Comparison of the hemocyanin beta-barrel with other Greek key beta-barrels: possible importance of the "beta-zipper" in protein structure and folding.

The Greek key beta-barrel topology is a folding motif observed in many proteins of widespread evolutionary origin. The arthropodan hemocyanins also have such a Greek key beta-barrel, which forms the core of the third domain of this protein. The hemocyanin beta-barrel was found to be structurally very similar to the beta-barrels of the immunoglobulin domains, Cu,Zn-superoxide dismutase and the chromophore carrying antitumor proteins. The structural similarity within this group of protein families is not accompanied by an evolutionary or functional relationship. It is therefore possible to study structure-sequence relations without bias from nonstructural constraints. The present study reports a conserved pattern of features in these Greek key beta-barrels that is strongly suggestive of a folding nucleation site. This proposed nucleation site, which we call a "beta-zipper," shows a pattern of well-conserved, large hydrophobic residues on two sequential beta-strands joined by a short loop. Each beta-zipper strand is near the center of one of the beta-sheets, so that the two strands face each other from opposite sides of the barrel and interact through their hydrophobic side chains, rather than forming a hydrogen-bonded beta-hairpin. Other protein families with Greek key beta-barrels that do not as strongly resemble the immunoglobulin fold--such as the azurins, plastocyanins, crystallins, and prealbumins--also contain the beta-zipper pattern, which might therefore be a universal feature of Greek key beta-barrel proteins.     

A comprehensive analysis of protein phosphatases in rice and Arabidopsis

Protein phosphatases play essential roles in many cellular processes through the reversible protein phosphorylation that dictates many signal transduction pathways among organisms. Based on an in silico analysis, we classified 163 and 164 non-redundant protein phosphatases in rice and Arabidopsis, respectively. Protein serine/threonine phosphatases make up 67% of the total in both plants, in contrast to those of human, where this fraction is about 27%. Based on domain organization and intron composition analyses, we found that protein phosphatases in the two plants are highly conserved in structure. Evolutionary analysis suggests that segmental duplications occurring 40–70 million years ago, contributed to the limited expansion of protein phosphatases. Gene expression analysis suggests that most phosphatases have broad expression spectra, with high abundance in four surveyed tissues (root, leaf, inflorescence, and seedling); only 46 and 12 phosphatases expressed in a single tissue of rice and Arabidopsis, respectively, regardless of their expression levels. Promoter analysis among different phosphatase subfamilies demonstrates a variable distribution of the w-box (a cis-element involved in disease resistance) between rice and Arabidopsis.     

The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate

Folding of the 123 amino acid residue Greek key protein apo-pseudo azurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15 degrees C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U <==> I <==> N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I <==> U <==> N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 microseconds). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.     

A comprehensive analysis of 40 blind protein structure predictions

Background  

We thoroughly analyse the results of 40 blind predictions for which an experimental answer was made available at the fourth meeting on the critical assessment of protein structure methods (CASP4). Using our comparative modelling and fold recognition methodologies, we made 29 predictions for targets that had sequence identities ranging from 50% to 10% to the nearest related protein with known structure. Using our ab initio methodologies, we made eleven predictions for targets that had no detectable sequence relationships.     

Alternate routes to conformational specificity in a Greek key beta barrel protein.

The N-terminal domain of protein S, a Greek key calcium-binding protein from Myxococcus xanthus, forms an atypical molten globule in the calcium-free state. The structure of this state is characterized by significant conformational fluctuations, which are localized to a subdomain that is not contiguous along the polypeptide chain. The conformational instability of this subdomain appears to arise from repulsive electrostatic interactions of four acidic side chains that are clustered together but are removed from the calcium-binding sites. This domain can be induced to form a native-like state through two different routes, calcium binding or reduction of pH. Acid-induced folding stabilizes the locally unfolded subdomain by selectively removing repulsive interactions without significantly affecting global stability. In contrast, calcium binding appears to increase local stability indirectly by causing global stabilization.     

A comprehensive analysis of non-sequential alignments between all protein structures

Background  

The majority of relations between proteins can be represented as a conventional sequential alignment. Nevertheless, unusual non-sequential alignments with different connectivity of the aligned fragments in compared proteins have been reported by many researchers. It is interesting to understand those non-sequential alignments; are they unique, sporadic cases or they occur frequently; do they belong to a few specific folds or spread among many different folds, as a common feature of protein structure. We present here a comprehensive large-scale study of non-sequential alignments between available protein structures in Protein Data Bank.     

Mutational analysis of the conserved motifs of influenza A virus polymerase basic protein 1.

Influenza virus polymerase complex is a heterotrimer consisting of polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Of these, only PB1, which has been implicated in RNA chain elongation, possesses the four conserved motifs (motifs I, II, III, and IV) and the four invariant amino acids (one in each motif) found among all viral RNA-dependent RNA or RNA-dependent DNA polymerases. We have modified an assay system developed by Huang et al. (T.-J. Huang, P. Palese, and M. Krystal, J. Virol. 64:5669-5673, 1990) to reconstitute the functional polymerase activity in vivo. Using this assay, we have examined the requirement of each of these motifs of PB1 in polymerase activity. We find that each of these invariant amino acids is critical for PB1 activity and that mutation in any one of these residues renders the protein nonfunctional. We also find that in motif III, which contains the SSDD sequence, the signature sequence of influenza virus RNA polymerase, SDD is essentially invariant and cannot accommodate sequences found in other RNA viral polymerases. However, conserved changes in the flanking sequences of SDD can be partially tolerated. These results provide the experimental evidence that influenza virus PB1 possesses a similar polymerase module as has been proposed for other RNA viruses and that the core SDD sequence of influenza virus PB1 represents a sequence variant of the GDN in negative-stranded nonsegmented RNA viruses, GDD in positive-stranded RNA virus and double-stranded RNA viruses, or MDD in retroviruses.     

Discovery of protein phosphorylation motifs through exploratory data analysis

Background

The need for efficient algorithms to uncover biologically relevant phosphorylation motifs has become very important with rapid expansion of the proteomic sequence database along with a plethora of new information on phosphorylation sites. Here we present a novel unsupervised method, called Motif Finder (in short, F-Motif) for identification of phosphorylation motifs. F-Motif uses clustering of sequence information represented by numerical features that exploit the statistical information hidden in some foreground data. Furthermore, these identified motifs are then filtered to find “actual” motifs with statistically significant motif scores.

Results and Discussion

We have applied F-Motif to several new and existing data sets and compared its performance with two well known state-of-the-art methods. In almost all cases F-Motif could identify all statistically significant motifs extracted by the state-of-the-art methods. More importantly, in addition to this, F-Motif uncovers several novel motifs. We have demonstrated using clues from the literature that most of these new motifs discovered by F-Motif are indeed novel. We have also found some interesting phenomena. For example, for CK2 kinase, the conserved sites appear only on the right side of S. However, for CDK kinase, the adjacent site on the right of S is conserved with residue P. In addition, three different encoding methods, including a novel position contrast matrix (PCM) and the simplest binary coding, are used and the ability of F-motif to discover motifs remains quite robust with respect to encoding schemes.

Conclusions

An iterative algorithm proposed here uses exploratory data analysis to discover motifs from phosphorylated data. The effectiveness of F-Motif has been demonstrated using several real data sets as well as using a synthetic data set. The method is quite general in nature and can be used to find other types of motifs also. We have also provided a server for F-Motif at http://f-motif.classcloud.org/, http://bio.classcloud.org/f-motif/ or http://ymu.classcloud.org/f-motif/.     

Mutational analysis of conserved sequence motifs in the budding yeast Cdc6 protein

The Cdc6 protein is required to load a complex of Mcm2-7 family members (the MCM complex) into prereplicative complexes at budding yeast origins of DNA replication. Cdc6p is a member of the AAA(+) superfamily of proteins, which includes the prokaryotic and eukaryotic clamp loading proteins. These proteins share a number of conserved regions of homology and a common three-dimensional architecture. Two of the conserved sequence motifs are the Walker A and B motifs that are involved in nucleotide metabolism and are essential for Cdc6p function in vivo. Here, we analyse mutants in the other conserved sequence motifs. Several of these mutants are temperature-sensitive for growth and are unable to recruit the MCM complex to chromatin at the restrictive temperature. In one such temperature-sensitive mutant, a highly conserved asparagine residue in the sensor I motif was changed to alanine. Overexpression of this mutant protein is lethal. This phenotype is very similar to the phenotype previously described for a mutation in the Walker B motif, suggesting a common role for sensor I and the Walker B motif in Cdc6 function.     

Genomic analysis of membrane protein families: abundance and conserved motifs

Background

Polytopic membrane proteins can be related to each other on the basis of the number of transmembrane helices and sequence similarities. Building on the Pfam classification of protein domain families, and using transmembrane-helix prediction and sequence-similarity searching, we identified a total of 526 well-characterized membrane protein families in 26 recently sequenced genomes. To this we added a clustering of a number of predicted but unclassified membrane proteins, resulting in a total of 637 membrane protein families.

Results

Analysis of the occurrence and composition of these families revealed several interesting trends. The number of assigned membrane protein domains has an approximately linear relationship to the total number of open reading frames (ORFs) in 26 genomes studied. Caenorhabditis elegans is an apparent outlier, because of its high representation of seven-span transmembrane (7-TM) chemoreceptor families. In all genomes, including that of C. elegans, the number of distinct membrane protein families has a logarithmic relation to the number of ORFs. Glycine, proline, and tyrosine locations tend to be conserved in transmembrane regions within families, whereas isoleucine, valine, and methionine locations are relatively mutable. Analysis of motifs in putative transmembrane helices reveals that GxxxG and GxxxxxxG (which can be written GG4 and GG7, respectively; see Materials and methods) are among the most prevalent. This was noted in earlier studies; we now find these motifs are particularly well conserved in families, however, especially those corresponding to transporters, symporters, and channels.

Conclusions

We carried out a genome-wide analysis on patterns of the classified polytopic membrane protein families and analyzed the distribution of conserved amino acids and motifs in the transmembrane helix regions in these families.

     

Trafficking motifs in the SARS-coronavirus nucleocapsid protein

The severe acute respiratory syndrome-coronavirus nucleocapsid (N) protein is involved in virus replication and modulation of cell processes. In this latter respect control may in part be achieved through the sub-cellular localisation of the protein. N protein predominately localises in the cytoplasm (the site of virus replication and assembly) but also in the nucleus/nucleolus. Using a combination of live-cell and confocal microscopy coupled to mutagenesis we identified a cryptic nucleolar localisation signal in the central part of the N protein. In addition, based on structural comparison to the avian coronavirus N protein, a nuclear export signal was identified in the C-terminal region of the protein.     

A comprehensive phylogenetic analysis of termites (Isoptera) illuminates key aspects of their evolutionary biology

The first comprehensive combined molecular and morphological phylogenetic analysis of the major groups of termites is presented. This was based on the analysis of three genes (cytochrome oxidase II, 12S and 28S) and worker characters for approximately 250 species of termites. Parsimony analysis of the aligned dataset showed that the monophyly of Hodotermitidae, Kalotermitidae and Termitidae were well supported, while Termopsidae and Rhinotermitidae were both paraphyletic on the estimated cladogram. Within Termitidae, the most diverse and ecologically most important family, the monophyly of Macrotermitinae, Foraminitermitinae, Apicotermitinae, Syntermitinae and Nasutitermitinae were all broadly supported, but Termitinae was paraphyletic. The pantropical genera Termes, Amitermes and Nasutitermes were all paraphyletic on the estimated cladogram, with at least 17 genera nested within Nasutitermes, given the presently accepted generic limits. Key biological features were mapped onto the cladogram. It was not possible to reconstruct the evolution of true workers unambiguously, as it was as parsimonious to assume a basal evolution of true workers and subsequent evolution of pseudergates, as to assume a basal condition of pseudergates and subsequent evolution of true workers. However, true workers were only found in species with either separate- or intermediate-type nests, so that the mapping of nest habit and worker type onto the cladogram were perfectly correlated. Feeding group evolution, however, showed a much more complex pattern, particularly within the Termitidae, where it proved impossible to estimate unambiguously the ancestral state within the family (which is associated with the loss of worker gut flagellates). However, one biologically plausible optimization implies an initial evolution from wood-feeding to fungus-growing, proposed as the ancestral condition within the Termitidae, followed by the very early evolution of soil-feeding and subsequent re-evolution of wood-feeding in numerous lineages.     

Domains,motifs and clusters in the protein universe

The rapid growth of bio-sequence information has resulted in an increasing demand for reliable methods that group proteins. A few databases with curated alignments of protein families have demonstrated that expert-driven repositories can keep up with the data deluge in the genome era. These original resources implicitly identify domain-like modules in proteins. An increasing number of automatic methods have sprouted over the past few years that cluster the protein universe. Many of these implicitly dissect proteins into structural domain-like fragments. In a very coarse-grained evaluation, some of the automatic methods appear to be on par with expert-driven approaches. However, neither automatic nor manual methods are currently entirely up to the challenges of tasks such as target selection in structural genomics. Thus, we urgently need refined and sustained automatic clustering tools.     

Conformational and geometrical properties of idealized beta-barrels in proteins

An equation for calculating the distances between the atoms involved in forming an idealized hydrogen bond in a parallel or antiparallel beta-barrel has been derived by adjusting the corresponding data given by Pauling and Corey for a beta-sheet. Based on these distances, a geometrical optimization method was developed, by which one can generate various idealized beta-barrels: parallel or antiparallel, tilted or non-tilted, right-tilted or left-tilted. For each type of idealized beta-barrel thus obtained, the corresponding conformation and characteristic geometric parameters as well as their relationship are analyzed and discussed. Since the strand in a tilted beta-barrel traces a curve rather than a straight line on a cylinder-like surface, a regular chain in which the dihedral angles of each residue are the same cannot form a tilted beta-barrel but only a non-tilted beta-barrel. As observed, the strands of a right-tilted beta-barrel possess a very strong right-handed twist. The radii of the idealized tilted parallel and antiparallel beta-barrels are greater than those of the corresponding non-tilted ones by approximately 1 A and approximately 1.5 A, respectively. Consequently, there is relatively more room for a tilted beta-barrel to accommodate the internal side-chains, suggesting that a conformational change from a non-tilted beta-barrel to a tilted one would ease the repulsion among the crowded internal side-chains so as to make the structure more stable. The values of root-mean-square fits indicate that the idealized right-tilted beta-barrels coincide quite well with the observed beta-barrels in both parallel and antiparallel cases.     

Evolution of CuZn superoxide dismutase and the Greek key beta-barrel structural motif

Detailed analysis of the CuZn superoxide dismutase (SOD) structure provides new results concerning the significance and molecular basis for sequence conservation, intron-exon boundary locations, gene duplication, and Greek key beta-barrel evolution. Using 15 aligned sequences, including a new mouse sequence, specific roles have been assigned to all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stereochemistry, supporting a primary biological function of superoxide dismutation. The beta-strands have no sequence insertions and deletions, whereas insertions occur within the loops connecting the beta-strands and at both termini. Thus, the beta-barrel with only four invariant residues is apparently over-determined, but dependent on multiple cooperative side chain interactions. The regions encoded by exon I, a proposed nucleation site for protein folding, and exon III, the Zn loop involved in stability and catalysis, are the major structural subdomains not included in the internal twofold axis of symmetry passing near the catalytic Cu ion. This provides strong confirmatory evidence for gene evolution by duplication and fusion followed by the addition of these two exons. The proposed evolutionary pathway explains the structural versatility of the Greek key beta-barrel through functional specialization and subdomain insertions in new loop connections, and provides a rationale for the size of the present day enzyme.     

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.