Functional diversity of the plant glycine-rich proteins superfamily

The first plant glycine-rich proteins (GRPs) have been isolated more than 20 years ago based on their specific expression pattern and/or modulation by several biotic and abiotic factors. This superfamily is characterized by the presence of a glycine-rich domain arranged in (Gly)_n-X repeats. The presence of additional motifs, as well as the nature of the glycine repeats, groups them in different classes. The diversity in structure as well as in expression pattern, modulation and sub cellular localization have always indicated that these proteins, although classified as members of the same superfamily, would perform different functions in planta. Only now, two decades later, with the first functional characterizations of plant GRPs their involvement in diverse biological and biochemical processes are being uncovered. Here, we review the so far ascribed functions of plant GRPs.Key words: glycine-rich protein, cold-shock protein, RNA-binding protein, plant defense, flowering, cell elongation, RNA chaperone, signal transduction, oleosin, pollen competition     

Brain mineralocorticoid receptor diversity: Functional implications

Mineralocorticoid receptors (MRs) in neurons of the anterior hypothalamus and the periventricular brain regions mediate aldosterone-selective actions on sodium hemeostasis, salt appetite and cardiovascular regulation. Corticosterone is not effective in these neurons, possibly because it is enzymatically inactivated. However, MRs in limbic brain regions, notably in the hippocampal neurons, do already respond to very low concentrations of both corticosterone and aldosterone. The MR-mediated effects stabilize neuronal transmission and appear critical for neuronal integrity of a sub-region of the hippocampus: the dentate gyrus. Higher concentrations of corticosterone induced by stress and the circadian rise progressively activate the lower affinity glucocorticoid receptors (GRs), which in coordination with MR-mediated actions then facilitate adaptive processes required for recovery of homeostasis. It is postulated that this balanced MR- and GR-mediated action of corticosterone is of critical importance for regulation of the stress response and behavioural adaptation.     

Functional implications of structure-based sequence alignment of proteins in the extracellular pectate lyase superfamily.

Pectate lyases are plant virulence factors that degrade the pectate component of the plant cell wall. The enzymes share considerable sequence homology with plant pollen and style proteins, suggesting a shared structural topology and possibly functional relationships as well. The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, C and E, have been superimposed and the structurally conserved amino acids have been identified. There are 232 amino acids that superimpose with a root-mean-square deviation of 3 A or less. These amino acids have been used to correct the primary sequence alignment derived from evolution-based techniques. Subsequently, multiple alignment techniques have allowed the realignment of other extracellular pectate lyases as well as all sequence homologs, including pectin lyases and the plant pollen and style proteins. The new multiple sequence alignment reveals amino acids likely to participate in the parallel beta helix motif, those involved in binding Ca2+, and those invariant amino acids with potential catalytic properties. The latter amino acids cluster in two well-separated regions on the pectate lyase structures, suggesting two distinct enzymatic functions for extracellular pectate lyases and their sequence homologs.     

Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes

The HAD (haloacid dehalogenase) superfamily includes phosphoesterases, ATPases, phosphonatases, dehalogenases, and sugar phosphomutases acting on a remarkably diverse set of substrates. The availability of numerous crystal structures of representatives belonging to diverse branches of the HAD superfamily provides us with a unique opportunity to reconstruct their evolutionary history and uncover the principal determinants that led to their diversification of structure and function. To this end we present a comprehensive analysis of the HAD superfamily that identifies their unique structural features and provides a detailed classification of the entire superfamily. We show that at the highest level the HAD superfamily is unified with several other superfamilies, namely the DHH, receiver (CheY-like), von Willebrand A, TOPRIM, classical histone deacetylases and PIN/FLAP nuclease domains, all of which contain a specific form of the Rossmannoid fold. These Rossmannoid folds are distinguished from others by the presence of equivalently placed acidic catalytic residues, including one at the end of the first core beta-strand of the central sheet. The HAD domain is distinguished from these related Rossmannoid folds by two key structural signatures, a "squiggle" (a single helical turn) and a "flap" (a beta hairpin motif) located immediately downstream of the first beta-strand of their core Rossmanoid fold. The squiggle and the flap motifs are predicted to provide the necessary mobility to these enzymes for them to alternate between the "open" and "closed" conformations. In addition, most members of the HAD superfamily contains inserts, termed caps, occurring at either of two positions in the core Rossmannoid fold. We show that the cap modules have been independently inserted into these two stereotypic positions on multiple occasions in evolution and display extensive evolutionary diversification independent of the core catalytic domain. The first group of caps, the C1 caps, is directly inserted into the flap motif and regulates access of reactants to the active site. The second group, the C2 caps, forms a roof over the active site, and access to their internal cavities might be in part regulated by the movement of the flap. The diversification of the cap module was a major factor in the exploration of a vast substrate space in the course of the evolution of this superfamily. We show that the HAD superfamily contains 33 major families distributed across the three superkingdoms of life. Analysis of the phyletic patterns suggests that at least five distinct HAD proteins are traceable to the last universal common ancestor (LUCA) of all extant organisms. While these prototypes diverged prior to the emergence of the LUCA, the major diversification in terms of both substrate specificity and reaction types occurred after the radiation of the three superkingdoms of life, primarily in bacteria. Most major diversification events appear to correlate with the acquisition of new metabolic capabilities, especially related to the elaboration of carbohydrate metabolism in the bacteria. The newly identified relationships and functional predictions provided here are likely to aid the future exploration of the numerous poorly understood members of this large superfamily of enzymes.     

Functional differentiation of proteins: implications for structural genomics

Structural genomics is a broad initiative of various centers aiming to provide complete coverage of protein structure space. Because it is not feasible to experimentally determine the structures of all proteins, it is generally agreed that the only viable strategy to achieve such coverage is to carefully select specific proteins (targets), determine their structure experimentally, and then use comparative modeling techniques to model the rest. Here we suggest that structural genomics centers refine the structure-driven approach in target selection by adopting function-based criteria. We suggest targeting functionally divergent superfamilies within a given structural fold so that each function receives a structural characterization. We have developed a method to do so, and an itemized survey of several functionally rich folds shows that they are only partially functionally characterized. We call upon structural genomics centers to consider this approach and upon computational biologists to further develop function-based targeting methods.     

Retroviral integrase superfamily: the structural perspective

The retroviral integrase superfamily (RISF) comprises numerous important nucleic acid‐processing enzymes, including transposases, integrases and various nucleases. These enzymes are involved in a wide range of processes such as transposition, replication and repair of DNA, homologous recombination, and RNA‐mediated gene silencing. Two out of the four enzymes that are encoded by the human immunodeficiency virus—RNase H1 and integrase—are members of this superfamily. RISF enzymes act on various substrates, and yet show remarkable mechanistic and structural similarities. All share a common fold of the catalytic core and the active site, which is composed primarily of carboxylate residues. Here, I present RISF proteins from a structural perspective, describing the individual members and the common and divergent elements of their structures, as well as the mechanistic insights gained from the structures of RNase H1 enzyme complexes with RNA/DNA hybrids.     

The 2-A crystal structure of 6-oxo camphor hydrolase. New structural diversity in the crotonase superfamily

6-Oxo camphor hydrolase (OCH) is an enzyme of the crotonase superfamily that catalyzes carbon-carbon bond cleavage in bicyclic beta-diketones via a retro-Claisen reaction (Grogan, G., Roberts, G. A., Bougioukou, D., Turner, N. J., and Flitsch, S. L. (2001) J. Biol. Chem. 276, 12565-12572). The native structure of OCH has been solved at 2.0-A resolution with selenomethionine multiple wave anomalous dispersion and refined to a final R(free) of 19.0. The structure of OCH consists of a dimer of trimers that resembles the "parent" enzyme of the superfamily, enoyl-CoA hydratase. In contrast to enoyl-CoA hydratase, however, two octahedrally coordinated sodium atoms are found at the 3-fold axis of the hexamer of OCH, and the C-terminal helix of OCH does not form a discrete domain. Models of the substrate, 6-oxo camphor, and a proposed enolate intermediate in the putative active site suggest possible mechanistic roles for Glu-244, Asp-154, His-122, His-45, and His-145.     

Functional and structural diversity of the human Dickkopf gene family

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.     

The cation/Ca(2+) exchanger superfamily: phylogenetic analysis and structural implications

Cation/Ca(2+) exchangers are an essential component of Ca(2+) signaling pathways and function to transport cytosolic Ca(2+) across membranes against its electrochemical gradient by utilizing the downhill gradients of other cation species such as H(+), Na(+), or K(+). The cation/Ca(2+) exchanger superfamily is composed of H(+)/Ca(2+) exchangers and Na(+)/Ca(2+) exchangers, which have been investigated extensively in both plant cells and animal cells. Recently, information from completely sequenced genomes of bacteria, archaea, and eukaryotes has revealed the presence of genes that encode homologues of cation/Ca(2+) exchangers in many organisms in which the role of these exchangers has not been clearly demonstrated. In this study, we report a comprehensive sequence alignment and the first phylogenetic analysis of the cation/Ca(2+) exchanger superfamily of 147 sequences. The results present a framework for structure-function relationships of cation/Ca(2+) exchangers, suggesting unique signature motifs of conserved residues that may underlie divergent functional properties. Construction of a phylogenetic tree with inclusion of cation/Ca(2+) exchangers with known functional properties defines five protein families and the evolutionary relationships between the members. Based on this analysis, the cation/Ca(2+) exchanger superfamily is classified into the YRBG, CAX, NCX, and NCKX families and a newly recognized family, designated CCX. These findings will provide guides for future studies concerning structures, functions, and evolutionary origins of the cation/Ca(2+) exchangers.     

Functional characterization of thioesterase superfamily member 1/Acyl-CoA thioesterase 11: implications for metabolic regulation

Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is highly expressed in brown adipose tissue and limits energy expenditure in mice. Them1 is a putative fatty acyl-CoA thioesterase that comprises tandem hot dog-fold thioesterase domains and a lipid-binding C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. To better define its role in metabolic regulation, this study examined the biochemical and enzymatic properties of Them1. Purified recombinant Them1 dimerized in solution to form an active fatty acyl-CoA thioesterase. Dimerization was induced by fatty acyl-CoAs, coenzyme A (CoASH), ATP, and ADP. Them1 hydrolyzed a range of fatty acyl-CoAs but exhibited a relative preference for long-chain molecular species. Thioesterase activity varied inversely with temperature, was stimulated by ATP, and was inhibited by ADP and CoASH. Whereas the thioesterase domains of Them1 alone were sufficient to yield active recombinant protein, the START domain was required for optimal enzyme activity. An analysis of subcellular fractions from mouse brown adipose tissue and liver revealed that Them1 contributes principally to the fatty acyl-CoA thioesterase activity of microsomes and nuclei. These findings suggest that under biological conditions, Them1 functions as a lipid-regulated fatty acyl-CoA thioesterase that could be targeted for the management of metabolic disorders.     

Phosphoribosyltransferase superfamily: A comparative structural analysis

Purine phosphoribosyltransferases are members of a group of enzymes that are responsible for the formation of purine, pyrimidine and pyridine nucleotides. One feature of this family is a flexible loop, which is involved in the catalytic mechanism. This loop is variable both in sequence and structure in the phosphoribosyltransferase family. This paper describes the modelling and validation of a model of Plasmodium falciparum hypoxanthine-guanidine- phosphoribosyltransferase in an open conformation. A comparison of this model with 3D-structures of other members of the phosphorybosyltransferase family has allowed an assessment of the role of the open and closed conformations of the loop in the catalytic mechanism.     

Evolutionary history,structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes

Background  

Peptidoglycan is hydrolyzed by a diverse set of enzymes during bacterial growth, development and cell division. The N1pC/P60 proteins define a family of cell-wall peptidases that are widely represented in various bacterial lineages. Currently characterized members are known to hydrolyze D-γ-glutamyl-meso-diaminopimelate or N-acetylmuramate-L-alanine linkages.     

Functional and structural diversity in the Als protein family of Candida albicans

The human fungal pathogen Candida albicans colonizes and invades a wide range of host tissues. Adherence to host constituents plays an important role in this process. Two members of the C. albicans Als protein family (Als1p and Als5p) have been found to mediate adherence; however, the functions of other members of this family are unknown. In this study, members of the ALS gene family were cloned and expressed in Saccharomyces cerevisiae to characterize their individual functions. Distinct Als proteins conferred distinct adherence profiles to diverse host substrates. Using chimeric Als5p-Als6p constructs, the regions mediating substrate-specific adherence were localized to the N-terminal domains in Als proteins. Interestingly, a subset of Als proteins also mediated endothelial cell invasion, a previously unknown function of this family. Consistent with these results, homology modeling revealed that Als members contain anti-parallel beta-sheet motifs interposed by extended regions, homologous to adhesins or invasins of the immunoglobulin superfamily. This finding was confirmed using circular dichroism and Fourier transform infrared spectrometric analysis of the N-terminal domain of Als1p. Specific regions of amino acid hypervariability were found among the N-terminal domains of Als proteins, and energy-based models predicted similarities and differences in the N-terminal domains that probably govern the diverse function of Als family members. Collectively, these results indicate that the structural and functional diversity within the Als family provides C. albicans with an array of cell wall proteins capable of recognizing and interacting with a wide range of host constituents during infection.     

Functional implications of skeletal diversity in two South American tamarins

This study examines the relationship between positional behavior and morphology for two closely related South American tamarin species, Saguinus fuscicollis illigeri and Saguinus oedipus oedipus. Although never systematically documented, the two species are suspected of exhibiting highly similar yet subtly different locomotor behaviors with a greater propensity for climbing, springing, and more powerful leaping in S. oedipus. Fourteen measurements reflecting biomechanical structuring were taken from the postcranial skeleton of 28 individuals of each species. The data were reduced to principal component scores and subjected to a multiple analysis of variance to determine the extent of intergroup skeletal variation. Areas of significant skeletal diversity were interpreted functionally. The results suggest that the differences in functional patterning among the two species is consistent with the behavioral hypothesis. It is concluded that subtle changes in positional behavior may be capable of altering skeletal morphology.     

Evolution of functional diversity in the cupin superfamily

The cupin superfamily of proteins is among the most functionally diverse of any described to date. It was named on the basis of the conserved β-barrel fold (‘cupa’ is the Latin term for a small barrel), and comprises both enzymatic and non-enzymatic members, which have either one or two cupin domains. Within the conserved tertiary structure, the variety of biochemical function is provided by minor variation of the residues in the active site and the identity of the bound metal ion. This review discusses the advantages of this particular scaffold and provides an evolutionary analysis of 18 different subclasses within the cupin superfamily.