawn that the number of sequence variants of torsion angles phi, psi in globular proteins is limited and is essentially less than the number of possible amino acid sequences for this chain length. Along with alpha-helix and beta-structure, the distribution analysis assigning every maximum of distribution of amino acid conformations on Ramachandran map to a certain type of the secondary structure exposed a third type of the secondary structure that was previously neglected. This type of the structure is extended left-handed helical conformation, designated as mobile (M-) conformation. A full set of M-conformation fragments that seems to play a major role in protein globule dynamics has been obtained, a small radius of correlation for the polypeptide chain in M-conformation is demonstrated. It explains a prevalence of short segments of mobile conformation revealed in globular proteins. For secondary structure types, the frequency of occurrence of amino acid residues has been computed.     

Investigation of Domain Motions in Bacteriophage T4 Lysozyme

Abstract

Hinge-bending in T4 lysozyme has been inferred from single amino acid mutant crystalline allomorphs by Matthews and coworkers. This raises an important question: are the different conformers in the unit cell artifacts of crystal packing forces, or do they represent different solution state structures? The objective of this theoretical study is to determine whether domain motions and hinge-bending could be simulated in T4 lysozyme using molecular dynamics. An analysis of a 400 ps molecular dynamics simulation of the 164 amino acid enzyme T4 lysozyme is presented. Molecular dynamics calculations were computed using the Discover software package (Biosym Technologies). All hydrogen atoms were modeled explicitly with the inclusion of all 152 crystallographic waters at a temperature of 300 K. The native T4 lysozyme molecular dynamics simulation demonstrated hinge-bending in the protein. Relative domain motions between the N-terminal and C-terminal domains were evident. The enzyme hinge bending sites resulted from small changes in backbone atom conformations over several residues rather than rotation about a single bound. Two hinge loci were found in the simulation. One locus comprises residues 8–14 near the C-terminal of the A helix; the other site, residues 77–83 near the C-terminal of the C helix. Comparison of several snapshot structures from the dynamics trajectory clearly illustrates domain motions between the two lysozyme lobes. Time correlated atomic motions in the protein were analyzed using a dynamical cross-correlation map. We found a high degree of correlated atomic motions in each of the domains and, to a lesser extent, anticorrelated motions between the two domains. We also found that the hairpin loop in the N-terminal lobe (residues 19–24) acted as a mobile ‘flap’ and exhibited highly correlated dynamic motions across the cleft of the active site, especially with residue 142.     

Sequence evolution correlates with structural dynamics

Biochemical activity and core stability are essential properties of proteins, maintained usually by conserved amino acids. Structural dynamics emerged in recent years as another essential aspect of protein functionality. Structural dynamics enable the adaptation of the protein to binding substrates and to undergo allosteric transitions, while maintaining the native fold. Key residues that mediate structural dynamics would thus be expected to be conserved or exhibit coevolutionary patterns at least. Yet, the correlation between sequence evolution and structural dynamics is yet to be established. With recent advances in efficient characterization of structural dynamics, we are now in a position to perform a systematic analysis. In the present study, a set of 34 enzymes representing various folds and functional classes is analyzed using information theory and elastic network models. Our analysis shows that the structural regions distinguished by their coevolution propensity as well as high mobility are predisposed to serve as substrate recognition sites, whereas residues acting as global hinges during collective dynamics are often supported by conserved residues. We propose a mobility scale for different types of amino acids, which tends to vary inversely with amino acid conservation. Our findings suggest the balance between physical adaptability (enabled by structure-encoded motions) and chemical specificity (conferred by correlated amino acid substitutions) underlies the selection of a relatively small set of versatile folds by proteins.     

A recurring two-hydrogen-bond motif incorporating a serine or threonine residue is found both at alpha-helical N termini and in other situations

Side-chain hydroxyl residues in protein crystal structures often form hydrogen bonds with main-chain atoms. The most common bond arrangement is a four to five residue motif in which a serine or threonine is the first residue forming two characteristic hydrogen bonds to residues ahead of it in sequence. We call them ST-motifs, by analogy with the term Asx-motif we suggested for the related motifs with aspartate and asparagine residues. ST-motifs are common, there being just under one and a half in a typical protein subunit. Asx-motifs are even more common, such that 9 % of the residues of an average protein consist of Asx or ST-motifs. Of the ST-motifs, three-quarters are at helical N termini, and the rest occur by themselves or in conjunction with beta-bulge loops. A third of all alpha-helices have either ST-motifs or Asx-motifs at their N termini. Previous work has emphasised the occurrence of the capping box at alpha-helical N termini, but the capping box occurs in only 5 % of alpha-helical N termini; also, we point out that it can be regarded as a subset of the ST-motif (or, occasionally, of the Asx-motif). By comparing related sequences, the rates which amino acid residues at the first position of ST or Asx-motifs interchange during evolution are examined. Serine <==> threonine, and aspartate <==> asparagine, interchange is rapid; inter-pair exchange is slower, but much faster than exchange with other amino acid residues. This is consistent with the general similarity of ST-motifs and Asx-motifs combined with some subtle structural differences between them that are described.     

Conformational propensities and residual structures in unfolded peptides and proteins

Ample evidence gathered over the last ten years indicates that unfolded and naturally disordered proteins and peptides can show local order in that short segments can adopt turn or polyproline II-like conformations. These findings show that unfolded states cannot be described by the so-called random coil model which assumes that individual amino acid residues sample the entire sterically accessible parts of the Ramachandran with very similar probabilities. This article reviews the experimental evidence for the notion that amino acid residues have different propensities for polyproline II, β-strand, helical and turn conformations in water. These propensities are changed by interactions with nearest neighbours. We show that for a substantial number of residues the conformational propensities in the unfolded state correlate with values for helix propagation and the Chou-Fasman propensities for β-strands. Based on the presented results we hypothesize that the conformational distributions of a representative set of short peptides could be used for predicting structural distributions of disordered peptides and proteins in the future.     

Conformational preferences of amino acid side chains in collagen

Conformational energy computations were carried out on collagenlike triple-stranded conformations of several poly(tripeptide)s with the general structure CH₃CO? (Gly? X? Y)₃? NHCH₃. The sequences considered had various amino acid residues in position X or Y of the central tripeptide, with either Pro or Ala as a neighbor, i.e., Gly-X-Pro, Gly-X-Ala, Gly-Pro-Y, and Gly-Ala-Y. Minimum-energy conformations were computed for the side chains, and their distributions were compared for the four sequences. The residues used were Abu (= α-aminobutyric acid), Leu, Phe, Ser, Asp, Asn, Val, Ile, and Thr. The conformational energy of a ? Ch₂? CH₃ side chain in Abu was mapped as a function of the dihedral angle χ¹. Intrastrand interactions with neighboring residues do not affect the conformations of a side chain in position Y, and they have a minor effect on it in the X-Ala sequence, but they strongly restrict the conformational freedom of the side chain in the X-Pro sequence. Conversely, interstrand interactions do not affect side chains in position X, but they strongly restrict the conformational freedom of a side chain in position Y if there is a nearby Pro residue in a neighboring strand. Hydrogen bonds with the backbone can be formed in some conformations of long polar side chains, such as Asp, Asn, or Gln. All amino acid residues can be accommodated in collagen. Because of the interactions mentioned above, steric and energetic constraints can be correlated with observed preferences of certain amino acids for positions X or Y in collagen. Hence, these preferences may be explained, in part, in terms of differences in the conformational freedom of the side chains in the triple-stranded structure.     

Snorkeling preferences foster an amino acid composition bias in transmembrane helices

By analyzing transmembrane (TM) helices in known structures, we find that some polar amino acids are more frequent at the N terminus than at the C terminus. We propose the asymmetry occurs because most polar amino acids are better able to snorkel their polar atoms away from the membrane core at the N terminus than at the C terminus. Two findings lead us to this proposition: (1) side-chain conformations are influenced strongly by the N or C-terminal position of the amino acid in the bilayer, and (2) the favored snorkeling direction of an amino acid correlates well with its N to C-terminal composition bias. Our results suggest that TM helix predictions should incorporate an N to C-terminal composition bias, that rotamer preferences of TM side-chains are position-dependent, and that the ability to snorkel influences the evolutionary selection of amino acids for the helix N and C termini.