Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. Exposure of Hsp33 to oxidizing conditions like H(2)O(2), however, rapidly converts Hsp33 into an efficient molecular chaperone. Activated Hsp33 binds tightly to refolding intermediates of chemically denatured luciferase and suppresses efficiently their aggregation in vitro. Matrix-assisted laser desorption/ionization-mass spectrometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accompanied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(232)-S-S-Cys(234) and Cys(265)-S-S-Cys(268). Cys(141), although not involved in disulfide bond formation, was found highly reactive toward chemical modifications. In contrast, Cys(239) is readily accessible under reducing conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.     

A pulse--radiolysis approach to fast reductive cleavage of a disulfide bond to uncage enzyme activity

The essential thiol of the enzyme papain has been caged by linking to an aromatic thiol. The resulting caged protein is inactive but enzymatic activity is fully restored upon chemical cleavage of the protective disulfide bond. We have exploited the chemistry of this disulfide bond to uncage papain by pulse radiolysis. We have shown that up to 10% of the enzyme activity can be restored by reductive pulse radiolysis. This approach has been tested on a small-molecule model system, and experiments on this model compound show that pulse radiolysis of the mixed cysteine-aromatic disulfide results in selective reduction of the disulfide bond to generate a thiol in 10-20% yield, consistent with the radiolytically restored activity of the caged papain quantified by the biochemical assay.     

Static and dynamic roles of extracellular loops in G-protein-coupled receptors: a mechanism for sequential binding of thyrotropin-releasing hormone to its receptor.

Small ligands generally bind within the seven transmembrane-spanning helices of G-protein-coupled receptors, but their access to the binding pocket through the closely packed loops has not been elucidated. In this work, a model of the extracellular loops of the thyrotropin-releasing hormone (TRH) receptor (TRHR) was constructed, and molecular dynamics simulations and quasi-harmonic analysis have been performed to study the static and dynamic roles of the extracellular domain. The static analysis based on curvature and electrostatic potential on the surface of TRHR suggests the formation of an initial recognition site between TRH and the surface of its receptor. These results are supported by experimental evidence. A quasi-harmonic analysis of the vibrations of the extracellular loops suggest that the low-frequency motions of the loops will aid the ligand to access its transmembrane binding pocket. We suggest that all small ligands may bind sequentially to the transmembrane pocket by first interacting with the surface binding site and then may be guided into the transmembrane binding pocket by fluctuations in the extracellular loops.     

Distinct molecular mechanisms for agonist peptide binding to types A and B cholecystokinin receptors demonstrated using fluorescence spectroscopy

Fluorescence spectroscopy provides a direct method for evaluating the environment of a fluorescent ligand bound to its receptor. We utilized this methodology to determine the environment of Alexa within a cholecystokinin (CCK)-like probe (Alexa488-Gly-[(Nle(28,31))CCK-26-33]; CCK-8 probe) bound to the type A CCK receptor (Harikumar, K. G., Pinon, D. L., Wessels, W. S., Prendergast, F. G., and Miller, L. J. (2002) J. Biol. Chem. 277, 18552-18560). Here, we study this probe at the type B CCK receptor and develop another probe with its fluorophore closer to the carboxyl-terminal pharmacophore of type B receptor ligands (Alexa488-Trp-Nle-Asp-Phe-NH2; CCK-4 probe). Both probes bound to type B CCK receptors in a saturable and specific manner and represented full agonists. Similar to the type A receptor, at the type B receptor these probes exhibited shorter lifetimes and lower anisotropy when the receptor was in the active conformation than when it was shifted to its inactive, G protein-uncoupled state using guanosine 5'-[beta,gamma-imido]-triphosphate trisodium salt. Absolute values for lifetime and anisotropy were lower for the CCK-8 probe bound to the type B receptor than for this probe bound to the type A receptor, and Alexa fluorescence was more easily quenched by iodide at the type B receptor. This represents the first direct evidence that, despite having identical affinities for binding and potencies for activating type A and B receptors, CCK is docked via distinct mechanisms, with the amino terminus more exposed to the aqueous milieu when bound to the type B CCK receptor than to the type A CCK receptor. Of interest, despite this difference in binding, activation of both receptors results in analogous direction of movement of the fluorescent indicator probes.     

Study of the molecular mechanism of interleukin-2 mutein D10 binding to IL-2 receptors by molecular simulations

Interleukin-2 (IL-2) was originally used therapeutically as an immune stimulatory agent due to its characteristics of immune regulation. Previous work showed that binding of IL2 to its α-subunit receptor induced low blood pressure, vascular leak syndrome and cardiac toxicity. It was reported that there would be no side effects if the mutein of IL-2 could bind directly with the IL-2β/IL-2Rγ complex, and without mediating with IL-2Rα. With the aim to understand the differences in the binding affinity between IL-2 and the mutein to IL-2Rβ/IL-2Rγ complex at an atomic level, we constructed the theoretical binding models of a wild type and a mutated type (D10) of IL-2 utilising homology modelling. We analysed the interactive differences between the two systems after the MD simulations were completed. Our results suggested that (1) the mutation Q74H caused the conformational change of the loop and led to a new binding mode, which consequently caused an increased binding affinity for IL-2Rβ; (2) the residue mutation of I92F provided more favourable interactions; (3) the mutation of R81D provided an increased binding affinity, a contribution by both direct and indirect interactions. The newly designed mutants, D10_D81E, D10_F92 W and D10_N119E, were predicted to have a better potency, especially at the residue 119, which was firstly found to improve the binding activity of IL-2Rγ. These results are expected to facilitate the discovery and rational design of novel IL-2 stimulatory agents, a potential treatment for cancer patients.     

From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors.

Transmembrane signaling systems relay information from the exterior to the interior of a cell, through a series of complex protein-protein interactions and second messenger cascades. One such system consists of the G-protein-coupled receptors, which interact with G proteins upon ligand binding, and in turn activate an effector molecule. The receptor is the first component in this signaling cascade and is subject to considerable regulation. Recent studies have shown that these regulatory events can occur at the levels of receptor protein modification and receptor gene expression. Interestingly, some of these processes appear to be mediated by the same second messenger systems that these receptors activate, which leads to various forms of positive and negative feedback regulation.     

A divalent recombinant immunotoxin formed by a disulfide bond between the extension peptide chains.

Recombinant immunotoxin for the treatment of cancer was made by connecting toxins to 'carcinoma-specific' antibodies that selectively bind to cancer cells, then kills them without harming the normal cells. The divalent recombinant immunotoxin, [B3(Fab)-ext-PE38]2, is a derivative of B3(Fab)-PE38. B3(Fab)-PE38 was made by fusing the Fab domain of the monoclonal antibody (MAb) B3 to PE38, a truncated mutant form of Pseudomonas exotoxin (PE). In this study, B3(Fab)-ext-PE38 was constructed, which has the hinge region of the B3(Fab)-PE38 extended with the peptide extension, G4C(G4S)2, and connected to the C3 connector. The Cys residue of the extension peptide chain makes the disulfide bond between the two Fab domains. The extension sequence (ext) makes the dimerization of B3(Fab)-ext-PE38 easier to form the divalent immunotoxin, because it decreases the steric hindrance between the two PE38s. The constructed genes were expressed in E. coli as inclusion bodies. Polypeptides that were obtained from the inclusion body were refolded, and the active forms were purified. The ID50 values of the divalent molecule, [B3(Fab)-ext-PE38]2, were about 4 ng/ml on A431 cell lines, about 1 ng/ml on CRL1739 cell lines, and 5 ng/ml on MCF-7 cell lines. The [B3(Fab)-ext-PE38]2 showed about a 12-fold higher cytotoxicity on CRL1739 cell lines than B3(scFv)-PE40 did.     

New covalent capture probes for imaging and therapy, based on a combination of binding affinity and disulfide bond formation

We describe the synthesis and development of new reactive DOTA-metal complexes for covalently targeting engineered receptors in vivo, which have superior tumor uptake and clearance properties for biomedical applications. These probes are found to clear efficiently through the kidneys and minimally through other routes, but bind persistently in the tumor target. We also explore the new technique of Cerenkov luminescence imaging to optically monitor radiolabeled probe distribution and kinetics in vivo. Cerenkov luminescence imaging uniquely enables sensitive noninvasive in vivo imaging of a β(-) emitter such as (90)Y with an optical imager.     

A critical interpretation of experiments of binding of peptide hormone to specific receptors by computer modelling

Many investigations dealing with the interaction of peptide hormones and specific cell membrane receptors imply the existence of two classes of independent binding sites. One class is characterized by high affinity and low capacity, the other one by low affinity and high capacity. This conclusion has been derived from the fact that the Scatchard plots of binding data show a significant upward curvature. Using a more precise and critical method of evaluation these findings probably must be revised in some cases. Other conclusions taken from linear plots concerning the regulation of hormone receptors should be discussed more carefully. Some sources of errors caused by an uncritical interpretation of Scatchard plots are demonstrated.     

ATP analogue binding to the A subunit induces conformational changes in the E subunit that involves a disulfide bond formation in plant V-ATPase.

Vacuolar H+-ATPase (V-ATPase) consists of a catalytic head, a stalk part and a membrane domain. We indirectly investigated the interaction between the A subunit (catalytic head) and the E subunit (stalk part) using an ATP analogue, adenosine 5'-[beta,gamma-imino]triphosphate (AMP-PNP), which holds the enzyme in the substrate-binding state. AMP-PNP treatment caused a mobility shift of the E subunit with a faster migration in SDS/polyacrylamide gel electrophoresis without a reductant, while ATP treatment did not. A mobility shift of the E subunit has been detected in several plants. As polypeptides with intramolecular disulfide bonds migrate faster than those without disulfide bonds, the mobility shift may be due to the formation of an intramolecular disulfide bond by two cysteine residues conserved among several plant species. The mobility shift may be involved in the binding of AMP-PNP to the ATP-binding site, which exists in the A and B subunits, as it was inhibited by the addition of ATP. Pretreatment with 2'-3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), which modifies the ATP-binding site of the B subunit under UV illumination, did not inhibit the mobility shift of the E subunit caused by AMP-PNP treatment. The response of V-ATPase following the AMP-PNP binding may cause a conformational change in the E subunit into a form that is susceptible to oxidation of cysteine residues. This is the first demonstration of interaction between the A and E subunits in the substrate-binding state of a plant V-ATPase.     

Functional consequences of the loss of high affinity agonist binding to gamma-aminobutyric acid type A receptors. Implications for receptor desensitization

We reported previously that tyrosine 62 of the beta2 subunit of the gamma-aminobutyric acid, type A (GABA(A)) receptor is an important determinant of high affinity agonist binding and that recombinant alpha1beta2gamma2(L) receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 approximately 0.7 microm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the beta2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA(A) receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.     

Insights into a highly conserved network of hydrogen bonds in the agonist binding site of nicotinic acetylcholine receptors: A structural and theoretical study

Structural and theoretical studies on the geometrical features of a hydrogen‐bond network occurring in the binding site of nicotinic acetylcholine receptors (nAChRs) and composed of interconnected WxPD (Trp‐x‐Pro‐Asp) and SWyz (Ser‐Trp‐yz) sequences from loops A and B, respectively, have been carried out. Multiple sequence alignments using as template the sequence of the apoform of Aplysia californica acetylcholine binding protein (Ac‐AChBP) show the strict conservation of serine and tryptophan residues of the loop B SWyz sequence. Considering a sample of 19 high resolution AChBP structures, the strong conformational preferences of the key tryptophan residue has been pointing out, whatever the form, free or bounded, of AChBP. The geometry of the motif hydrogen‐bond network has been characterized through the analyses of seven distances. The robustness of the various hydrogen‐bond interactions is pointed out, the one involving the aspartate carboxylate group and the serine residue being the shortest of the network. The role of a cooperative effect involving a NH(His145)…OH (Ser142) hydrogen bond is highlighted. Density functional theory calculations on several simplified models based on the motif hydrogen‐bond network allow probing the importance of the various hydrogen‐bond interactions. The removal of the Ser142 hydroxyl group induces strong structural rearrangements, in agreement with the structural observations. Molecular electrostatic potential calculations on model systems highlight the importance of a cooperative effect in the whole hydrogen‐bond network. More precisely, the key role of the Ser142 hydroxyl group, involved in several hydrogen bonds, is underlined. Proteins 2014; 82:2303–2317. © 2014 Wiley Periodicals, Inc.     

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-[3H]piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. In contrast, the major differences between the kinetic binding parameters of agonists and antagonists to the low affinity agonist binding sites are in the association rate constants, which were 2-5 orders of magnitude lower for agonists. This demonstrates that there are basic differences in the interactions of agonists with the low and high affinity sites. Our findings also suggest that isomerization of the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.     

Acceleration of cleavage of the carbon-cobalt bond of sterically hindered alkylcobalamins by binding to apoprotein of diol dehydrase

Cleavage of the C-Co bond of sterically hindered alkylcobalamins bearing neither an adenine moiety nor functional groups, such as isobutylcobalamin, neopentylcobalamin, and cyclohexylcobalamin, was markedly accelerated by their interaction with apoprotein of diol dehydrase, although these cobalamins do not function as coenzyme. Acceleration of the conversion of alkylcobalamins to enzyme-bound hydroxocobalamin was stoichiometric and obeyed first-order reaction kinetics. These results, together with strong competitive inhibition by these alkylcobalamins with respect to adenosylcobalamin, indicate that acceleration of the C-Co bond cleavage by the apoenzyme is due to labilization of their C-Co bond by binding to the active site of the enzyme. This labilization is considered to be caused by a steric distortion of the corrin ring which is induced by specific tight interaction of the cobalamin moiety with apoprotein. The importance of such a labilizing effect for activation of the C-Co bond of adenosylcobalamin in enzymatic reactions is discussed.     

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700–3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.     

Density functional calculations on the effect of sulfur substitution for 2'-hydroxypropyl-p-nitrophenyl phosphate: C-O vs. P-O bond cleavage

Density functional theory calculations have been used to investigate the intra-molecular attack of 2'-hydroxypropyl-p-nitrophenyl phosphate (HPpNP) and its analogous compound 2-thiouridyl-p-nitrophenyl phosphate (s-2'pNP). Bulk solvent effect has been tested at the geometry optimization level with the polarized continuum model. It is found that the P-path involving the intra-molecular attack at the phosphorus atom and C-path involving the attack at the beta carbon atom proceed through the S(N)2-type mechanism for HPpNP and s-2'pNP. The calculated results indicate that the P-path with the free energy barrier of about 11 kcal/mol is more accessible than the C-path for the intra-molecular attack of HPpNP, which favors the formation of the five-membered phosphate diester. While for s-2'pNP, the C-path with the free energy barrier of about 21 kcal/mol proceeds more favorably than the P-path. The calculated energy barriers of the favorable pathways for HPpNP and s-2'pNP are both in agreement with the experimental results.     

Rate limiting P-O(5'') bond cleavage of RNA fragment: ab initio molecular orbital calculations on the base-catalyzed hydrolysis of phosphate.

In order to examine the energetics in base-catalyzed hydrolysis of RNA, a tentative pentacoordinated intermediate (3) has been characterized by molecular orbital calculations. Ab initio studies at the level of 3-21G* indicate that, under the Cs symmetry restricted conditions, the P-O(2) bond possessing antiperiplanar (app) lone pair electrons (Ip) on the equatorial oxygen (O(3)) can be cleaved with almost no barrier (TS1 transition state; 0.08 kcal mol-1), from the pentacoordinated intermediate (3) of base-catalyzed hydrolysis of phosphate, compared to the P-O(5) bond (TS2 transition state; 28.9 kcal mol-1) which lacks app lp assistance from O(3). The dianionic intermediate, however, loses the TS1 transition state thus its property as an intermediate when the Cs restriction is removed. The analysis of the entire potential energy surface enables us to conclude that, in a related system examined by Lim and Karplus [1990) J. Am. Chem. Soc., 112, 5872-5873) for attack by OH- on ethylene phosphate monoanion, the TS1 transition state had also been lost and thus no intermediate had been found. These results further support our earlier conclusions (Taira et al. (1990) Protein Engineering, 3, 691-701) of rate limiting transition state possessing extended P-O(5') bond breaking character (the TS2 transition state) in the base-catalyzed hydrolysis of RNA. Finally, although the lack of 2',3' -migration of phosphate moieties in basic condition appears to be in accord with the short-lived intermediate, it really does not prove the absence of the intermediate. The detail will be discussed in the text.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.