Putative Binding Residues Research Articles

Characterisation of lepidopteran geranylgeranyl diphosphate synthase as a putative pesticide target.

Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.

Accurate Sequence-Based Prediction of Deleterious nsSNPs with Multiple Sequence Profiles and Putative Binding Residues.

Non-synonymous single nucleotide polymorphisms (nsSNPs) may result in pathogenic changes that are associated with human diseases. Accurate prediction of these deleterious nsSNPs is in high demand. The existing predictors of deleterious nsSNPs secure modest levels of predictive performance, leaving room for improvements. We propose a new sequence-based predictor, DMBS, which addresses the need to improve the predictive quality. The design of DMBS relies on the observation that the deleterious mutations are likely to occur at the highly conserved and functionally important positions in the protein sequence. Correspondingly, we introduce two innovative components. First, we improve the estimates of the conservation computed from the multiple sequence profiles based on two complementary databases and two complementary alignment algorithms. Second, we utilize putative annotations of functional/binding residues produced by two state-of-the-art sequence-based methods. These inputs are processed by a random forests model that provides favorable predictive performance when empirically compared against five other machine-learning algorithms. Empirical results on four benchmark datasets reveal that DMBS achieves AUC > 0.94, outperforming current methods, including protein structure-based approaches. In particular, DMBS secures AUC = 0.97 for the SNPdbe and ExoVar datasets, compared to AUC = 0.70 and 0.88, respectively, that were obtained by the best available methods. Further tests on the independent HumVar dataset shows that our method significantly outperforms the state-of-the-art method SNPdryad. We conclude that DMBS provides accurate predictions that can effectively guide wet-lab experiments in a high-throughput manner.

Homology Modelling and in Silico Substrate Binding Analysis of a Rhizobium sp. RC1 Haloalkanoic Acid Permease

Rhizobium sp. RC1 grows on haloalkanoic acid (haloacid) pollutants and expresses a haloacid permease (DehrP) which mediates the uptake of haloacids into the cells. For the first time, we report the homology model and docking analysis of DehrP and proposed its putative binding residues. The Protein Data Bank for protein of similar sequence. Ligand structures were retrieved from the ChemSpider database. The 3-dimensional (3-D) structure of DehrP was modelled based on the structure of Staphylococcus epidermidis glucose: H+ symporter (GlcPse) by Phyre2, refined by 3Drefine and evaluated by ProSA z-score, ERRAT and RAMPAGE. Docking of monobromoacetate, monochloroacetate, dibromoacetate, dichloroacetate, trichloroacetate, and 2,2-dichloropropionate ligands was done with AutoDock vina1.1.2. The 3-D structure of DehrP protein has twelve transmembrane helices. The overall quality factor of the model is ∼91%, with 93.6% of the residues in the favored region and the z-score is within the ≤ 10 limit. The putative H+ binding site residues are Gln133, Asp36, and Arg130. Docking analysis showed that Glu33, Trp34, Phe37, Phe38, Gln165, and Glu370 are potential haloacid interacting residues. DehrP-haloacid complexes had a binding affinity between -2.9 to -4.0 kcal/mol. DehrP has both putative H+ and haloacid binding sites that are most likely involved in the co-transport of H+ and haloacids. DehrP interacts with haloacids majorly through van der Waals and halogen bond interactions and has greater affinity for 2,2-dichloropropionate and could be a specialized chloropropionate uptake system. Site-directed mutagenasis of DehrP binding residues could improve its haloacid binding affinity.

Homology Modelling and in Silico Substrate Binding Analysis of a Rhizobium sp. RC1 Haloalkanoic Acid Permease

Rhizobium sp. RC1 grows on haloalkanoic acid (haloacid) pollutants and expresses a haloacid permease (DehrP) which mediates the uptake of haloacids into the cells. For the first time, we report the homology model and docking analysis of DehrP and proposed its putative binding residues. The Protein Data Bank for protein of similar sequence. Ligand structures were retrieved from the ChemSpider database. The 3-dimensional (3-D) structure of DehrP was modelled based on the structure of Staphylococcus epidermidis glucose: H+ symporter (GlcPse) by Phyre2, refined by 3Drefine and evaluated by ProSA z-score, ERRAT and RAMPAGE. Docking of monobromoacetate, monochloroacetate, dibromoacetate, dichloroacetate, trichloroacetate, and 2,2-dichloropropionate ligands was done with AutoDock vina1.1.2. The 3-D structure of DehrP protein has twelve transmembrane helices. The overall quality factor of the model is ∼91%, with 93.6% of the residues in the favored region and the z-score is within the ≤ 10 limit. The putative H+ binding site residues are Gln133, Asp36, and Arg130. Docking analysis showed that Glu33, Trp34, Phe37, Phe38, Gln165, and Glu370 are potential haloacid interacting residues. DehrP-haloacid complexes had a binding affinity between -2.9 to -4.0 kcal/mol. DehrP has both putative H+ and haloacid binding sites that are most likely involved in the co-transport of H+ and haloacids. DehrP interacts with haloacids majorly through van der Waals and halogen bond interactions and has greater affinity for 2,2-dichloropropionate and could be a specialized chloropropionate uptake system. Site-directed mutagenasis of DehrP binding residues could improve its haloacid binding affinity.

A Biochemical and Structural Look into the Functional Role of Transferrin in D. melanogaster

Transferrins (TFs) are a family of vertebrate and invertebrate proteins that have high affinity for ferric iron. The extensively studied mammalian TFs are typically bilobal in structure and have iron binding sites in both their lobes—critical to their essential role in proper iron sequestration and transport in blood. These sites coordinate iron binding through four conserved residues—aspartate, tyrosine, tyrosine and histidine—and a carbonate ion cofactor. Aside from their function of transporting iron to cells, their ability to sequester and tightly bind iron is a crucial defense against invading pathogens. Many insect TFs show less sequence conservation and have only one iron binding site. They have not been adequately studied for their roles in iron metabolism and immunity. Our research aims to determine the function(s) of insect TF via biochemical and structural analysis using Drosophila melanogaster transferrin (DmTsf1) as a model. We purified DmTsf1 from a baculovirus expression system using ammonium sulfate precipitation, and ion exchange and gel filtration chromatography, and generated apo‐ and holo‐forms. Apo‐DmTsf1 but not holo‐DmTsf1 inhibited Escherichia coli growth, suggesting an immune function that relies on iron sequestration. A homology model of DmTsf1 showed a similar bilobal structure but iron binding in only one lobe. The putative binding residues are glutamate, tyrosine, and tyrosine. If the model is correct, a loss of the histidine residue in DmTsf1 should result in a lower binding affinity for iron. We are using crystallography to identify the actual iron binding residues. Initial protein‐ligand binding kinetics using equilibrium dialysis have shown Apo‐DmTsf1 to indeed having one binding site for ferric iron and a lower Kd of 10−18. Further biochemical pursuits into the role of insect TFs will include examining DmTsf1's ability to bind other metals.Support or Funding InformationNIH NGMS R37 GM041247, NSF IOS 1656388This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Homology modelling and in silico substrate-binding analysis of a Rhizobium sp. RC1 haloalkanoic acid permease

ABSTRACTRhizobium sp. RC1 grows on haloalkanoic acid (haloacid) pollutants and expresses a haloacid permease (DehrP), which mediates the uptake of haloacids into the cells. For the first time, we report the homology model and docking analysis of DehrP and propose its putative binding residues. Ligand structures were retrieved from the ChemSpider database. The three-dimensional (3D) structure of DehrP was modelled based on the structure of Staphylococcus epidermidis glucose:H+ symporter (GlcPse) by Phyre2, refined by 3Drefine and evaluated by ProSA z-score, ERRAT and RAMPAGE. The 3D structure of the DehrP protein has 12 transmembrane helices. The overall quality factor of the model is ∼91%, with 93.6% of the residues in the favoured region and the z-score (−2.86) falls within the range (≤10) for a good model. Subsequent docking of monobromoacetate, monochloroacetate, dibromoacetate, dichloroacetate, trichloroacetate and 2,2-dichloropropionate ligands via AutoDock Vina1.1.2 showed that residues Gln133, Asp36 and Arg130 are the putative H+-binding site, while the probable haloacid interacting residues are Glu33, Trp34, Phe37, Phe38, Gln165 and Glu370. The DehrP-haloacid complexes exhibited binding affinities between −2.9 and −4.0 kcal/mol. Both the putative H+ and haloacid-binding sites of DehrP possibly aided in co-transportation of substrates H+ and haloacids into the bacterial cells through the alternating access mechanism, which occurs by formation of halogen bonds and van der Waals interactions with the substrates. Hence, site-directed mutagenesis on the DehrP binding residues could improve the haloacid-binding affinity for efficient haloacid degradation.

Molecular Recognition of Muramyl Dipeptide Occurs in the Leucine-rich Repeat Domain of Nod2.

Genetic mutations in the innate immune receptor nucleotide-binding oligomerization domain-containing 2 (Nod2) have demonstrated increased susceptibility to Crohn's disease, an inflammatory bowel disease that is hypothesized to be accompanied by changes in the gut microbiota. Nod2 responds to the presence of bacteria, specifically a fragment of the bacterial cell wall, muramyl dipeptide (MDP). The proposed site of this interaction is the leucine-rich repeat (LRR) domain. Surface plasmon resonance and molecular modeling were used to investigate the interaction of the LRR domain with MDP. A functional and pure LRR domain was obtained from Escherichia coli expression in high yield. The LRR domain binds to MDP with high affinity, with a KD of 212 ± 24 nM. Critical portions of the receptor were determined by mutagenesis of putative binding residues. Fragment analysis of MDP revealed that both the peptide and carbohydrate portion contribute to the binding interaction.

Flavones Inhibit the Activity of AKR1B10, a Promising Therapeutic Target for Cancer Treatment.

AKR1B10 is an NADPH-dependent reductase that plays an important function in several physiological reactions such as the conversion of retinal to retinol, reduction of isoprenyl aldehydes, and biotransformation of procarcinogens and drugs. A growing body of evidence points to the important role of the enzyme in the development of several types of cancer (e.g., breast, hepatocellular), in which it is highly overexpressed. AKR1B10 is regarded as a therapeutic target for the treatment of these diseases, and potent and specific inhibitors may be promising therapeutic agents. Several inhibitors of AKR1B10 have been described, but the area of natural plant products has been investigated sparingly. In the present study almost 40 diverse phenolic compounds and alkaloids were examined for their ability to inhibit the recombinant AKR1B10 enzyme. The most potent inhibitors-apigenin, luteolin, and 7-hydroxyflavone-were further characterized in terms of IC50, selectivity, and mode of action. Molecular docking studies were also conducted, which identified putative binding residues important for the interaction. In addition, cellular studies demonstrated a significant inhibition of the AKR1B10-mediated reduction of daunorubicin in intact cells by these inhibitors without a considerable cytotoxic effect. Although these compounds are moderately potent and selective inhibitors of AKR1B10, they constitute a new structural type of AKR1B10 inhibitor and may serve as a template for the development of better inhibitors.

Novel Yeast-based Strategy Unveils Antagonist Binding Regions on the Nuclear Xenobiotic Receptor PXR

Ketoconazole binds to and antagonizes pregnane X receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. Ketoconazole genetically interacts with specific PXR surface residues. A yeast-based genetic method to discover novel nuclear receptor interactions with ligands that associate with surface binding sites is suggested. The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple "gain-of-function" mutants that were "resistant" to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications.

A molecular dynamics study of C1r and C1s dimers: Implications for the structure of the C1 complex

AbstractComplement is an important part of the immune system. It is initiated through three different pathways known as the classical, lectin, and alternative pathway. The multimolecular C1 complex of the classical pathway consists of a subcomponent, C1q, which binds to a tetramer comprising two C1r and two C1s proteases. A detailed description of the structure of the C1 complex is essential to fully understand how the complex acts on pathogens. A variety of different models have been proposed, which differ mainly in the way the proteases interact with C1q. In this study, we have used a combination of homology‐based structure prediction and molecular dynamics to predict a partial structure of the C1s/C1r/C1r/C1s tetramer. For computational expediency the study was restricted to the CUB1‐EGF‐CUB2 domains which are directly involved in the formation of the tetramer and its interaction with C1q; the catalytic fragments (CCP1‐CCP2‐SP), which mediate C1 activation and subsequent cleavage of substrates, were omitted. A systematic molecular dynamics (MD) study of several possible dimeric combinations suggest that the tetramer is formed when a pair of C1r/C1s dimers form a “doughnut” via a C1s/C1s head‐to‐tail interaction, which is stabilized by several putative salt bridges at the dimer interface. This result is consistent with biochemical data which have shown that self assembly requires the formation of C1r‐C1s contacts and that electrostatic interactions play a key role. Furthermore, it identifies a number of putative binding residues that can be tested using site‐directed mutagenesis. Proteins 2012. © 2012 Wiley Periodicals, Inc

Inhibition of Human Aldose Reductase-Like Protein (AKR1B10) by α- and γ-Mangostins, Major Components of Pericarps of Mangosteen

A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was recently identified as both diagnostic marker and therapeutic target in the treatment of several types of cancer. In this study, we have examined AKR1B10 inhibition by five xanthone derivatives, components of pericarps of mangosteen, of which α- and γ-mangostins show potential anti-cancer properties. Among the five xanthones, γ-mangostin was found to be the most potent competitive inhibitor (inhibition constant, 5.6 nM), but its 7-methoxy derivative, α-mangostin, was the second potent inhibitor (inhibition constant, 80 nM). Molecular docking of the two mangostins in AKR1B10 and site-directed mutagenesis of the putative binding residues revealed that Phe123, Trp220, Val301 and Gln303 are important for the tight binding of γ-mangostin, and suggested that the 7-methoxy group of α-mangostin impairs the inhibitory potency by altering the orientation of the inhibitor molecule in the substrate-binding site of the enzyme.

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM.

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.

Selective Inhibition of the Tumor Marker AKR1B10 by Antiinflammatory N-Phenylanthranilic Acids and Glycyrrhetic Acid

A human aldose reductase-like protein, AKR1B10 in the aldo-keto reductase (AKR) superfamily, was recently identified as a tumor marker of several types of cancer. Tolrestat, an aldose reductase inhibitor (ARI), is known to be the most potent inhibitor of the enzyme. In this study, we compared the inhibitory effects of other ARIs including flavonoids on AKR1B10 and aldose reductase to evaluate their specificity. However, ARIs showed lower inhibitory potency for AKR1B10 than for aldose reductase. In the search for potent and selective inhibitors of AKR1B10 from other drugs used clinically, we found that non-steroidal antiinflammatory N-phenylanthranilic acids, diclofenac and glycyrrhetic acid competitively inhibited AKR1B10, showing K(i) values of 0.35-2.9 microM and high selectivity to this enzyme (43-57 fold versus aldose reductase). Molecular docking studies of mefenamic acid and glycyrrhetic acid in the AKR1B10-nicotinamide adenine dinucleotide phosphate (NADP(+)) complex and site-directed mutagenesis of the putative binding residues suggest that the side chain of Val301 and a hydrogen-bonding network among residues Val301, Gln114 and Ser304 are important for determining the inhibitory potency and selectivity of the non-steroidal antiinflammatory drugs. Thus, the potent and selective inhibition may be related to the cancer chemopreventive roles of the drugs, and their structural features may facilitate the design of new anti-cancer agents targeting AKR1B10.

Potent and selective inhibition of the tumor marker AKR1B10 by bisdemethoxycurcumin: Probing the active site of the enzyme with molecular modeling and site-directed mutagenesis

A human member of the aldo–keto reductase (AKR) superfamily, AKR1B10, shares high sequence identity with aldose reductase (AR), and was recently identified as a therapeutic target in the treatment of several types of cancer. We have compared the inhibitory effects of plant components on recombinant AKR1B10 and AR. AKR1B10 was inhibited by curcuminoids, magnolol, honokiol and resveratrol, with IC50 values of 0.06–5μM, which were lower than their values for AR. Among them, bisdemethoxycurcumin was the most potent competitive inhibitor (Ki=22nM) with the highest selectivity (85-fold versus AR), and acted as an effective inhibitor in cellular level. In contrast, demethoxycurcumin and curcumin showed >3-fold less potency and selectivity. Molecular docking studies of the curcuminoids in the AKR1B10–NADP+ complex and site-directed mutagenesis of the putative binding residues suggest that Gln114, Val301 and Gln303 are important for determining the inhibitory potency and selectivity of the curcuminoids.

Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin

To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP 95–98/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg 2+-ATPase ( V max and K ATPase) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007–0.030 s − 1 ) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP ( K ATPase) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V max was 64% and the actin-sliding velocity was ∼ 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.

Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor

We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

Probing the structure of Lhca3 by mutation analysis

Lhc proteins constitute a family of transmembrane proteins which share homology in sequence and similarity in the general organisation although members can be strongly differentiated such as in the case of PsbS and ELIPs. In this work, we report on the structure of Lhca3, a pigment-protein subunit component of the antenna system of higher plants Photosystem I, through the effect of point mutations in critical sites. Based on the structure of PSI-LHCI (Ben Shem et al., PDB file 1QZV remark 999) it has been suggested that Lhca3 may have different folding as compared to other members of the Lhc family. In particular, it was proposed that the two central helices may be swapped and chlorophylls in sites 1013 and 1023 are not present. This different folding would imply that the chlorophylls coordinated to the two central helices have different ligands in Lhca3 with respect to the other Lhc complexes. The structural model was tested by substituting the putative binding residues with residues unable to coordinate chlorophyll and the spectroscopic properties of the individual pigments were used as structural probes. The results indicate that Lhca3 folds in the same way as the other antenna proteins. Moreover, the low-energy absorption form originates from interaction between chlorophylls in site 1015 and 1025, like for the other PSI antenna subunits. Evidence is also shown for the presence in Lhca3 of chlorophylls in sites 1013 and 1023.

Escherichia coli thioredoxin inhibition by cadmium: two mutually exclusive binding sites involving Cys32 and Asp26.

Observations of thioredoxin inhibition by cadmium and of a positive role for thioredoxin in protection from Cd(2+) led us to investigate the thioredoxin-cadmium interaction properties. We used calorimetric and spectroscopic methods at different pH values to explore the relative contribution of putative binding residues (Cys32, Cys35, Trp28, Trp31 and Asp26) within or near the active site. At pH 8 or 7.5 two binding sites were identified by isothermal titration calorimetry with affinity constants of 10 x 10(6) m(-1) and 1 x 10(6) m(-1). For both sites, a proton was released upon Cd(2+) binding. One mole of Cd(2+) per mole of reduced thioredoxin was measured by mass spectrometry at these pH values, demonstrating that the two binding sites were partially occupied and mutually exclusive. Cd(2+) binding at either site totally inhibited the thiol-disulfide transferase activity of Trx. The absence of Cd(2+) interaction detected for oxidized or alkylated Trx and the inhibition of the enzymatic activity of thioredoxin by Cd(2+) supported the role of Cys32 at the first site. The fluorescence profile of Cd(2+)-bound thioredoxin differed, however, from that of oxidized thioredoxin, indicating that Cd(2+) was not coordinated with Cys32 and Cys35. From FTIR spectroscopy, we inferred that the second site might involve Asp26, a buried residue that deprotonates at a rather high and unusual pK(a) for a carboxylate (7.5/9.2). The pK(a) of the two residues Cys32 and Asp26 have been shown to be interdependent [Chivers, T. P. (1997) Biochemistry36, 14985-14991]. A mechanism is proposed in which Cd(2+) binding at the solvent-accessible thiolate group of Cys32 induces a decrease of the pK(a) of Asp26 and its deprotonation. Conversely, interaction between the carboxylate group of Asp26 and Cd(2+) at a second binding site induces Cys32 deprotonation and thioredoxin inhibition, so that Cd(2+) inhibits thioredoxin activity not only by binding at the Cys32 but also by interacting with Asp26.

Characterization of Lactogen Receptor-binding Site 1 of Human Prolactin

Prolactin (PRL) binds to two molecules of PRL receptor (PRLR) through two regions referred to as binding sites 1 and 2. Although binding site 1 has been generally assigned to the pocket delimited by helix 1, helix 4, and the second half of loop 1, the residues involved in receptor binding have not yet all been precisely identified. In an earlier alanine-scanning mutational study, we identified three major binding determinants in loop 1 of human PRL (hPRL) (Goffin, V., Norman, M. & Martial, J. A.(1992) Mol. Endocrinol. 6, 1381-1392). Here we focus on the two other regions that form binding site 1, namely helices 1 and 4. Putative binding residues, selected on the basis of a three-dimensional model of hPRL constructed in this laboratory, were mutated to alanine, and recombinant hPRL mutants produced in Escherichia coli were tested for their ability to bind to the PRLR and to stimulate Nb2 cell proliferation. We thus identified nine single mutations (three in helix 1 and six in helix 4) whose effect was to reduce both binding and mitogenic activity by more than half as compared with wild-type hPRL, indicating the functional involvement of the corresponding residues. Adding these to the three binding determinants identified in loop 1, we now propose a complete picture of PRLR-binding site 1 of hPRL. As we earlier hypothesized, the binding site 1 determinants of hPRL differ from those of human growth hormone, a hPRL homolog.

The envelope glycoprotein of HIV-1 may have incorporated the CD4 binding site from HLA-DQβ1

An hypothesis is presented which states that the increased binding for CD4 by the envelope glycoprotein (gp120) from HIV-1 compared with that from HIV-2 is due to the env gene from HIV-1 having at some stage incorporated exon 2 of the gene coding for the β subunit of a class II MHC protein, possibly HLA-DQ, which contains part of the CD4 binding site. Evidence is presented from amino acid sequence analysis and consideration of putative binding residues from gp120 and HLA-DQ.