Structural and molecular determinants of Candida glabrata metacaspase maturation and activation by calcium.

Metacaspases are caspase-like homologs which undergo a complex maturation process involving multiple intra-chain cleavages resulting in a composite enzyme made of a p10 and a p20 domain. Their proteolytic activity involving a cysteine-histidine catalytic dyad, show peptide bond cleavage specificity in the C-terminal to lysine and arginine, with both maturation- and catalytic processes being calcium-dependent. Here, we present the structure of a metacaspase from the yeast Candida glabrata, CgMCA-I, in complex with a unique calcium along with a structure in which three magnesium ions are bound. We show that the Ca2+ ion interacts with a loop in the vicinity of the catalytic site. The reorganization of this cation binding loop, by bringing together the two catalytic residues, could be one of the main structural determinants triggering metacaspase activation. Enzymatic exploration of CgMCA-I confirmed that the maturation process implies a trans mechanism with sequential cleavages.

Dynamics of the secreted frizzled related protein Sizzled and potential implications for binding to bone morphogenetic protein-1 (BMP-1).

Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl. When compared with known structures of BMP-1 in complex with small molecule inhibitors, this reveals features that may be helpful in the design of new inhibitors to prevent the excessive accumulation of extracellular matrix that is the hallmark of fibrotic diseases.

Tertiary and Quaternary Structure Organization in GMP Synthetases: Implications for Catalysis.

Glutamine amidotransferases, enzymes that transfer nitrogen from Gln to various cellular metabolites, are modular, with the amidotransferase (GATase) domain hydrolyzing Gln, generating ammonia and the acceptor domain catalyzing the addition of nitrogen onto its cognate substrate. GMP synthetase (GMPS), an enzyme in the de novo purine nucleotide biosynthetic pathway, is a glutamine amidotransferase that catalyzes the synthesis of GMP from XMP. The reaction involves activation of XMP though adenylation by ATP in the ATP pyrophosphatase (ATPPase) active site, followed by channeling and attack of NH3 generated in the GATase pocket. This complex chemistry entails co-ordination of activity across the active sites, allosteric activation of the GATase domain to modulate Gln hydrolysis and channeling of ammonia from the GATase to the acceptor active site. Functional GMPS dimers associate through the dimerization domain. The crystal structure of the Gln-bound complex of Plasmodium falciparum GMPS (PfGMPS) for the first time revealed large-scale domain rotation to be associated with catalysis and leading to the juxtaposition of two otherwise spatially distal cysteinyl (C113/C337) residues. In this manuscript, we report on an unusual structural variation in the crystal structure of the C89A/C113A PfGMPS double mutant, wherein a larger degree of domain rotation has led to the dissociation of the dimeric structure. Furthermore, we report a hitherto overlooked signature motif tightly related to catalysis.

Structure and catalytic regulation of Plasmodium falciparum IMP specific nucleotidase.

Plasmodium falciparum (Pf) relies solely on the salvage pathway for its purine nucleotide requirements, making this pathway indispensable to the parasite. Purine nucleotide levels are regulated by anabolic processes and by nucleotidases that hydrolyse these metabolites into nucleosides. Certain apicomplexan parasites, including Pf, have an IMP-specific-nucleotidase 1 (ISN1). Here we show, by comprehensive substrate screening, that PfISN1 catalyzes the dephosphorylation of inosine monophosphate (IMP) and is allosterically activated by ATP. Crystal structures of tetrameric PfISN1 reveal complex rearrangements of domain organization tightly associated with catalysis. Immunofluorescence microscopy and expression of GFP-fused protein indicate cytosolic localization of PfISN1 and expression in asexual and gametocyte stages of the parasite. With earlier evidence on isn1 upregulation in female gametocytes, the structures reported in this study may contribute to initiate the design for possible transmission-blocking agents.

Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase.

GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.

Lead optimization and biological evaluation of fragment-based cN-II inhibitors.

The development of cytosolic 5'-nucleotidase II (cN-II) inhibitors is essential to validate cN-II as a potential target for the reversion of resistance to cytotoxic nucleoside analogues. We previously reported a fragment-based approach combined with molecular modelling, herein, the selected hit-fragments were used again in another computational approach based on the Ilib-diverse (a software enabling to build virtual molecule libraries through fragment based de novo design) program to generate a focused library of potential inhibitors. A molecular scaffold related to a previously identified compound was selected and led to a novel series of compounds. Ten out of nineteen derivatives showed 50-75% inhibition on the purified recombinant protein at 200 µM and among them three derivatives (12, 13 and 18) exhibited Ki in the sub-millimolar range (0.84, 2.4 and 0.58 mM, respectively). Despite their only modest potency, the cN-II inhibitors showed synergistic effects when used in combination with cytotoxic purine nucleoside analogues on cancer cells. Therefore, these derivatives represent a family of non-nucleos(t)idic cN-II inhibitors with potential usefulness to overcome cancer drug resistance especially in hematological malignancies in which cN-II activity has been described as an important parameter.

Structural basis of homo- and heterotrimerization of collagen I.

Fibrillar collagen molecules are synthesized as precursors, procollagens, with large propeptide extensions. While a homotrimeric form (three α1 chains) has been reported in embryonic tissues as well as in diseases (cancer, fibrosis, genetic disorders), collagen type I usually occurs as a heterotrimer (two α1 chains and one α2 chain). Inside the cell, the role of the C-terminal propeptides is to gather together the correct combination of three α chains during molecular assembly, but how this occurs for different forms of the same collagen type is so far unknown. Here, by structural and mutagenic analysis, we identify key amino acid residues in the α1 and α2 C-propeptides that determine homo- and heterotrimerization. A naturally occurring mutation in one of these alters the homo/heterotrimer balance. These results show how the C-propeptide of the α2 chain has specifically evolved to permit the appearance of heterotrimeric collagen I, the major extracellular building block among the metazoa.

Identification of Noncompetitive Inhibitors of Cytosolic 5'-Nucleotidase II Using a Fragment-Based Approach.

We used a combined approach based on fragment-based drug design (FBDD) and in silico methods to design potential inhibitors of the cytosolic 5'-nucleotidase II (cN-II), which has been recognized as an important therapeutic target in hematological cancers. Two subgroups of small compounds (including adenine and biaryl moieties) were identified as cN-II binders and a fragment growing strategy guided by molecular docking was considered. Five compounds induced a strong inhibition of the 5'-nucleotidase activity in vitro, and the most potent ones were characterized as noncompetitive inhibitors. Biological evaluation in cancer cell lines showed synergic effect with selected anticancer drugs. Structural studies using X-ray crystallography lead to the identification of new binding sites for two derivatives and of a new crystal form showing important domain swapping. Altogether, the strategy developed herein allowed identifying new original noncompetitive inhibitors against cN-II that act in a synergistic manner with well-known antitumoral agents.

Active site coupling in Plasmodium falciparum GMP synthetase is triggered by domain rotation.

GMP synthetase (GMPS), a key enzyme in the purine biosynthetic pathway performs catalysis through a coordinated process across two catalytic pockets for which the mechanism remains unclear. Crystal structures of Plasmodium falciparum GMPS in conjunction with mutational and enzyme kinetic studies reported here provide evidence that an 85° rotation of the GATase domain is required for ammonia channelling and thus for the catalytic activity of this two-domain enzyme. We suggest that conformational changes in helix 371-375 holding catalytic residues and in loop 376-401 along the rotation trajectory trigger the different steps of catalysis, and establish the central role of Glu374 in allostery and inter-domain crosstalk. These studies reveal the mechanism of domain rotation and inter-domain communication, providing a molecular framework for the function of all single polypeptide GMPSs and form a solid basis for rational drug design targeting this therapeutically important enzyme.

Identification of critical residues for the activity and thermostability of Streptomyces sp. SK glucose isomerase.

The role of residue 219 in the physicochemical properties of D-glucose isomerase from Streptomyces sp. SK strain (SKGI) was investigated by site-directed mutagenesis and structural studies. Mutants G219A, G219N, and G219F were generated and characterized. Comparative studies of their physicochemical properties with those of the wild-type enzyme highlighted that mutant G219A displayed increased specific activity and thermal stability compared to that of the wild-type enzyme, while for G219N and G219F, these properties were considerably decreased. A double mutant, SKGI F53L/G219A, displayed a higher optimal temperature and a higher catalytic efficiency than both the G219A mutant and the wild-type enzyme and showed a half-life time of about 150 min at 85 °C as compared to 50 min for wild-type SKGI. Crystal structures of SKGI wild-type and G219A enzymes were solved to 1.73 and 2.15 Å, respectively, and showed that the polypeptide chain folds into two structural domains. The larger domain consists of a (ß/α)8 unit, and the smaller domain forms a loop of α helices. Detailed analyses of the three-dimensional structures highlighted minor but important changes in the active site region as compared to that of the wild-type enzyme leading to a displacement of both metal ions, and in particular that in site M2. The structural analyses moreover revealed how the substitution of G219 by an alanine plays a crucial role in improving the thermostability of the mutant enzyme.

Identification and characterization of inhibitors of cytoplasmic 5'-nucleotidase cN-II issued from virtual screening.

Clinical and preclinical observations have lead to the hypothesis that 5'-nucleotidase cN-II could constitute a therapeutic target in oncology, either per se or to increase the activity of cytotoxic nucleoside analogs. To identify potential cN-II inhibitors, we performed in silico screening of freely available chemical databases, in vitro enzymatic assays with recombinant cN-II, soaking experiments with crystals of truncated cN-II as well as biological evaluation of selected compounds, alone or in combination with cytotoxic nucleoside analogs, on cancer cells. The top ranked compounds from virtual screening included an anthraquinone derivative (AdiS) that were shown to block the enzyme activity with a K(i) of 2.0mM. Soaking experiments performed with crystals of truncated cN-II allowed to obtain crystallographic data at a resolution of 2.9 Å and indicating interaction between AdiS and F354/I152 situated in the effector site 1 of cN-II. In addition, this compound exhibited different levels of cytotoxicity in vitro on several cancer cell lines and increased the induction of apoptosis in RL cells incubated with 0.5 or 1.5 µM cladribine, 0.05 µM clofarabine or 30 µM fludarabine. Finally, AdiS showed synergy with cladribine and additivity with clofarabine. This study showed that virtual screening is a useful tool for the identification of potent cN-II inhibitors, and our biological results indicated interesting activity for one lead compound that can be further developed as therapeutics.

Structural basis of fibrillar collagen trimerization and related genetic disorders.

The C propeptides of fibrillar procollagens have crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils. Mutations in C propeptides are associated with several, often lethal, genetic disorders affecting bone, cartilage, blood vessels and skin. Here we report the crystal structure of a C-propeptide domain from human procollagen III. It reveals an exquisite structural mechanism of chain recognition during intracellular trimerization of the procollagen molecule. It also gives insights into why some types of collagen consist of three identical polypeptide chains, whereas others do not. Finally, the data show striking correlations between the sites of numerous disease-related mutations in different C-propeptide domains and the degree of phenotype severity. The results have broad implications for understanding genetic disorders of connective tissues and designing new therapeutic strategies.

Structuring detergents for extracting and stabilizing functional membrane proteins.

BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.

C-terminal residues regulate localization and function of the antiapoptotic protein Bfl-1.

Unlike other antiapoptotic members of the Bcl-2 family, Bfl-1 does not contain a well defined C-terminal transmembrane domain, and whether the C-terminal tail of Bfl-1 functions as a membrane anchor is not yet clearly established. The molecular modeling study of the full-length Bfl-1 performed within this work suggests that Bfl-1 may co-exist in two distinct conformational states: one in which its C-terminal helix alpha9 is inserted in the hydrophobic groove formed by the BH1-3 domains of Bfl-1 and one with its C terminus. Parallel analysis of the subcellular localization of Bfl-1 indicates that even if Bfl-1 may co-exist in two distinct conformational states, most of the endogenous protein is tightly associated with the mitochondria by its C terminus in both healthy and apoptotic peripheral blood lymphocytes as well as in malignant B cell lines. However, the helix alpha9 of Bfl-1, and therefore the binding of Bfl-1 to mitochondria, is not absolutely required for the antiapoptotic activity of Bfl-1. A particular feature of Bfl-1 is the amphipathic character of its C-terminal helix alpha9. Our data clearly indicate that this property of helix alpha9 is required for the anchorage of Bfl-1 to the mitochondria but also regulates the antiapoptotic function Bfl-1.

The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium.

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.