Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha.

A purified preparation of human estrogen receptor alpha (hERalpha) ligand-binding domain (LBD) involving mainly the Ser(309)Ala(569) (approximately 30%) and Ser(309)Ala(571) (approximately 63%) ER portions was used to identify the covalent attachment sites of two closely related estrogenic ER affinity labels 17alpha-bromoacetamidopropylestradiol (17BAPE(2)) and 17alpha-bromoacetamidomethylestradiol (17BAME(2)). To identify and quantify the electrophile covalent attachment sites, [(14)C]17BAPE(2)- and [(14)C]17BAME(2)-alkylated hLBD preparations were trypsinized and submitted to HPLC. In each case, two radioactive fractions were obtained. Mass spectrometry analyses of the two fractions showed signals, which closely matched the molecular masses of alkylated Cys(530)Lys(531) and Cys(417)Arg(434) hLBD tryptic peptides. The covalent attachment of the two electrophiles on hLBD was assigned to the S atoms of Cys(530) and Cys(417). However, the balance between Cys(530) and Cys(417) labeling markedly differed according to the affinity label used, with the Cys(530)/Cys(417) ratio being 2.1 for 17BAPE(2), and 20 for 17BAME(2). We attempted to interpret the covalent attachment of electrophiles by molecular modeling using the crystallographic structure of LBD bound to E(2). In agreement with the different levels of Cys(417) alkylation, the LBD model with unchanged helices could not easily account for Cys(417) labeling by 17BAME(2), whereas favorable results were obtained through 17BAPE(2) docking. Moreover, labeling at Cys(530) by the two electrophiles could not be interpreted using the LBD model. This indicates that some states of solute LBD bound to the estrogenic E(2) 17alpha-derivatives differ from the structure of crystallized LBD bound to E(2).

Identification of covalent attachment site of antiestrogenic estradiol 11 beta-derivatives on human estrogen receptor alpha ligand-binding domain.

Affinity labeling of human estrogen receptor alpha (ERalpha) by high affinity and antiestrogenic estradiol (E(2)) 11 beta-derivatives, 11 beta-bromoacetamidoethoxyphenylE(2) (11BAEOPE(2)) and 11 beta-bromoacetamidopentoxyphenylE(2) (11BAPOPE(2)) was studied using glutathione-S-transferase (GST) fused to the ligand-binding domain (LBD) of human ERalpha. To identify and quantify the electrophile covalent attachment sites on LBD, [(14)C]11BAEOPE(2)- and [(14)C]11BAPOPE(2)-alkylated LBD were separated from GST, purified, and then trypsinized. HPLC of LBD tryptic fragments afforded one and two radioactive peaks (the ratio of the two latter peaks was 84/16) in the chromatograms related to LBD alkylated by 11BAEOPE(2) and 11BAPOPE(2), respectively. Mass spectrometry (MS) analyses of the fractions related to the single peak and to the major one of the two peaks showed signals which accurately matched the mass of electrophile-alkylated Cys(530)Lys(531) LBD tryptic peptide, whereas no signal compatible with an alkylated form of an LBD tryptic peptide was detected in the MS analysis of the minor peak-related fractions. MS/MS analysis of alkylated CysLys dipeptide revealed the presence of fragments that unambiguously designated the Cys S as the covalent attachment site of the electrophiles. We attempted to interpret the biochemical data by molecular modeling using various crystallographic structures of human LBD-ligand complexes. In agreement with the endocrine properties of electrophiles, labeling at Cys(530) could be accounted for by a LBD structure derived from LBD bound to 4-hydroxytamoxifen, a triphenylethylene antiestrogen. The common attachment to Cys(530) of estrogenic E(2) 17 alpha-derivatives [H. Mattras, S. Aliau, E. Demey, J. Poncet, J.L. Borgna, Mass spectrometry identification of covalent attachment sites of two related estrogenic ligands on human estrogen receptor alpha, J. Steroid Biochem. Mol. Biol. 98 (4-5), in press] and antiestrogenic E(2) 11 beta-derivatives suggests that the LBD portion encompassing this amino acid possesses a marked plasticity.

Requirements for reliable determination of binding affinity constants by kinetic approach.

The nonspecific binding (equilibrium coefficient kn) of ligand (L) and/or the incomplete recovery (alpha < 1) of the receptor-ligand (RL) complex in binding measurements, could hamper accurate determination of the association and dissociation rate constants of the R/L system. For the simplest model of R/L interaction, characterized by a bimolecular association process (rate constant k1) and a monomolecular dissociation process (rate constant k2), the consequences of kn and/or alpha neglect on k1 and k2 determination were investigated. Various situations that are especially relevant for k1 determination, were examined in which nonspecific binding was: (i) negligible relative to specific binding, or (ii) developed progressively or very rapidly in association kinetics. When only the initial kinetic phase was used, according to the situation (i.e. the nonspecific binding characteristics, and the fact that kn and/or alpha were or were not taken into account to correct the binding measurements), k1 could be accurately determined or generally slightly overestimated or slightly underestimated (in the two latter cases by factors involving mainly kn and/or alpha but not the R concentration or the R/L equilibrium association constant, K), whereas k2 should always be fairly well estimated. Consequently, for the simplest R/L systems, the k1/k2 ratio derived from such kinetic experiments should be much less susceptible to substantial underestimation than K derived from R saturation experiments [Borgna, J. Steroid Biochem. Mol. Biol. (2004)]. Kinetic experiments could also be more appropriate than R saturation experiments to detect cooperative--positive or negative--binding of L to R.

Requirements for reliable determination of binding affinity constants by saturation analysis approach.

Accurate calculation of the equilibrium association constant (K) and binding site concentration (N) related to a receptor (R)/ligand (L) interaction, via R saturation analysis, requires exact determination of the specifically bound L concentration (B(S)) and the unbound L concentration (U) at equilibrium. However, most binding determinations involve a procedure for separation of bound and unbound L. In such situations, it was previously shown that correct calculation of B(S) and U from binding data requires prior determination of alpha, i.e. the procedure parameter representing the proportion of equilibrium B(S) recovered after running the separation process, and of kn, i.e. the equilibrium nonspecific binding coefficient. For the simplest model of R/L interaction, the consequences of alpha neglect and/or kn neglect on determination of K and N, via R saturation analysis, are investigated. When alpha but not kn has been determined, B(S) can be accurately calculated, whereas U is overestimated by factor (kn + 1). Consequently the type (linear or hyperbolic) of theoretic curves obtained by usual representations (such as the Scatchard, the Lineweaver-Burk or the Michaelis-Menten plot) of the R/L binding is unchanged; these curves afford correct N and underestimation of K by factor (kn + 1). When alpha (alpha < 1) has not been determined B(S) and U are underestimated and overestimated, respectively. Then erroneous representations of the R/L binding result (e.g. instead of regular straight line segments, Scatchard plot and Lineweaver-Burk plot involve convex-upward and convex-downward hyperbola portions, respectively, suggestive of positive cooperativity of L binding), which leads to incorrect N and K. Errors in N and K would depend on (i) the binding (K, N and kn) and method (alpha) parameters and (ii) the expressions used to calculate approximate B(S) and U values. Simulations involving variable alpha, KN and kn values indicate that: (1) the magnitude of error in N determination (mainly involving moderate underestimation) directly depends on the alpha value; (2) the magnitude of K underestimation mainly depends on the KN value; it is moderate (usually < two-fold) with KN values < 1, but could become very high (e.g. > 100-fold), when KN > 10(2). In this case, the K underestimation is modulated by the alpha and kn values. Practical situations which afford high KN and thus might result in very marked underestimation of K are discussed. A single R dilution method is proposed to assess the validity of K determinations using the R saturation analysis approach.

Identification by MALDI-TOF mass spectrometry of 17 alpha-bromoacetamidopropylestradiol covalent attachment sites on estrogen receptor alpha.

Mass spectrometry was used to identify the sites of covalent attachment of [(14)C]-17alpha-bromoacetamidopropylestradiol ([(14)C]17BAPE(2), an estradiol agonist) to the ligand-binding domain (LBD) of mouse estrogen receptor alpha (ERalpha). A glutathione S-transferase (GST)-LBD chimera protein was overexpressed in Escherichia coli, using a vector encoding GST fused with a C-terminal portion of mouse ERalpha (Ser(313)-Ile(599)), via a sequence enclosing a thrombin cleavage site (located 14 amino acids ahead of Ser313). [(14)C]17BAPE(2) covalent labeling experiments were carried out on the GST-LBD chimera immobilized on glutathione-Sepharose. After thrombin cleavage of the chimeric LBD, two major [(14)C]17BAPE(2)-labeled species of 34 ( approximately 75%) and 30 kDa ( approximately 25%) were detected by SDS-PAGE and autoradiography. Their identity was assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS): two main signals were consistent with the mass of the full-length (Ser(313)-Ile(599)) and truncated LBD (Ser(313)-Ala(573)), both comprising the extra 14 N-terminal amino acids and covalently bound [(14)C]17BAPE(2) (via HBr elimination). A purified (14)C-labeled LBD preparation was trypsinized to identify the covalent attachment sites of 17BAPE(2). HPLC of tryptic fragments only revealed two discrete and practically equivalent radioactive fractions. MALDI-TOF MS analysis of these two fractions showed only two signals which exactly matched the molecular masses of the [(14)C]17BAPE(2)-alkylated Cys(534)Lys(535) and Cys(421)-Arg(438) peptides, respectively. Hydrolysis of the second (14)C-labeled fraction by Staphylococcus aureus V8 Glu-C endoproteinase generated signals typical of alkylated the Cys(421)-Glu(423) tripeptide. We concluded that Cys421 and Cys534 were equivalent alternative covalent attachment sites of 17BAPE(2) on the LBD. These biochemical data were interpreted using the crystallographic structures of estradiol-LBD and raloxifene- or 4-hydroxytamoxifen-LBD complexes. The covalent attachment to Cys421, Cys534, or both could be interpreted according to the starting structure. Various hypotheses based on the biochemical results and molecular modeling simulations are discussed, with the likely involvement of dynamic interconversion between multiple conformational states of the LBD-17BAPE(2) complex.

Differential interactions of estrogens and antiestrogens at the 17 beta-hydroxyl or counterpart hydroxyl with histidine 524 of the human estrogen receptor alpha.

We investigated the role of H524 of the human estrogen receptor alpha (ERalpha) for the binding of various estrogens [estradiol (E(2)), 3-deoxyestradiol (3-dE(2)), and 17beta-deoxyestradiol (17beta-dE(2))] and antiestrogens [4-hydroxytamoxifen (OHT), RU 39 411 (RU), and raloxifene (Ral)], which possess the 17beta-hydroxyl or counterpart hydroxyl (designated: 17beta/c-OH), with the exception of 17beta-dE(2) and OHT. The work involved a comparison of the binding affinities of these ligands for wild-type and H524 mutant ERs, modified or not with diethyl pyrocarbonate (DEPC), a selective histidine reagent. Alanine substitution of H524 did not significantly change the association affinity constant (relative to OHT) of 17beta-dE(2), whereas those of RU, Ral, E(2), and 3-dE(2) were decreased 3-fold, 14-fold, 24-fold, and 49-fold, respectively. Values of the two ligands available in radiolabeled form (E(2) and OHT) were correlated with the dissociation rate constants, which were increased 250-fold and 2-fold, respectively. The action of DEPC on wild-type ER led to a homogeneous ER population which still bound antiestrogens and 17beta-dE(2) with practically unchanged affinities (less than 4-fold decreases in relative affinity constants), while E(2) and 3-dE(2) displayed markedly decreased affinities (56-fold decrease for E(2)). Conversely, DEPC treatment of H524A mutant ER did not induce marked decreases in the relative affinities of any of the checked compounds (decreases wild-type ER) and very weakly protected H524A ER. Molecular modeling was tentatively used to interpret the biochemical results.