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 the Greek key motifs in protein beta-barrels and beta-sandwiches

The Greek key motifs are the topological signature of many beta-barrels and a majority of beta-sandwich structures. An updated survey of these structures integrates many early observations and newly emerging patterns and provides a better understanding of the unique role of Greek keys in protein structures. A stereotypical Greek key beta-barrel accommodates five or six strands and can have 12 possible topologies. All except one six-stranded topologies have been observed, and only one five-stranded topologies have been seen in actual structures. Of the representative beta-barrel structures analyzed here, half have left-handed Greek keys. This result challenges the empirical claim of the handedness regularity of Greek keys in beta-barrels. One of the five-stranded topologies that has not been observed in beta-barrels comprises two overlapping Greek keys. The two three-dimensional forms of this topology constitute a structural unit that is present in a vast majority of known beta-sandwich structures. Using this unit as the root, we have built a new taxonomy tree for the beta-sandwich folds and deduced a set of rules that appear to constrain how other beta-strands adjoin the unit to form a larger double-layered structure. These rules, though derived from a larger data set, are essentially the same as those drawn from earlier studies, suggesting that they may reflect the true topological constraints in the design of beta-sandwich structures. Finally, a novel variant of the Greek key motif (defined here as the twisted Greek key) has emerged which introduces loop crossings into the folded structures. Proteins 2000;40:409-419.     

Methionine adenosyltransferase as a useful molecular systematics tool revealed by phylogenetic and structural analyses

Structural and phylogenetic relationships among Bacteria and Eukaryota were analyzed by examining 292 methionine adenosyltransferase (MAT) amino acid sequences with respect to the crystal structure of this enzyme established for Escherichia coli and rat liver. Approximately 30% of MAT residues were found to be identical in all species. Five highly conserved amino acid sequence blocks did not vary in the MAT family. We detected specific structural features that correlated with sequence signatures for several clades, allowing taxonomical identification by sequence analysis. In addition, the number of amino acid residues in the loop connecting beta-strands A2 and A3 served to clearly distinguish sequences between eukaryotes and eubacteria. The molecular phylogeny of MAT genes in eukaryotes can be explained in terms of functional diversification coupled to gene duplication or alternative splicing and adaptation through strong structural constraints. Sequence analyses and intron/exon junction positions among nematodes, arthropods and vertebrates support the traditional Coelomata hypothesis. In vertebrates, the liver MAT I isoenzyme has gradually adapted its sequence towards one providing a more specific liver function. MAT phylogeny also served to cluster the major bacterial groups, demonstrating the superior phylogenetic performance of this ubiquitous, housekeeping gene in reconstructing the evolutionary history of distant relatives.     

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.     

Probing the Structural Basis for Enzyme-Substrate Recognition In Cu,Zn Superoxide Dismutase

A full understanding of enzyme-substrate interactions requires a detailed knowledge of their structural basis at atomic resolution. Crystallographic and biochemical data have been analyzed with coupled computational and computer graphic approaches to characterize the molecular basis for recognition of the superoxide anion substrate by Cu. Zn superoxide dismutase (SOD). Detailed analysis of the bovine SOD structure aligned with SOD sequences from 15 species provides new results concerning the significance and molecular basis for sequence conservation. Specific roles have been assigned for all 23 invariant residues and additional residues exhibiting functional equivalence. Sequence invariance is dominated by 15 residues that form the active site stcreochemistry. supporting a primary biological function of superoxide dismutation. Using data from crystallographic structures and site-directed mutants, we are testing the role of individual residues in the active site channel, including (in human SOD) Glu132, Glu133, Lys136, Thr137, and Arg 143. Electrostatic calculations incorporating molecular flexibility suggest that the region of positive electrostatic potential in and over the active site channel above the Cu ion sweeps through space during molecular motion to enhance the facilitated diffusion responsible for the enzyme's rapid catalytic rate.     

Functional expression in Escherichia coli and membrane topology of porin HopE, a member of a large family of conserved proteins in Helicobacter pylori

HopE is one of the smallest members of a family of 31 outer membrane proteins in Helicobacter pylori and has been shown to function as a porin. In this study it was cloned into Escherichia coli where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from E. coli reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from H. pylori. A model of the membrane topology of HopE was constructed and indicated that this protein formed a beta-barrel with 16 transmembrane amphipathic beta-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane beta-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.     

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.     

Evolutionary Aspects of Superoxide Dismutase: The Copper/Zinc Enzyme

Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes. The presence of the enzyme in the ponyfish symbiont Photobocterium leiognothi and some free living bacteria does not have an immediate explanation. Amino acid sequence alignment of 19 Cu/Zn superoxide disrnutases shows 21 invariant residues in key positions related to maintenance of the β-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts. Nineteen other residues are invariant in 18 of the 19 sequences. Thirteen of these nearly invariant residues show substitutions in Photobocterium Cu/Zn superoxide dismutase. Copper/zinc superoxide disrnutase from the trematode Schisiosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide. as in human extracellular superoxide dismutase which is a Cu/Zn enzyme. The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues. The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase. including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of β-barrel fold and active site structure for the extracellular enzyme.     

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins.

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides.The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear ((15)N) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel.This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action.     

Dynamic Monte Carlo simulations of globular protein folding/unfolding pathways. I. Six-member, Greek key beta-barrel proteins

In the context of a simplified diamond lattice model of a six-member, Greek key beta-barrel protein that is closely related in topology to plastocyanin, the nature of the folding and unfolding pathways have been investigated using dynamic Monte Carlo techniques. The mechanism of Greek key assembly is best described as punctuated "on site construction". Folding typically starts at or near a beta-turn, and then the beta-strands sequentially form by using existing folded structure as a scaffold onto which subsequent tertiary structure assembles. On average, beta-strands tend to zip up from one tight bend to the next. After the four-member, beta-barrel assembles, there is a long pause as the random coil portion of the chain containing the long loop thrahes about trying to find the native state. Thus, there is an entropic barrier that must be surmounted. However, while a given piece of the protein may be folding, another section may be unfolding. A competition therefore exists to assemble a fairly stable intermediate before it dissolves. Folding may initiate at any of the tight turns, but the turn closer to the N terminus seems to be preferred due to well-known excluded volume effects. When the protein first starts to fold, there are a multiplicity of folding pathways, but the number of options is reduced as the system gets closer to the native state. In the early stages, the excluded volume effect exerted by the already assembled protein helps subsequent assembly. Then, near the native conformation, the folded parts reduce the accessible conformational space available to the remaining unfolded sections. Unfolding essentially occurs in reverse. Employing a simple statistical mechanical theory, the configurational free energy along the reaction co-ordinate for this model has been constructed. The free energy surface, in agreement with the simulations, provides the following predictions. The transition state is quite near the native state, and consists of five of the six beta-strands being fully assembled, with the remaining long loop plus sixth beta-strand in place, but only partially assembled. It is separated from the beta-barrel intermediate by a free energy barrier of mainly entropic origin and from the native state by a barrier that is primarily energetic in origin. The latter feature is in agreement with the "Cardboard Box" model described by Goldenberg and Creighton but, unlike their model, the transition state is not a high-energy distorted form of the native state.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding.

The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity.     

The hypothetical protein Atu4866 from Agrobacterium tumefaciens adopts a streptavidin-like fold

Atu4866 is a 79-residue conserved hypothetical protein of unknown function from Agrobacterium tumefaciens. Protein sequence alignments show that it shares > or =60% sequence identity with 20 other hypothetical proteins of bacterial origin. However, the structures and functions of these proteins remain unknown so far. To gain insight into the function of this family of proteins, we have determined the structure of Atu4866 as a target of a structural genomics project using solution NMR spectroscopy. Our results reveal that Atu4866 adopts a streptavidin-like fold featuring a beta-barrel/sandwich formed by eight antiparallel beta-strands. Further structural analysis identified a continuous patch of conserved residues on the surface of Atu4866 that may constitute a potential ligand-binding site.     

Sequential reorganization of beta-sheet topology by insertion of a single strand

Insertions, duplications, and deletions of sequence segments are thought to be major evolutionary mechanisms that increase the structural and functional diversity of proteins. Alternative splicing, for example, is an intracellular editing mechanism that is thought to generate isoforms for 30%-50% of all human genes. Whereas the inserted sequences usually display only minor structural rearrangements at the insertion site, recent observations indicate that they may also cause more dramatic structural displacements of adjacent structures. In the present study we test how artificially inserted sequences change the structure of the beta-sheet region in T4 lysozyme. Copies of two different beta-strands were inserted into two different loops of the beta-sheet, and the structures were determined. Not surprisingly, one insert "loops out" at its insertion site and forms a new small beta-hairpin structure. Unexpectedly, however, the second insertion leads to displacement of adjacent strands and a sequential reorganization of the beta-sheet topology. Even though the insertions were performed at two different sites, looping out occurred at the C-terminal end of the same beta-strand. Reasons as to why a non-native sequence would be recruited to replace that which occurs in the native protein are discussed. Our results illustrate how sequence insertions can facilitate protein evolution through both local and nonlocal changes in structure.     

Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies

The crystal structure of a complex of methyl-alpha-D-mannoside with banana lectin from Musa paradisiaca reveals two primary binding sites in the lectin, unlike in other lectins with beta-prism I fold which essentially consists of three Greek key motifs. It has been suggested that the fold evolved through successive gene duplication and fusion of an ancestral Greek key motif. In other lectins, all from dicots, the primary binding site exists on one of the three motifs in the three-fold symmetric molecule. Banana is a monocot, and the three motifs have not diverged enough to obliterate sequence similarity among them. Two Greek key motifs in it carry one primary binding site each. A common secondary binding site exists on the third Greek key. Modelling shows that both the primary sites can support 1-2, 1-3, and 1-6 linked mannosides with the second residue interacting in each case primarily with the secondary binding site. Modelling also readily leads to a bound branched mannopentose with the nonreducing ends of the two branches anchored at the two primary binding sites, providing a structural explanation for the lectin's specificity for branched alpha-mannans. A comparison of the dimeric banana lectin with other beta-prism I fold lectins, provides interesting insights into the variability in their quaternary structure.     

Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter

Autotransporter proteins are defined by the ability to drive their own secretion across the bacterial outer membrane. The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adhesion to the respiratory epithelium. In this report, we present the crystal structure of the C-terminal end of Hia, corresponding to the entire Hia translocator domain and part of the passenger domain (residues 992-1098). This domain forms a beta-barrel with 12 transmembrane beta-strands, including four strands from each subunit. The beta-barrel has a central channel of 1.8 nm in diameter that is traversed by three N-terminal alpha-helices, one from each subunit. Mutagenesis studies demonstrate that the transmembrane portion of the three alpha-helices and the loop region between the alpha-helices and the neighboring beta-strands are essential for stability of the trimeric structure of the translocator domain, and that trimerization of the translocator domain is a prerequisite for translocator activity. Overall, this study provides important insights into the mechanism of translocation in trimeric autotransporters.     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

Three-dimensional structure determined for a subunit of human tRNA splicing endonuclease (Sen15) reveals a novel dimeric fold

Splicing of eukaryal intron-containing tRNAs requires the action of the heterotetrameric splicing endonuclease, which is composed of two catalytic subunits, Sen34 and Sen2, and two structural subunits, Sen15 and Sen54. Here we report the solution structure of the human tRNA splicing endonuclease subunit HsSen15. To facilitate the structure determination, we removed the disordered 35 N-terminal and 14 C-terminal residues of the full-length protein to produce HsSen15(36-157). The structure of HsSen15(36-157), the first for a subunit of a eukaryal splicing endonuclease, revealed that the protein possesses a novel homodimeric fold. Each monomer consists of three alpha-helices and a mixed antiparallel/parallel beta-sheet, arranged in a topology similar to that of the C-terminal domain of Methanocaldococcus jannaschii endonuclease. The dimeric interface is dominated by a beta-barrel structure, formed by face-to-face packing of two, three-stranded beta-sheets. Each of the beta-sheets results from reciprocal parallel pairing of one beta-strand from one subunit with two other beta-strands from the symmetric subunit. The structural model provides insights into the functional assembly of the human tRNA splicing endonuclease.