Deciphering the Kinetic Binding Mechanism of Dimeric Ligands Using a Potent Plasma-stable Dimeric Inhibitor of Postsynaptic Density Protein-95 as an Example

Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity toward their targets due to their ability to bind two binding sites simultaneously and are therefore attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide and PDZ1–2 of postsynaptic density protein-95 (PSD-95) using protein engineering in combination with fluorescence polarization, isothermal titration calorimetry, and stopped-flow fluorimetry. We demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and found evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.     

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.     

Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.     

Activation of the Ghrelin Receptor is Described by a Privileged Collective Motion: A Model for Constitutive and Agonist-induced Activation of a Sub-class A G-Protein Coupled Receptor (GPCR)

Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser _[octa]-Phe-NH ₂ and Gly-Ser-Ser _[octa]-Phe-Leu-NH ₂ allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.     

The Helicobacter pylori GroES Cochaperonin HspA Functions as a Specialized Nickel Chaperone and Sequestration Protein through Its Unique C-Terminal Extension

The transition metal nickel plays a central role in the human gastric pathogen Helicobacter pylori because it is required for two enzymes indispensable for colonization, the nickel metalloenzyme urease and [NiFe] hydrogenase. To sustain nickel availability for these metalloenzymes while providing protection from the metal''s harmful effects, H. pylori is equipped with several specific nickel-binding proteins. Among these, H. pylori possesses a particular chaperone, HspA, that is a homolog of the highly conserved and essential bacterial heat shock protein GroES. HspA contains a unique His-rich C-terminal extension and was demonstrated to bind nickel in vitro. To investigate the function of this extension in H. pylori, we constructed mutants carrying either a complete deletion or point mutations in critical residues of this domain. All mutants presented a decreased intracellular nickel content measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced nickel tolerance. While urease activity was unaffected in the mutants, [NiFe] hydrogenase activity was significantly diminished when the C-terminal extension of HspA was mutated. We conclude that H. pylori HspA is involved in intracellular nickel sequestration and detoxification and plays a novel role as a specialized nickel chaperone involved in nickel-dependent maturation of hydrogenase.Helicobacter pylori is a Gram-negative, microaerophilic bacterium that is the only persistent inhabitant of the human stomach. Its presence in humans is associated with a variety of pathologies, ranging from gastric and duodenal peptic ulcers to the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (1, 39). Indeed, H. pylori is the only formally recognized bacterial carcinogen for humans (17), infecting half of the world''s population (19).In H. pylori, metal ions play a central role, since the transition metal nickel is the cofactor of the urease enzyme and is also required for [NiFe] hydrogenase. Urease catalyzes the hydrolysis of urea into the buffering compounds bicarbonate and ammonia, enabling H. pylori to persist in the acidic environment of the stomach. This enzyme accounts for up to 6% of the soluble cellular proteins and requires 24 nickel ions per active enzymatic complex (16). The uptake-type hydrogenase of H. pylori is a nickel-dependent enzyme containing a binuclear [NiFe] active site. This [NiFe] hydrogenase catalyzes the oxidation of molecular hydrogen and permits the utilization of hydrogen as an energy source during respiration-based energy production in the mucosa (21). Both enzymes are important for host colonization, as shown with several animal models (9, 10, 28, 42, 43). To sustain nickel availability for urease and hydrogenase while providing protection from the metal''s harmful effects, H. pylori possesses an elaborate and strictly controlled nickel metabolism.The incorporation of nickel ions into apohydrogenase requires the participation of the HypAB (HP0869 and HP0900) accessory proteins; for apourease, both the UreEFGH (HP0070-0067) accessory proteins and HypAB are necessary (4, 29). Besides these widely distributed accessory proteins, H. pylori possesses several specific proteins that are present in all H. pylori strains, namely, the histidine-rich proteins Hpn (HP1427) and Hpn-like (HP1432). These cytoplasmic and abundant proteins (Hpn represents 2% of the total protein content) bind nickel ions (five Ni²⁺ ions per monomer; dissociation constant [K_d] for nickel of 7.1 μM) and protect H. pylori against metal overload (15). Furthermore, it has recently been proposed that Hpn and Hpn-like can compete for nickel ions with the urease enzyme and thus regulate its enzymatic activity. In vivo and in vitro experiments indicate that Hpn and Hpn-like sequester nickel ions at neutral pH but donate them for urease activation under acidic pH conditions (14, 35, 44). Hydrogenase activity was unchanged in the Δhpn and Δhpn-like mutants (35).In addition to these proteins, H. pylori possesses a particular chaperone, HspA (HP0011), that is a homolog of the highly conserved and essential bacterial heat shock protein GroES (40). No other gene encoding a GroES homolog is found in the genome of H. pylori. GroES is the cochaperonin of the heptameric GroEL-GroES barrel complex, which mediates the correct folding of a variety of cellular proteins and which is conserved and essential in prokaryotes and eukaryotes (30). In addition to the conserved GroES chaperonin domain (domain A, amino acids 1 to 90) (Fig. ​(Fig.1A),1A), HspA contains a C-terminal extension of 28 amino acids (domain B, amino acids 91 to 118) (Fig. 1A and B) that contains 8 His and 4 Cys residues. Based on this high number of His and Cys residues known to bind transition metal ions, the purified recombinant HspA protein specifically binds two nickel ions per molecule (K_d of 1.1 to 1.8 μM) (7, 18). This domain also contains an HX₄DH motif (boxed in Fig. ​Fig.1B)1B) that is considered to be a nickel-binding signature sequence in the nickel-cobalt (NiCoT) transporter family (11). In addition, Loguercio et al. (20) observed that in vitro, the HspA C-terminal domain is folding into two vicinal disulfide bounds engaging two cysteine pairs that form a unique closed-loop structure. However, since HspA is a cytoplasmic protein, the in vivo relevance of this structure is uncertain.Open in a separate windowFIG. 1.(A) Representation of the HspA protein of H. pylori with the GroES-like domain A and the nickel-binding domain B. (B) Amino acid sequence of domain B of wild-type HspA and of three mutants: HspA-ΔC, with a complete deletion of this domain, and HspA-NB and -CC, each carrying two substitutions that are underlined. Cysteine and histidine residues are in blue and red, respectively. The HX₄DH motif, which in the nickel-cobalt (NiCoT) transporter family is considered to be a nickel-binding signature sequence, is boxed. (C) Immunoblot experiment with whole-cell lysates from the H. pylori wild-type strain and from the three hspA mutants after denaturing SDS-PAGE and using the monoclonal antibody P1-1, which specifically recognizes a conserved epitope of HspA domain A. The predicted molecular mass of the wild-type HspA monomer is 13 kDa, and that of HspA-ΔC is 9.8 kDa. The monomeric (M) and dimeric (D) forms of the HspA wild type (WT) are indicated on the left side of the blot. A cross-reacting unspecific protein band is marked with a star (*) and served as a loading control. Molecular mass standards are indicated at right.The domain B sequence is conserved in and restricted to H. pylori and the closely related Helicobacter acinonychis species but is absent from all other available sequenced Helicobacter species (see Fig. S1 in the supplemental material). When expressed in Escherichia coli, HspA protected bacteria from nickel overload (7) and increased urease activity 4-fold from the coexpressed H. pylori urease gene cluster (18). Therefore, HspA was hypothesized to function in nickel sequestration and as a specialized nickel donor protein for urease (18). However, no functional characterization of the C terminus was carried out for H. pylori due to the essential nature of HspA (40).In this study, we investigated the role of the nickel-binding C terminus of HspA in H. pylori. We found that the unique C terminus of HspA is involved in nickel sequestration and protection against nickel overload. Contrary to previous data from heterologous studies of E. coli, HspA seemed not to provide nickel ions for urease activation. In contrast, we have found an unexpected and specific function of the HspA C-terminal region in the nickel-dependent maturation of the important colonization factor hydrogenase.     

Identification and Heterologous Expression of Genes Involved in Anaerobic Dissimilatory Phosphite Oxidation by Desulfotignum phosphitoxidans

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO₂ reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.Phosphorus (P) is an important nutrient for all living organisms. The predominant forms of phosphorus in biological systems are inorganic phosphate and its organic esters and acid anhydrides in which P is at its highest oxidation state (+V). The P requirements of living cells can be fulfilled with phosphate in various forms, including reduced organic and inorganic phosphorus compounds (23). Several aerobic bacteria were shown to be able to oxidize hypophosphite (+I) and phosphite (+III) to phosphate (+V) and to incorporate the last into their biomass (5, 15-17, 31, 34). Phosphite can also be oxidized under anaerobic conditions, as shown for an anaerobic Bacillus strain (7) and for Pseudomonas stutzeri which can use phosphite under denitrifying conditions (17, 21). The only bacterium known to oxidize phosphite as the sole source of electrons in lithoautotrophic energy metabolism is Desulfotignum phosphitoxidans (24, 25).Three different metabolic pathways for the use of phosphite as a single P source have been characterized so far. Two of them were discovered and characterized with Escherichia coli and one with Pseudomonas stutzeri. The first pathway in E. coli is mediated by the enzyme carbon phosphorus lyase (C-P lyase), and the second one by the alkaline phosphatase encoded by phoA (16, 34). This alkaline phosphatase not only hydrolyzes phosphate esters but also hydrolyzes phosphite to phosphate and molecular hydrogen (32). This is a particular property only of the E. coli alkaline phosphatase and is not observed with alkaline phosphatases of other bacteria. The third pathway is encoded by the ptxABCDE gene cluster in P. stutzeri (17). In this system, phosphite is transported into the cell by a binding protein-dependent phosphite transporter at the expense of ATP (PtxABC). Phosphite is oxidized by a phosphite:NAD⁺ oxidoreductase (encoded by ptxD), a new member of the 2-hydroxy acid dehydrogenases (8). The ptx operon of P. stutzeri is regulated in response to phosphate starvation by the two-component regulatory system phoBR (28, 29). Furthermore, in Alcaligenes faecalis WM2072, another gene cluster involved in hypophosphite and phosphite uptake and oxidation was characterized: the htxABCD-ptxDE locus (31). The htxABCD-ptxDE genes and their products in A. faecalis WM 2072 have high nucleotide and amino acid sequence identities with those found in the htx and ptx operons in P. stutzeri WM88, which are required for the oxidation of hypophosphite and phosphite, respectively. This unique genetic arrangement of hypophosphite- and phosphite-oxidizing genes in A. faecalis WM2072 suggests a horizontal gene transfer and an ancient evolution of phosphite oxidation.The diversity of pathways used for assimilatory phosphite oxidation and the fact that D. phosphitoxidans is so far the only bacterium known to use phosphite as an electron source caused us to investigate the phosphite uptake and oxidation gene cluster of this bacterium. The aims of our study were (i) to establish enzymatic assays for measurement of phosphite oxidation activity in cell extracts, (ii) to identify the genes involved in phosphite uptake and oxidation, and (iii) to characterize these genes physiologically.     

Identification and Regulation of the N-Acetylglucosamine Utilization Pathway of the Plant Pathogenic Bacterium Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, is known for its ability to catabolize a wide range of plant compounds. This ability is correlated with the presence of specific carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) devoted to scavenging specific carbohydrates. In this study, we demonstrate that there is an X. campestris pv. campestris CUT system involved in the import and catabolism of N-acetylglucosamine (GlcNAc). Expression of genes belonging to this GlcNAc CUT system is under the control of GlcNAc via the LacI family NagR and GntR family NagQ regulators. Analysis of the NagR and NagQ regulons confirmed that GlcNAc utilization involves NagA and NagB-II enzymes responsible for the conversion of GlcNAc-6-phosphate to fructose-6-phosphate. Mutants with mutations in the corresponding genes are sensitive to GlcNAc, as previously reported for Escherichia coli. This GlcNAc sensitivity and analysis of the NagQ and NagR regulons were used to dissect the X. campestris pv. campestris GlcNAc utilization pathway. This analysis revealed specific features, including the fact that uptake of GlcNAc through the inner membrane occurs via a major facilitator superfamily transporter and the fact that this amino sugar is phosphorylated by two proteins belonging to the glucokinase family, NagK-IIA and NagK-IIB. However, NagK-IIA seems to play a more important role in GlcNAc utilization than NagK-IIB under our experimental conditions. The X. campestris pv. campestris GlcNAc NagR regulon includes four genes encoding TonB-dependent active transporters (TBDTs). However, the results of transport experiments suggest that GlcNAc passively diffuses through the bacterial envelope, an observation that calls into question whether GlcNAc is a natural substrate for these TBDTs and consequently is the source of GlcNAc for this nonchitinolytic plant-associated bacterium.Xanthomonas campestris pv. campestris, the causal agent of black rot disease of brassicas, produces extracellular plant cell wall-degrading enzymes which contribute to its pathogenicity by facilitating its spread through plant tissues and give the bacterium access to a ready source of nutrients via the carbohydrate utilization loci containing TonB-dependent transporters (CUT loci) (7, 16, 35). The CUT loci are characterized by the presence of genes encoding regulators, degradative enzymes, inner membrane transporters, and outer membrane TonB-dependent transporters (TBDTs), which have been identified as active carbohydrate transporters (7, 33, 44). However, recently, an example of passive diffusion through a TBDT in Caulobacter crescentus was described (17). X. campestris pv. campestris has 72 TBDTs and belongs to a class of bacteria in which TBDTs are overrepresented (7). Our previous study suggested that there are several CUT loci or systems in this bacterium (7).N-Acetylglucosamine (GlcNAc) is an amino sugar that is used for the synthesis of cell surface structures in bacteria and plays an important role in supplying carbon and energy by entering the glycolytic pathway after it is converted into fructose-6-phosphate (fructose-6P) (1, 9). In a recent comparative study of bacterial GlcNAc utilization pathways and regulatory networks, Yang and coworkers identified conserved and distinct features of the GlcNAc utilization pathway in proteobacteria (48). The expression of X. campestris pv. campestris GlcNAc-specific genes was proposed to be controlled by NagR and NagQ regulators belonging to the LacI and GntR families, respectively. In X. campestris pv. campestris strain ATCC 33913, one predicted binding motif specific for NagQ (designated the NagQ box) consists of two imperfect repeats of the TGGTATT sequence separated by 4 bp and is located upstream of the nagQ gene (XCC3414) (Fig. ​(Fig.1A)1A) (48). This gene is part of the nag cluster and is followed by genes encoding the major facilitator superfamily (MFS) inner membrane transporter NagP (XCC3413), the regulator NagR (XCC3412), the GlcN-6P deaminase NagB-II (XCC3411), and the GlcNAc-6P deacetylase NagA (XCC3410) (Fig. ​(Fig.1A).1A). NagR boxes contain the palindromic sequence AATGACARCGYTGTCATT (bold type indicates less highly conserved nucleotides) and are upstream of genes encoding two glucokinase-like NagK-II proteins (XCC2886 [nagK-IIA] and XCC2943 [nagK-IIB]), as well as 5 genes encoding TBDTs (XCC0531, XCC2887, XCC3045, XCC3408, and XCC2944 located downstream of XCC2943) (Fig. ​(Fig.1A).1A). All of the X. campestris pv. campestris genes located downstream of NagR or NagQ boxes were proposed to belong to a GlcNAc utilization pathway involved in uptake of GlcNAc through the bacterial envelope and subsequent phosphorylation, deacetylation, and deamination, which finally leads to the common metabolic intermediate fructose-6-phosphate (Fig. ​(Fig.1B)1B) (48). It was recently demonstrated that in C. crescentus the TBDT CC0446 gene, which is clustered with other nag genes, is responsible for the uptake of GlcNAc (17). The presence of TBDTs in the GlcNAc regulon, which has been observed in Alteromonadales and Xanthomonadales (48), suggests that genes belonging to the GlcNAc utilization pathway define a new CUT system.Open in a separate windowFIG. 1.X. campestris pv. campestris N-acetylglucosamine (GlcNAc) utilization pathway. (A) Organization of genes in the proposed GlcNAc utilization pathway. NagR boxes are indicated by filled circles, and the NagQ box is indicated by an open circle. (B) GlcNAc is proposed to be transported through the outer membrane by TBDTs and then transported across the inner membrane by the MFS transporter NagP. GlcNAc would then be phosphorylated by nagK-II-encoded enzymes. Subsequent metabolism via the nagA-encoded (GlcNAc-6P deacetylase) and nagB-II-encoded (GlcN-6P deaminase) enzymes results in fructose 6-phosphate (Fru-6P) (48). MFS, major facilitator superfamily; PP, periplasm; TBDT, TonB-dependent transporter.Here we describe characterization of the X. campestris pv. campestris GlcNAc utilization pathway and regulatory network, which involves at least the repressors NagR and NagQ. TBDTs are associated with this pathway, confirming the presence of a GlcNAc CUT system in X. campestris pv. campestris. In this bacterium, GlcNAc entry and catabolism imply that novel families containing a GlcNAc inner membrane transporter and GlcNAc kinases are involved.     

A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic

The mammalian class III phosphatidylinositol 3-kinase (PI3K-III) complex regulates fundamental cellular functions, including growth factor receptor degradation, cytokinesis and autophagy. Recent studies suggest the existence of distinct PI3K-III sub-complexes that can potentially confer functional specificity. While a substantial body of work has focused on the roles of individual PI3K-III subunits in autophagy, functional studies on their contribution to endocytic receptor downregulation and cytokinesis are limited. We therefore sought to elucidate the specific nature of the PI3K-III complexes involved in these two processes. High-content microscopy-based assays combined with siRNA-mediated depletion of individual subunits indicated that a specific sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates both receptor degradation and cytokinesis, whereas ATG14L, a PI3K-III subunit involved in autophagy, is not required. The unanticipated role of UVRAG and BIF-1 in cytokinesis was supported by a strong localisation of these proteins to the midbody. Importantly, while the tumour suppressive functions of Beclin 1, UVRAG and BIF-1 have previously been ascribed to their roles in autophagy, these results open the possibility that they may also contribute to tumour suppression via downregulation of mitogenic signalling by growth factor receptors or preclusion of aneuploidy by ensuring faithful completion of cell division.