Protein tyrosine phosphatases: structure-function relationships

Structural analysis of protein tyrosine phosphatases (PTPs) has expanded considerably in the last several years, producing more than 200 structures in this class of enzymes (from 35 different proteins and their complexes with ligands). The small-medium size of the catalytic domain of approximately 280 residues plus a very compact fold makes it amenable to cloning and overexpression in bacterial systems thus facilitating crystallographic analysis. The low molecular weight PTPs being even smaller, approximately 150 residues, are also perfect targets for NMR analysis. The availability of different structures and complexes of PTPs with substrates and inhibitors has provided a wealth of information with profound effects in the way we understand their biological functions. Developments in mammalian expression technology recently led to the first crystal structure of a receptor-like PTP extracellular region. Altogether, the PTP structural work significantly advanced our knowledge regarding the architecture, regulation and substrate specificity of these enzymes. In this review, we compile the most prominent structural traits that characterize PTPs and their complexes with ligands. We discuss how the data can be used to design further functional experiments and as a basis for drug design given that many PTPs are now considered strategic therapeutic targets for human diseases such as diabetes and cancer.     

Protein kinase C pseudosubstrate prototope: structure-function relationships

A structure-function study of the protein kinase C (PK-C) pseudosubstrate sequence (R19FARK-GALRQKNV31) has been undertaken. The role of specific residues was investigated using an alanine substitution scan. Arg-22 was the most important determinant in the inhibitor sequence, since substitution of this residue by alanine gave a 600-fold increase in the IC50 value to 81 +/- 9 microM. Substitutions of other basic residue also increased the IC50, 5-, 11- and 24-fold for the Ala-19, Ala-23 and Ala-27 substitutions, respectively. The importance of basic residues in determining the potency of the pseudosubstrate peptide reflects the requirements for these residues in peptide substrate phosphorylation. The residues Gly-24, Leu-26 and Gln-28 were also important for pseudosubstrate inhibitor potency. The large difference in the IC50 value for the [A22]PK-C(19-31) peptide makes it a valuable control in studies employing the pseudosubstrate peptide to explore functional roles of PK-C.     

Apolipoprotein A-I: structure-function relationships

The inverse relationship between high density lipoprotein (HDL) plasma levels and coronary heart disease has been attributed to the role that HDL and its major constituent, apolipoprotein A-I (apoA-I), play in reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apoA-I to promote cellular cholesterol efflux, bind lipids, activate lecithin:cholesterol acyltransferase (LCAT), and form mature HDL that interact with specific receptors and lipid transfer proteins. From the intensive analysis of apoA-I secondary structure has emerged our current understanding of its different classes of amphipathic alpha-helices, which control lipid-binding specificity. The main challenge now is to define apoA-I tertiary structure in its lipid-free and lipid-bound forms. Two models are considered for discoidal lipoproteins formed by association of two apoA-I with phospholipids. In the first or picket fence model, each apoA-I wraps around the disc with antiparallel adjacent alpha-helices and with little intermolecular interactions. In the second or belt model, two antiparallel apoA-I are paired by their C-terminal alpha-helices, wrap around the lipoprotein, and are stabilized by multiple intermolecular interactions. While recent evidence supports the belt model, other models, including hybrid models, cannot be excluded. ApoA-I alpha-helices control lipid binding and association with varying levels of lipids. The N-terminal helix 44-65 and the C-terminal helix 210-241 are recognized as important for the initial association with lipids. In the central domain, helix 100-121 and, to a lesser extent, helix 122-143, are also very important for lipid binding and the formation of mature HDL, whereas helices between residues 144 and 186 contribute little. The LCAT activation domain has now been clearly assigned to helix 144-165 with secondary contribution by helix 166-186. The lower lipid binding affinity of the region 144-186 may be important to the activation mechanism allowing displacement of these apoA-I helices by LCAT and presentation of the lipid substrates. No specific sequence has been found that affects diffusional efflux to lipid-bound apoA-I. In contrast, the C-terminal helices, known to be important for lipid binding and maintenance of HDL in circulation, are also involved in the interaction of lipid-free apoA-I with macrophages and specific lipid efflux. While much progress has been made, other aspects of apoA-I structure-function relationships still need to be studied, particularly its lipoprotein topology and its interaction with other enzymes, lipid transfer proteins and receptors important for HDL metabolism.     

Globins in nonvertebrate species: dispersal by horizontal gene transfer and evolution of the structure-function relationships

Using a new template based on an alignment of 145 nonvertebrate globins we examined several recently determined sequences of putative globins and globin-like hemeproteins. We propose that all globins have evolved from a family of ancestral, approx. 17-kDa hemeproteins, which displayed the globin fold and functioned as redox proteins. Once atmospheric O2 became available the acquisition of oxygen-binding properties was initiated, culminating in the various highly specialized functions known as present. During this evolutionary process, we suggest that (1) high oxygen affinity may have been acquired repeatedly and (2) the formation of chimeric proteins containing both a globin and a flavin binding domain was an additional and distinct evolutionary trend. Furthermore, globin-like hemeproteins encompass hemeproteins produced through convergent evolution from nonglobin ancestral proteins to carry out O2-binding functions as well as hemeproteins whose sequences exhibit the loss of some or all of the structural determinants of the globin fold. We also propose that there occurred two cases of horizontal globin gene transfer, one from an ancestor common to the ciliates Paramecium and Tetrahymena and the green alga Chlamydomonas to a cyanobacterium ancestor and the other, from a eukaryote ancestor of the yeasts Saccharomyces and Candida to a bacterial ancestor of the proteobacterial genera Escherichia, Alcaligenes, and Vitreoscilla.      

ATP synthase: structure-function relationships.

Recent work has focused on obtaining a better understanding of the three-dimensional structural relationships between the alpha and beta subunits of the F1 moiety and the location of nucleotide binding domains within these subunits. Four types of approach are currently being pursued: X-ray crystallographic, chemical, molecular biological and biochemical. Here we briefly review some of the major conclusions of these studies, and point out some of the problems that must be resolved before an adequate model that relates structure to function in the ATP synthase molecule can be formulated.     

The CCN family of proteins: structure-function relationships

The CCN proteins are key signalling and regulatory molecules involved in many vital biological functions, including cell proliferation, angiogenesis, tumourigenesis and wound healing. How these proteins influence such a range of functions remains incompletely understood but is probably related to their discrete modular nature and a complex array of intra- and inter-molecular interactions with a variety of regulatory proteins and ligands. Although certain aspects of their biology can be attributed to the four individual modules that constitute the CCN proteins, recent results suggest that some of their biological functions require cooperation between modules. Indeed, the modular structure of CCN proteins provides important insight into their structure-function relationships.     

The evolution of High Mobility Group Box (HMGB) chromatin proteins in multicellular animals

Mammalian HMGB proteins are abundant chromatin components, and are characterized by the presence of 2 HMG-box domains and an acidic tail. HMG boxes are present in a large number of DNA-binding proteins, and HMGB chromatin proteins represent a small and specific subset of HMG-box proteins. The comparison of DNA sequences that code for HMG-box proteins suggests that the ancestral HMG box was coded by an intronless gene, which picked up one or more introns during its radiation. Canonical HMGB proteins are only present in multicellular animals, from sponges onwards, and appear to have arisen through the fusion of two different genes, each coding for one of the boxes. The organization of HMGB genes was very conserved during Metazoan evolution, with the only deviations appearing in Caenorhabditis and Dipteran (Drosophila and Anopheles) species.     

Glucansucrases: mechanism of action and structure-function relationships

Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood. However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca. 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function.     

Leveraging enzyme structure-function relationships for functional inference and experimental design: the structure-function linkage database

The study of mechanistically diverse enzyme superfamilies-collections of enzymes that perform different overall reactions but share both a common fold and a distinct mechanistic step performed by key conserved residues-helps elucidate the structure-function relationships of enzymes. We have developed a resource, the structure-function linkage database (SFLD), to analyze these structure-function relationships. Unique to the SFLD is its hierarchical classification scheme based on linking the specific partial reactions (or other chemical capabilities) that are conserved at the superfamily, subgroup, and family levels with the conserved structural elements that mediate them. We present the results of analyses using the SFLD in correcting misannotations, guiding protein engineering experiments, and elucidating the function of recently solved enzyme structures from the structural genomics initiative. The SFLD is freely accessible at http://sfld.rbvi.ucsf.edu.     

Immunity proteins to pore-forming colicins: structure-function relationships

Colicin A and B immunity proteins (Cai and Cbi, respectively) are homologous integral membrane proteins that interact within the core of the lipid bilayer with hydrophobic transmembrane helices of the corresponding colicin channel. By using various approaches (exchange of hydrophilic loops between Cai and Cbi, construction of Cbi/Cai hybrids, production of Cai as two fragments), we studied the structure-function relationships of Cai and Cbi. The results revealed unexpectedly high structural constraints for the function of these proteins. The periplasmic loops of Cai and Cbi did not carry the determinants for colicin recognition although most of these loops were required for Cai function; the cytoplasmic loop of Cai was found to be Involved in topology and function of Cai. The immunity function did not seem to be confined to a particular region of the immunity proteins.     

Specificity and randomness: structure-function relationships in neural circuits

A fundamental but unsolved problem in neuroscience is how connections between neurons might underlie information processing in central circuits. Building wiring diagrams of neural networks may accelerate our understanding of how they compute. But even if we had wiring diagrams, it is critical to know what neurons in a circuit are doing: their physiology. In both the retina and cerebral cortex, a great deal is known about topographic specificity, such as lamination and cell-type specificity of connections. Little, however, is known about connections as they relate to function. Here, we review how advances in functional imaging and electron microscopy have recently allowed the examination of relationships between sensory physiology and synaptic connections in cortical and retinal circuits.     

Simulation modeling of the origin of multicellular animals

A simulation model of the evolution of a free-living bilayered animal is proposed. The model object developed two layers of cells. The inner layer was able to produce digestive enzymes, to split and absorb organic substances. The evolution of these model objects was accompanied by mutations resembling real adaptations in some coelenterates and placozoans. It was observed that the outer layer of the model produced cells capable of secretion of digestive ferments. This mutation was a principal apomorphy leading to the appearance of organisms with extraorganismal digestion. Visual Basic and STELLA modeling software were used for simulations.     

Vasoconstrictor effects of sarafotoxins in rabbit aorta: structure-function relationships

The sarafotoxins SRTX-a, b and c from the venom of the snake Atractaspis engaddensis are 21-amino acid peptides that affect the cardiovascular system. They are strong vasoconstrictors, the potency of which may be in correlation with their primary structure: SRTX-a, which differs from SRTX-b in a single amino acid residue (Asn instead of Tyr), shows about half of its maximal vasoconstriction, while SRTX-c, which differs in 3 additional residues is a very weak vasoconstrictor and, at high doses, shows vasodilatory effects. Sequential application of the three isotoxins result in a summated response.     

The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.