Elongation factor-dependent affinity labeling of Escherichia coli ribosomes

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes was obtained using a functional analogue of aminoacyl-tRNA. Since elongation factor Tu (EF-Tu) screens both the modified aminoacyl-tRNAs and the ribosomal complexes for active particles, only functional macromolecular complexes are examined. This approach also provides an unequivocal identification of the transfer RNA binding site from which affinity labeling occurs. N^ε-bromoacetyl-Lys-tRNA was prepared by covalently attaching an electrophilic group to the side-chain of the amino acid. This chemical modification did not interfere with function, since the ?BrAcLys-tRNA participated successfully in EF-Tu and poly(rA)-dependent binding to ribosomes, peptide bond formation, and elongation factor G (EF-G)-mediated translocation. Affinity labeling of ribosomal RNA was observed only in those incubations which contained both EF-Tu and EF-G. The crosslinking of ?BrAcLys-tRNA to 23 S rRNA was found even if fusidic acid was added to the incubation before EF-G. The dependence of the covalent reaction on EF-G demonstrates, unambiguously, that a reactive residue of 23 S rRNA is located adjacent to the 3′ end of the functionally defined P site. Similarly, the affinity labeling of proteins L13/14/15, L2, L32/33, and L24 required EF-G-dependent translocation of ?BrAcLys-tRNA into the P site. Protein L27 was alkylated following the EF-Tu-dependent binding of ?BrAcLys-tRNA to the ribosome, and the extent of affinity labeling was stimulated by the addition of EF-G to the incubation. Double-label dipeptide experiments confirmed that affinity labeling occurred from functional tRNA binding sites by demonstrating that the same ?BrAcLys-tRNA which reacted covalently with 23 S rRNA or a ribosomal protein could also participate in peptide bond formation. Finally, the ribosome affinity labeling obtained with ?BrAcLys-tRNA · EF-Tu · guanylylimidodiphosphate differed little from that obtained with ?BrAcLys-tRNA · EF-Tu · GTP. This work constitutes the first direct examination of the aminoacyl ends of the EF-Tu-dependent conformational states of the ribosomal complex, and demonstrates the potential value of functional Lys-tRNA analogues with different probes attached to the lysine side-chain.     

Assay of the acetylcholine receptor by affinity labeling

An accurate and sensitive assay for nicotinic acetylcholine receptor binding sites is described which is based on the specificities of receptor both for an affinity label, 4-(N-maleimido)benzyltrimethylammonium iodide (MBTA), and for α-neurotoxins from Naja venoms. It has been demonstrated that MBTA reacts exclusively with one type of subunit of the acetylcholine receptors isolated from the electric tissue of Electrophorus electricus and Torpedo californica and that this reaction is blocked in the presence of Naja naja siamensis α-neurotoxin and of other ligands of the acetylcholine binding site. Thus, in this assay the difference in the extent of labeling by MBTA in the absence and presence of N. n. siamensis toxin is considered the specific labeling of receptor. Although this assay is more complicated than direct α-neurotoxin binding, it is justified by the wellestablished site specificity of the labeling. The specific activities of several different receptor preparations determined using this assay are one-half of those determined using toxin binding. It is possible to assay accurately as little as 0.25 μg of receptor in the presence of 100-fold as much other protein.     

Capturing of the free cysteine residue in the ligand-binding site by affinity labeling of the ORL1 nociceptin receptor

All of the δ, μ, and κ opioid receptors have a free thiol group of the Cys residue in the ligand-binding site, although its functional role is not yet known. In order to examine whether or not a similar Cys is also present in the ORL1 nociceptin receptor, we attempted to identify it by affinity labeling using a specific antagonist peptide. We first treated ORL1-expressing COS-7 cell membrane preparations with the thiol-alkylation reagent N-ethylmaleimide (NEM) to perform a binding assay using [³H]nociceptin as a tracer and nociceptin, an ORL1 agonist, or Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂, a nociceptin/ORL1 antagonist, as a competitor. It was suggested that ORL1 has a free Cys in its ligand-binding site, since the NEM treatment reduced the population of ligand-binding sites. This was further confirmed by affinity labeling using Cys(Npys)-Arg-Tyr-Tyr-Arg-Ile-Lys-NH₂ with the SNpys group that can react with a free thiol group, resulting in the formation of a disulfide bond. This affinity labeling was approximately 23 times more specific than NEM alkylation. The results revealed that the ORL1 nociceptin receptor does contain a free Cys residue in the ligand-binding site.     

In vitro binding to and in vivo effect on the cytosol and nuclear progesterone receptors of various progestins,and their relationship to synthesis of uteroglobin in rabbit uterus

In vitro binding affinities of various progestins to cytosol and nuclear progesterone receptors of rabbit uterus were determined and correlated with the biological potency of these steroids. In addition, cytosol and nuclear progesterone receptor levels were measured after a 5-day administration of different progestins (0.5 mg/kg daily) with variable biologic activites. The receptor levels were compared with the bilological response; the induction of uteroglobin synthesis. Cytosol and nuclear progesterone receptors had identical steroid binding properties (r = 0.98). The correlation between the in vitro binding affinity (cytosol or nuclear) and the in vivo biologic activity of the steroids was good (r = 0.73). After a 5-day treatment with progestins, the nuclear receptor concentration correlated in an inverse manner (r = ?0.84) with the uterine fluid unteroglobin concentration. A similar, but slightly weaker correlation (r = ?0.81) was also found for the cytosol receptor content and uteroglobin secretion. These data indicate that not only nuclear, but also cytosol progesterone receptor levels decrease in the rabbit uterus during chronic hormone action. Decline in the nuclear progesterone receptor content seemed to occur during treatment with all progestational steroids, while onlyi progestins with high biological potency were capable of decreasing the cytosol receptor content.     

Purification of a progesterone receptor from rabbit uterus

A progesterone receptor has been purified to homogeneity from rabbit uterus by steroid affinity chromatography. The receptor was obtained in 5% yield, with a specific activity for [³H]progesterone binding of 14,580 pmol/mg protein. The pure receptor migrated as a single band on SDS-polyacrylamide electrophoresis, with a MW of 70,000. Progesterone binding to the receptor was heat labile and was displaced by an excess of R5020. Photoaffinity labeling of the pure receptor with [³H]R5020 corresponded to the major photoaffinity labeled species in crude cytosol.     

Ring A-stereoisomers of 1-hydroxyvitamin D3 and their relative binding affinities for the intestinal 1α,25-dihydroxyvitamin D3 receptor protein

A set of eight 1-hydroxyvitamin D₃ compounds comprising the four possible (5Z)-1,3-diol stereoisomers and the corresponding (5E)-double bond isomers, has been prepared in order to assess the effect of 1,3-diol stereochemistry and 5,6-double bond geometry on binding affinity for the intestinal 1,25-(OH)₂D₃-receptor protein. The compounds were synthesized from either vitamin D₃ or 3-epivitamin D₃ via 3,5-cyclovitamin D intermediates. Competitive receptor binding assays establish that all changes from the natural ring A-configuration (1S, 3R, 5Z) lead to decreased binding affinity, and confirm the importance of the 1-hydroxy function since the conversion of stereochemistry at that center from 1α(S) to 1β(R) has the most pronounced effect on binding affinity (attenuation by more than three orders of magnitude). Other modifications (i.e., conversion at C-3, or cis to trans isomerization of the 5,6-double bond) decrease binding affinity by more moderate (ca. 10-fold) but cumulative factors.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Selective covalent labeling of cysteines in bovine serum albumin and in hepatoma tissue culture cell glucocorticoid receptors by dexamethasone 21-mesylate

The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.     

Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling

Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.     

Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor

The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes has been identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate or ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N epsilon-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with Mr 50 000-65 000. The receptor has been solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same molecular weight material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Therefore, although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a two saturable sites model fitted the data significantly better (p less than 0.01) than did a one saturable site model. The characteristics of the two saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 +/- 0.45 nM and a low-affinity site with a KD value of 19.77 +/- 3.28 nM. A total of 35% of the two sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 +/- 0.12 was observed.     

α2-Macroglobulin-protease reactions: Relationship of covalent bond formation,methylamine reactivity,and specific proteolysis

Experiments were performed to define the relation between covalent binding of enzymes to β₂-macroglobulin (α₂M), the specific proteolysis of α₂M subunits to 85K fragments, and the reactivity of the methylamine site on α₂M. We studied the reaction of α₂M with native trypsin, anhydrotrypsin, and two active lysyl-blocked derivatives, methyl-trypsin and dimethylmaleyl-trypsin, the last with reversibly modified amino groups that can be regenerated at low pH. The results were: (1) All enzymes tested reacted with α₂M but only native trypsin formed covalent complexes (not dissociable by sodium dodecyl sulfate). Trypsin and the lysyl-blocked enzymes caused complete proteolysis of the α₂M subunits, in agreement with previous studies. (2) The dimethyl-maleyl-trypsin became covalently bound to α₂M only after removing the blocking groups of the bound enzyme, indicating that sequential proteolysis and covalent bond formation is possible. Under the conditions used for deblocking, there was no change in the covalent/noncovalent binding ratio of native trypsin, anhydrotrypsin, or the other lysyl-blocked derivative, methyl-trypsin. (3) Native trypsin or anhydrotrypsin displaced methyl- or dimethylmaleyl-trypsin from their α₂M complexes but the newly bound enzymes with free amino groups did not form covalent bonds indicating that enzymes must remain in association with the inhibitor for the bond to form. (4) Methylamine reacts with noncovalent α₂M complexes but not with covalent complexes. (5) Methylamine-treated α₂M can still form complexes with trypsin but at a drastically reduced rate and only noncovalent complexes are formed. In summary, sequential proteolysis and covalent bond formation is possible under certain conditions, and there is a strong correlation between covalent binding and loss of methylamine reactivity. The latter observation is suggestive evidence for the identity of the covalent binding site of α₂M and the putative thiol ester of the methylamine site. The enzyme lysyl amino groups, are likewise possible candidates for attacking nucleophile at that site.     

Design of a binuclear Ni(II)–iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag fused proteins

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions under live-cell conditions. In this Letter, we report the design of the binuclear Ni(II)–iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag-fused proteins. We found that the Ni(II)–IDA complex 1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding affinity (K_d = 24 nM), the value of which is 16-fold higher than the conventional Ni(II)–NTA complex (K_d = 390 nM). The strong binding affinity of the Ni(II)–IDA complex was successfully used in the covalent labeling and fluorescence bioimaging of a His-tag fused GPCR (G-protein coupled receptor) located on the surface of living cells.     

4-Azidophlorizin,a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ($\text{2′-O-(β-}\text{d}\text{-}\text{glucopyranosyl}\text{)-4-}\text{azidophloretin}$, 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na⁺ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′_i, the constant for fully-competitive inhibition of (Na⁺, Δψ)-dependent d-glucose uptake, (2) K′_d, the dissociation constant of 4-azido[³H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″_i, the constant for fully-competitive inhibition of the specific ((Na⁺, Δψ)-dependent, d-glucose protectable) high-affinity [³H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (K′_i, K′_d and K″_i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′_i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Affinity labeling of eukaryotic elongation factors using N epsilon-bromoacetyl-Lys-tRNA.

eEF-T and eEF-Tu from rabbit reticulocyte and from Artemia were affinity labeled using N epsilon-bromoacetyl-Lys-tRNA prepared with either yeast or E. coli tRNA. Only the eEF-Tu polypeptide was crosslinked when eEF-T was incubated with the reactive aminoacyl-tRNA analogue, which indicates that at least part of the aminoacyl-tRNA binding site is the same in both eEF-Tu and the multisubunit eEF-T. Complex formation (eEF-Tu x aa-tRNA x GTP) was required for crosslinking, since no covalent reaction with eEF-Tu occurred in the absence of GTP. The yield of crosslinked product was greatly reduced by adding either unmodified rabbit liver aminoacyl-tRNA or unmodified E. coli Lys-tRNA to the incubation to compete for the aminoacyl-tRNA binding site on eEF-T or eEF-Tu, indicating that the covalent reaction occurs while the N epsilon-bromoacetyl-Lys-tRNA is bound in this site. The affinity labeling of a prokaryotic and two different eukaryotic elongation factors by the same reagent suggests that there may be conservation of structure in the region of the proteins which binds the aminoacyl end of the aminoacyl-tRNA.     

S-(4-Bromo-2,3-dioxobutyl)CoA: an affinity label for certain enzymes than bind acetyl-CoA.

S-(4-Bromo-2,3-dioxobutyl)-CoA, a potential affinity label for enzymes possessing a receptor site(s) for short-chain acyl-CoA, was synthesized by condensing CoA and 1,4-dibromo-2,3-butanedione in acidified methanol. The new reagent was tested as an active site-directed irreversible inhibitor with four enzymes that accept a short-chain acyl-CoA as substrate. With citrate synthase (pig heart) and acetyl-CoA hydrolase (beef kidney) irreversible inhibition was observed, and the rate of inactivation obeyed first-order kinetics. Benzoyl-CoA, a reversible competitive inhibitor versus acetyl-CoA with both citrate synthase and acetyl-CoA hydrolase, protected the active site of both enzymes against the irreversible inhibitor. The new reagent was an exceptionally potent irreversible inhibitor of acetoacetyl-CoA thiolase (beef liver). Relatively low concentrations of the reagent (≥1 μm) completely inhibited the thiolase in less than 2 min. Preincubation of thiolase with acetoacetyl-CoA protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. In contrast, irreversible inhibition of l-3-hydroxyacyl-CoA dehydrogenase (pig heart) was not observed. Instead, the new reagent appeared to be a weak alternate substrate for this dehydrogenase. In all cases, the new reagent exhibited tight reversible binding at the active site since the measured K_i's (and K_m) were in the range, 30 to 120 μm. It is anticipated that the new reagent will be suitable for investigating a number of acyl-CoA using enzymes by affinity labeling techniques.     

Native subunit composition of two insect nicotinic receptor subtypes with differing affinities for the insecticide imidacloprid

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively to control a variety of insect pest species. The brown planthopper (Nilaparvata lugens), an insect pest of rice crops throughout Asia, is an important target species for control with neonicotinoid insecticides such as imidacloprid. Studies with nAChRs purified from N. lugens have identified two [³H]imidacloprid binding sites with different affinities (K_d = 3.5 ± 0.6 pM and 1.5 ± 0.2 nM). Co-immunoprecipitation studies with native preparations of N. lugens nAChRs, using subunit-selective antisera, have demonstrated the co-assembly of Nlα1, Nlα2 and Nlβ1 subunits into one receptor complex and of Nlα3, Nlα8 and Nlβ1 into another. Immunodepletion of Nlα1 or Nlα2 subunits resulted in the selective loss of the lower affinity imidacloprid binding site, whereas immunodepletion of Nlα3 or Nlα8 caused the selective loss of the high-affinity site. Immunodepletion of Nlβ1 resulted in a complete absence of specific imidacloprid binding. In contrast, immunodepletion with antibodies selective for other N. lugens nAChR subunits (Nlα4, Nlα6, Nlα7 and Nlβ2) had no significant effect on imidacloprid binding. Taken together, these data suggest that nAChRs containing Nlα1, Nlα2 and Nlβ1 constitute the lower affinity binding site, whereas nAChRs containing Nlα3, Nlα8 and Nlβ1 constitute the higher affinity binding site for imidacloprid in N. lugens.     

Discovery of a novel covalent non-β-lactam inhibitor of the metallo-β-lactamase NDM-1

The inhibition of metallo-β-lactamases (MBL) can prevent the hydrolysis of β-lactam antibiotics and hence is a promising strategy for the treatment of antibiotic resistant infections. In this study, we present a novel reversible covalent inhibitor of the clinically relevant MBL New Delhi metallo-β-lactamase 1 (NDM-1). Electrospray ionization-mass spectrometry (ESI-MS) and single site directed mutagenesis were used to show that the inhibitor forms a covalent bond with Lys224 in the active site of NDM-1. The inhibitor was further characterized using an enzyme inhibition assay, a surface plasmon resonance (SPR) based biosensor assay and covalent docking. The determined inhibition constant (K_I¹) was 580 nM and the inhibition constant for the initial complex (K_I) was 76 μM. To our knowledge, this inhibitor is the first example for a reversible covalent non-β-lactam inhibitor targeting NDM-1 and a promising starting point for the design of potent covalent inhibitors.     

Development of covalent antagonists for β1- and β2-adrenergic receptors

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β₂-adrenergic receptor (β₂AR^H2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β₁- and β₂ receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants β₁AR^I2.64C and β₂AR^H2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the β₂AR^H2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the β₂AR^H2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the β₁AR^I2.64C and β₂AR^H2.64C mutant while retaining a 41-fold selectivity for the β₁AR wild type over β₂AR. These compounds may serve as valuable molecular tools for studying β₁/β₂ subtype selectivity or investigations on GPCR trafficking and dimerization.