Crystal structures of catrocollastatin/VAP2B reveal a dynamic, modular architecture of ADAM/adamalysin/reprolysin family proteins

Catrocollastatin/vascular apoptosis-inducing protein (VAP)2B is a metalloproteinase from Crotalus atrox venom, possessing metalloproteinase/disintegrin/cysteine-rich (MDC) domains that bear the typical domain architecture of a disintegrin and metalloproteinase (ADAM)/adamalysin/reprolysin family proteins. Here we describe crystal structures of catrocollastatin/VAP2B in three different crystal forms, representing the first reported crystal structures of a member of the monomeric class of this family of proteins. The overall structures show good agreement with both monomers of atypical homodimeric VAP1. Comparison of the six catrocollastatin/VAP2B monomer structures and the structures of VAP1 reveals a dynamic, modular architecture that may be important for the functions of ADAM/adamalysin/reprolysin family proteins.     

Autolysis at the disintegrin domain of patagonfibrase,a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 °C, primarily producing a ∼ 32.6 kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca²⁺, patagonfibrase was only partially autolysed, giving rise mainly to one fragment of ∼ 52.2 kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.     

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.     

Disintegrin-like/cysteine-rich domains of the reprolysin HF3: Site-directed mutagenesis reveals essential role of specific residues

Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein-protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain.     

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom

Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55 kDa as revealed by the reduced SDS-PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation (“Met-turn”), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala₁₄-Leu₁₅ peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aα-chain of fibrinogen and the α-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca²⁺ and Mg²⁺ but inhibited by Zn²⁺ and Cu²⁺. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD = 30 ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure-function relationship of a P-III snake venom metalloproteinase.     

Molecular characterisation of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localisation

Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilisation of TgOWP1–3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulphide bridges. Immunofluorescence analysis allowed the localisation of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.     

Structural basis for membrane-proximal proteolysis of substrates by ADAM10

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Structural basis for diversity of the EF-hand calcium-binding proteins

The calcium binding proteins of the EF-hand super-family are involved in the regulation of all aspects of cell function. These proteins exhibit a great diversity of composition, structure, Ca2+-binding and target interaction properties. Here, our current understanding of the Ca2+-binding mechanism is assessed. The structures of the EF-hand motifs containing 11-14 amino acid residues in the Ca2+-binding loop are analyzed within the framework of the recently proposed two-step Ca2+-binding mechanism. A hypothesis is put forward that in all EF-hand proteins the Ca2+-binding and the resultant conformational responses are governed by the central structure connecting the Ca2+-binding loops in the two-EF-hand domain. This structure, named EFbeta-scaffold, defines the position of the bound Ca2+, and coordinates the function of the N-terminal (variable and flexible) with the C-terminal (invariable and rigid) parts of the Ca2+-binding loop. It is proposed that the nature of the first ligand of the Ca2+-binding loop is an important determinant of the conformational change. Additional factors, including the interhelical contacts, the length, structure and flexibility of the linker connecting the EF-hand motifs, and the overall energy balance provide the fine-tuning of the Ca2+-induced conformational change in the EF-hand proteins.     

Structural basis for interaction of DivIVA/GpsB proteins with their ligands

DivIVA proteins and their GpsB homologues are late cell division proteins found in Gram‐positive bacteria. DivIVA/GpsB proteins associate with the inner leaflet of the cytosolic membrane and act as scaffolds for other proteins required for cell growth and division. DivIVA/GpsB proteins comprise an N‐terminal lipid‐binding domain for membrane association fused to C‐terminal domains supporting oligomerization. Despite sharing the same domain organization, DivIVA and GpsB serve different cellular functions: DivIVA plays diverse roles in division site selection, chromosome segregation and controlling peptidoglycan homeostasis, whereas GpsB contributes to the spatiotemporal control of penicillin‐binding protein activity. The crystal structures of the lipid‐binding domains of DivIVA from Bacillus subtilis and GpsB from several species share a fold unique to this group of proteins, whereas the C‐terminal domains of DivIVA and GpsB are radically different. A number of pivotal features identified from the crystal structures explain the functional differences between the proteins. Herein we discuss these structural and functional relationships and recent advances in our understanding of how DivIVA/GpsB proteins bind and recruit their interaction partners, knowledge that might be useful for future structure‐based DivIVA/GpsB inhibitor design.     

Structural basis for the ADP-specificity of a novel glucokinase from a hyperthermophilic archaeon

BACKGROUND: ATP is the most common phosphoryl group donor for kinases. However, certain hyperthermophilic archaea such as Thermococcus litoralis and Pyrococcus furiosus utilize unusual ADP-dependent glucokinases and phosphofructokinases in their glycolytic pathways. These ADP-dependent kinases are homologous to each other but show no sequence similarity to any of the hitherto known ATP-dependent enzymes. RESULTS: We solved the crystal structure at 2.3 A resolution of an ADP-dependent glucokinase from T. litoralis (tlGK) complexed with ADP. The overall structure can be divided into large and small alpha/beta domains, and the ADP molecule is buried in a shallow pocket in the large domain. Unexpectedly, the structure was similar to those of two ATP-dependent kinases, ribokinase and adenosine kinase. Comparison based on three-dimensional structure revealed that several motifs important both in structure and function are conserved, and the recognition of the alpha- and beta-phosphate of the ADP in the tlGK was almost identical with the recognition of the beta- and gamma-phosphate of ATP in these ATP-dependent kinases. CONCLUSIONS: Noticeable points of our study are the first structure of ADP-dependent kinase, the structural similarity to members of the ATP-dependent ribokinase family, its rare nucleotide specificity caused by a shift in nucleotide binding position by one phosphate unit, and identification of the residues that discriminate ADP- and ATP-dependence. The strict conservation of the binding site for the terminal and adjacent phosphate moieties suggests a common ancestral origin of both the ATP- and ADP-dependent kinases.     

Structural basis of folding cooperativity in model proteins: insights from a microcanonical perspective

Two-state cooperativity is an important characteristic in protein folding. It is defined by a depletion of states that lie energetically between folded and unfolded conformations. There are different ways to test for two-state cooperativity; however, most of these approaches probe indirect proxies of this depletion. Generalized-ensemble computer simulations allow us to unambiguously identify this transition by a microcanonical analysis on the basis of the density of states. Here, we present a detailed characterization of several helical peptides obtained by coarse-grained simulations. The level of resolution of the coarse-grained model allowed to study realistic structures ranging from small α-helices to a de novo three-helix bundle without biasing the force field toward the native state of the protein. By linking thermodynamic and structural features, we are able to show that whereas short α-helices exhibit two-state cooperativity, the type of transition changes for longer chain lengths because the chain forms multiple helix nucleation sites, stabilizing a significant population of intermediate states. The helix bundle exhibits signs of two-state cooperativity owing to favorable helix-helix interactions, as predicted from theoretical models. A detailed analysis of secondary and tertiary structure formation fits well into the framework of several folding mechanisms and confirms features that up to now have been observed only in lattice models.     

Structural basis for CO2 fixation by a novel member of the disulfide oxidoreductase family of enzymes, 2-ketopropyl-coenzyme M oxidoreductase/carboxylase

The NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is the terminal enzyme in a metabolic pathway that results in the conversion of propylene to the central metabolite acetoacetate in Xanthobacter autotrophicus Py2. This enzyme is an FAD-containing enzyme that is a member of the NADPH:disulfide oxidoreductase (DSOR) family of enzymes that include glutathione reductase, dihydrolipoamide dehydrogenase, trypanothione reductase, thioredoxin reductase, and mercuric reductase. In contrast to the prototypical reactions catalyzed by members of the DSOR family, the NADPH:2-ketopropyl-coenzyme M oxidoreductase/carboxylase catalyzes the reductive cleavage of the thioether linkage of 2-ketopropyl-coenzyme M, and the subsequent carboxylation of the ketopropyl cleavage product, yielding the products acetoacetate and free coenzyme M. The structure of 2-KPCC reveals a unique active site in comparison to those of other members of the DSOR family of enzymes and demonstrates how the enzyme architecture has been adapted for the more sophisticated biochemical reaction. In addition, comparison of the structures in the native state and in the presence of bound substrate indicates the binding of the substrate 2-ketopropyl-coenzyme M induces a conformational change resulting in the collapse of the substrate access channel. The encapsulation of the substrate in this manner is reminiscent of the conformational changes observed in the well-characterized CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxidase (Rubisco).     

Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins

The Cbl adapter proteins typically function to down-regulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the four-helix bundle of the TKB domain.     

Structural basis for copper transfer by the metallochaperone for the Menkes/Wilson disease proteins

The Hah1 metallochaperone protein is implicated in copper delivery to the Menkes and Wilson disease proteins. Hah1 and the N-termini of its target proteins belong to a family of metal binding domains characterized by a conserved MT/HCXXC sequence motif. The crystal structure of Hah1 has been determined in the presence of Cu(I), Hg(II), and Cd(II). The 1.8 A resolution structure of CuHah1 reveals a copper ion coordinated by Cys residues from two adjacent Hah1 molecules. The CuHah1 crystal structure is the first of a copper chaperone bound to copper and provides structural support for direct metal ion exchange between conserved MT/HCXXC motifs in two domains. The structures of HgHah1 and CdHah1, determined to 1.75 A resolution, also reveal metal ion coordination by two MT/HCXXC motifs. An extended hydrogen bonding network, unique to the complex of two Hah1 molecules, stabilizes the metal binding sites and suggests specific roles for several conserved residues. Taken together, the structures provide models for intermediates in metal ion transfer and suggest a detailed molecular mechanism for protein recognition and metal ion exchange between MT/HCXXC containing domains.     

Structural basis for product specificities of MLL family methyltransferases

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The YefM antitoxin defines a family of natively unfolded proteins: implications as a novel antibacterial target

Although natively unfolded proteins are being observed increasingly, their physiological role is not well understood. Here, we demonstrate that the Escherichia coli YefM protein is a natively unfolded antitoxin, lacking secondary structure even at low temperature or in the presence of a stabilizing agent. This conformation of the protein is suggested to have a key role in its physiological regulatory activity. Because of the unfolded state of the protein, a linear determinant rather than a conformational one is presumably being recognized by its toxin partner, YoeB. A peptide array technology allowed the identification and validation of such a determinant. This recognition element may provide a novel antibacterial target. Indeed, a pair-constrained bioinformatic analysis facilitated the definite determination of novel YefM-YoeB toxin-antitoxin systems in a large number of bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Mycobacterium tuberculosis. Taken together, the YefM protein defines a new family of natively unfolded proteins. The existence of a large and conserved group of proteins with a clear physiologically relevant unfolded state serves as a paradigm to understand the structural basis of this state.     

The direct p53 target gene, FLJ11259/DRAM, is a member of a novel family of transmembrane proteins

The tumor suppressor p53 regulates diverse biological processes primarily via activation of downstream target genes. Even though many p53 target genes have been described, the precise mechanisms of p53 biological actions are uncertain. In previous work we identified by microarray analysis a candidate p53 target gene, FLJ11259/DRAM. In this report we have identified three uncharacterized human proteins with sequence homology to FLJ11259, suggesting that FLJ11259 is a member of a novel family of proteins with six transmembrane domains. Several lines of investigation confirm FLJ11259 is a direct p53 target gene. p53 siRNA prevented cisplatin-mediated up-regulation of FLJ11259 in NT2/D1 cells. Likewise in HCT116 p53+/+ cells and MCF10A cells, FLJ11259 is induced by cisplatin treatment but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element was identified 22.3 kb upstream of the first coding exon of FLJ11259 and is shown to be active in reporter assays. In addition, chromatin immunoprecipitation assays indicate that p53 binds directly to this element in vivo and that binding is enhanced following cisplatin treatment. Confocal microscopy showed that an FLJ-GFP fusion protein localizes mainly in a punctate pattern in the cytoplasm. Overexpression studies in Cos-7, Saos2, and NT2/D1 cells suggest that FLJ11259 is associated with increased clonal survival. In summary, we have identified FLJ11259/DRAM as a p53-inducible member of a novel family of transmembrane proteins. FLJ11259/DRAM may be an important modulator of p53 responses in diverse tumor types.     

The direct p53 target gene, FLJ11259/DRAM, is a member of a novel family of transmembrane proteins

The tumor suppressor p53 regulates diverse biological processes primarily via activation of downstream target genes. Even though many p53 target genes have been described, the precise mechanisms of p53 biological actions are uncertain. In previous work we identified by microarray analysis a candidate p53 target gene, FLJ11259/DRAM. In this report we have identified three uncharacterized human proteins with sequence homology to FLJ11259, suggesting that FLJ11259 is a member of a novel family of proteins with six transmembrane domains. Several lines of investigation confirm FLJ11259 is a direct p53 target gene. p53 siRNA prevented cisplatin-mediated up-regulation of FLJ11259 in NT2/D1 cells. Likewise in HCT116 p53+/+ cells and MCF10A cells, FLJ11259 is induced by cisplatin treatment but to a much lesser extent in isogenic p53-suppressed cells. A functional p53 response element was identified 22.3 kb upstream of the first coding exon of FLJ11259 and is shown to be active in reporter assays. In addition, chromatin immunoprecipitation assays indicate that p53 binds directly to this element in vivo and that binding is enhanced following cisplatin treatment. Confocal microscopy showed that an FLJ-GFP fusion protein localizes mainly in a punctate pattern in the cytoplasm. Overexpression studies in Cos-7, Saos2, and NT2/D1 cells suggest that FLJ11259 is associated with increased clonal survival. In summary, we have identified FLJ11259/DRAM as a p53-inducible member of a novel family of transmembrane proteins. FLJ11259/DRAM may be an important modulator of p53 responses in diverse tumor types.