Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-alpha-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H(2)O(2) into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly(12)-Gly(13)-Gly(14)-Ile(15)-Ile(16)-Gly(17). Recombinant GlpO lacking these six amino acids (GlpODeltaFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpODeltaFAD, similarly to anti-GlpO antibodies, neutralised H(2)O(2) production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the β 2 subunit of GABA A receptors?

NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

The low abundance of U7 snRNA is partly determined by its Sm binding site.

In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4- to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Sm-mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.

Role of the glutathione S -transferase P1 (GSTP1) electrophile binding site (H-site) in protection from doxorubicin -mediated cytotoxicity

The hypothesis addressed in this project was that novel variants of naturally occurring human glutathione S-transferase P1 (GSTP1) can be created by random mutagenesis of the GSTP1 active site to yield polypeptides with increased enzymatic activity against electrophilic substrates. Specifically, the mutant proteins would metabolize and inactivate selected electrophiles more efficiently than wild-type GSTP1 and confer significant cytoprotection, as measured by reduced apoptosis and increased clonogenic survival. Glutathione S-transferase P1, a major electrophile metabolizing and detoxifying enzyme, is encoded by a polymorphic genetic locus. This locus contains nucleotide transitions in the region encoding the active site of the peptide that yields proteins with significant structural and functional differences. The method of Degenerate Oligonucleotide Mediated Random Mutagenesis (DOMRM) was used to generate cDNAs encoding unique GSTP1 polypeptides with mutations within electrophile binding site (H-site) while leaving the glutathione binding site unaffected. A prokaryotic expression library of the mutant GSTP1 polypeptides was created and screened for increased resistance to cisplatin. This screen resulted in the isolation of 96 clones representing 22 distinct mutant cDNA sequences. To investigate the effects of the changes in the H-site on the biological activity of GSTP1, the cDNA of wild-type GSTP1c and two of the identified mutants were stably transfected into human LNCaP-Pro5 prostate cancer cells that do not endogenously express GSTP1. Wild-type transfectants were resistant to doxorubicin-induced apoptosis and displayed increased clonogenic survival compared to vector controls. However, contrary to the hypothesis, in both assays the mutant transfectants were no more resistant to doxorubicin than the wild-type transfectants. To elucidate the mechanisms underlying GSTP1-mediated survival, an in-vitro assay was developed to determine whether active GSTP1 protein directly metabolizes doxorubicin by conjugation to reduced glutathione (GSH). Although GSH did promote the appearance of a unique doxorubicin conjugate, conjugate formation was not substantially increased by the addition of GSTP1 in a variety of reaction conditions. ^

Identification of a nucleotide binding site in HIV-1 integrase

HIV-1 integrase is essential for viral replication and can be inhibited by antiviral nucleotides. Photoaffinity labeling with the 3′-azido-3′-deoxythymidine (AZT) analog 3′,5-diazido-2′,3′-dideoxyuridine 5′-monophosphate (5N3-AZTMP) and proteolytic mapping identified the amino acid 153–167 region of integrase as the site of photocrosslinking. Docking of 5N3-AZTMP revealed the possibility for strong hydrogen bonds between the inhibitor and lysines 156, 159, and 160 of the enzyme. Mutation of these residues reduced photocrosslinking selectively. This report elucidates the binding site of a nucleotide inhibitor of HIV-1 integrase, and possibly a component of the enzyme polynucleotide binding site.

Manipulation of the membrane binding site of vitamin K-dependent proteins: Enhanced biological function of human factor VII

Recent studies suggested that modification of the membrane contact site of vitamin K-dependent proteins may enhance the membrane affinity and function of members of this protein family. The properties of a factor VII mutant, factor VII-Q10E32, relative to wild-type factor VII (VII, containing P10K32), have been compared. Membrane affinity of VII-Q10E32 was about 20-fold higher than that of wild-type factor VII. The rate of autoactivation VII-Q10E32 with soluble tissue factor was 100-fold faster than wild-type VII and its rate of activation by factor Xa was 30 times greater than that of wild-type factor VII. When combined with soluble tissue factor and phospholipid, activated factor VII-Q10E32 displayed increased activation of factor X. Its coagulant activity was enhanced in all types of plasma and with all sources of tissue factor tested. This difference in activity (maximum 50-fold) was greatest when coagulation conditions were minimal, such as limiting levels of tissue factor and/or phospholipid. Because of its enhanced activity, factor VII-Q10E32 and its derivatives may provide important reagents for research and may be more effective in treatment of bleeding and/or clotting disorders.

Epitopes close to the apolipoprotein B low density lipoprotein receptor-binding site are modified by advanced glycation end products

Advanced glycation end products (AGEs) are thought to contribute to the abnormal lipoprotein profiles and increased risk of cardiovascular disease of patients with diabetes and renal failure, in part by preventing apolipoprotein B (apoB)-mediated cellular uptake of low density lipoproteins (LDL) by LDL receptors (LDLr). It has been proposed that AGE modification at one site in apoB, almost 1,800 residues from the putative apoB LDLr-binding domain, may be sufficient to induce an apoB conformational change that prevents binding to the LDLr. To further explore this hypothesis, we used 29 anti-human apoB mAbs to identify other potential sites on apoB that may be modified by in vitro advanced glycation of LDL. Glycation of LDL caused a time-dependent decrease in its ability to bind to the LDLr and in the immunoreactivity of six distinct apoB epitopes, including two that flank the apoB LDLr-binding domain. ApoB appears to be modified at multiple sites by these criteria, as the loss of glycation-sensitive epitopes was detected on both native glycated LDL and denatured, delipidated glycated apoB. Moreover, residues directly within the putative apoB LDLr-binding site are not apparently modified in glycated LDL. We propose that the inability of LDL modified by AGEs to bind to the LDLr is caused by modification of residues adjacent to the putative LDLr-binding site that were undetected by previous immunochemical studies. AGE modification either eliminates the direct participation of the residues in LDLr binding or indirectly alters the conformation of the apoB LDLr-binding site.

Identification of the binding site for Gqα on its effector Bruton’s tyrosine kinase

Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqα binds directly to the nonreceptor Bruton’s tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both in vitro and in vivo. Only active GTP-bound Gqα, not inactive GDP-bound Gqα, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqα also eliminate Btk stimulation by Gqα, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqα to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.

Single prenyl-binding site on protein prenyl transferases

Three distinct protein prenyl transferases, one protein farnesyl transferase (FTase) and two protein geranylgeranyl transferases (GGTase), catalyze prenylation of many cellular proteins. One group of protein substrates contains a C-terminal CAAX motif (C is Cys, A is aliphatic, and X is a variety of amino acids) in which the single cysteine residue is modified with either farnesyl or geranylgeranyl (GG) by FTase or GGTase type-I (GGTase-I), respectively. Rab proteins constitute a second group of substrates that contain a C-terminal double-cysteine motif (such as XXCC in Rab1a) in which both cysteines are geranylgeranylated by Rab GG transferase (RabGGTase). Previous characterization of CAAX prenyl transferases showed that the enzymes form stable complexes with their prenyl pyrophosphate substrates, acting as prenyl carriers. We developed a prenyl-binding assay and show that RabGGTase has a prenyl carrier function similar to the CAAX prenyl transferases. Stable RabGGTase:GG pyrophosphate (GGPP), FTase:GGPP, and GGTase-I:GGPP complexes show 1:1 (enzyme:GGPP) stoichiometry. Chromatographic analysis of prenylated products after single turnover reactions by using isolated RabGGTase:GGPP complex revealed that Rab is mono-geranylgeranylated. This study establishes that all three protein prenyl transferases contain a single prenyl-binding site and suggests that RabGGTase transfers two GG groups to Rabs in independent and consecutive reactions.

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABAA) receptors. Coexpression of either rat γ2 or γ3, in combination with α1 and β2 subunits, results both in receptors displaying high [3H]Ro 15-1788 affinity. However, receptors containing a γ3 subunit display a 178-fold reduced affinity to zolpidem as compared with γ2-containing receptors. Eight chimeras between γ2 and γ3 were constructed followed by nine different point mutations in γ2, each to the homologous amino acid residue found in γ3. Chimeric or mutant γ subunits were coexpressed with α1 and β2 in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ2 (γ2M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [3H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ2M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ2M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ2M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABAA receptors. As the modulatory site in the GABAA receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ2M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.

Transfer of the HIV-1 cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can confer both cyclosporin sensitivity and cyclosporin dependence

HIV-1 specifically incorporates the peptidyl prolyl isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). HIV-1 replication is inhibited by CsA as well as by nonimmunosuppressive CsA analogues that bind to CyPA and interfere with its virion association. In contrast, the related simian immunodeficiency virus SIVmac, which does not interact with CyPA, is resistant to these compounds. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction between the active site of the enzyme and the capsid (CA) domain of the HIV-1 Gag polyprotein. We report here that the transfer of HIV-1 CA residues 86–93, which form part of an exposed loop, to the corresponding position in SIVmac resulted in the efficient incorporation of CyPA and conferred an HIV-1-like sensitivity to a nonimmunosuppressive cyclosporin. HIV-1 CA residues 86–90 were also sufficient to transfer the ability to efficiently incorporate CyPA, provided that the length of the CyPA-binding loop was preserved. However, the resulting SIVmac mutant required the presence of cyclosporin for efficient virus replication. The results indicate that the presence or absence of a type II tight turn adjacent to the primary CyPA-binding site determines whether CyPA incorporation enhances or inhibits viral replication. By demonstrating that CyPA-binding-site residues can induce cyclosporin sensitivity in a heterologous context, this study provides direct in vivo evidence that the exposed loop between helices IV and V of HIV-1 CA not merely constitutes a docking site for CyPA but is a functional target of this cellular protein.