General Binding Mode Research Articles

Crystal structure of the C-terminal SH2 domain of the p85α regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 Å resolution of the C-terminal SH2 domain (C-SH2) of the P85α regulatory subunit of phosphoinositide 3-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (αA2) and Arg36 (βB5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal.

Coordination chemistry of glutathione.

The metal ion coordination abilities of reduced and oxidized glutathione are reviewed. Reduced glutathione (GSH) is a very versatile ligand, forming stable complexes with both hard and soft metal ions. Several general binding modes of GSH are described. Soft metal ions coordinate exclusively or primarily through thiol sulfur. Hard ones prefer the amino acid-like moiety of the glutamic acid residue. Several transition metal ions can additionally coordinate to the peptide nitrogen of the gamma-Glu-Cys bond. Oxidized glutathione lacks the thiol function. Nevertheless, it proves to be a surprisingly efficient ligand for a range of metal ions, coordinating them primarily through the donors of the glutamic acid residue.

Human Phenylalanyl-tRNA Synthetase: Cloning, Characterization of the Deduced Amino Acid Sequences in Terms of the Structural Domains and Coordinately Regulated Expression of the α and β Subunits in Chronic Myeloid Leukemia Cells

Unlike the catalytic α-subunit, the β-subunit of heterodimeric (αβ)2phenylalanyl-tRNA synthetase (PheRS) has no invariant functional amino acids directly involved in the aminoacylation process as it is evident from the crystal structure of theT. thermophilusenzyme complexed with tRNAPhe. Having no catalytic function, the prokaryotic β-subunit comprises OB-, RNP-, SH3-, and DNA-binding-like domains involved in a variety of biological functions in other proteins. It was shown that the mRNA of the human α-subunit overexpressed in the tumorigenic versus the nontumorigenic variant of the same acute-phase chronic myeloid leukemia cell line (CML). We cloned, sequenced, and expressed human PheRS. The layout of the human sequence indicates that the general tRNA binding mode and anticodon recognition differ between prokaryotes and eukaryotes for the phenylalanine system. Northern blot hybridization analysis from malignant and normal human tissues enabled us to assess the relative expression levels of the α- and β-subunits independently, in view of the additional cellular role proposed for the β-subunit in tumorigenic events. The levels of mRNA corresponding to the α- and β-subunits were remarkably similar in all cell types and tissues examined, thus indicating the implication of the entire (αβ)2heterodimer in tumorigenic events.

Catechol(amine)s as probes of lactoperoxidase catalytic site structure: spectroscopic and modeling studies.

Binding affinities to lactoperoxidase (LPO) of a homologous series of substituted catechol(amine)s [such as catechol, 4-methylcatechol, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid; dopamine, noradrenaline, adrenaline; L-3,4-dihydroxyphenylalanine] were studied by UV-visible spectroscopy and docking simulations. Dissociation constant (Kd) values were calculated by direct fitting of the experimental data and fall in a range of 3-95 mM. Thermodynamic parameters are comparable with those reported for the interaction of LPO with p-substituted phenols, suggesting a similar general mode of binding. Furthermore, the relative contributions to binding energy, described by the unimolecular constant Ku, show that interaction between protein and ligands originates from a relatively large number of groups. Docking and molecular dynamics simulations, in agreement with experimental evidence, predict that the substrate is localized into the access channel in the vicinity of heme distal pocket. This channel is characterized by a hydrophobic patch (six Phe residues) and by a charged contribution (two Glu and one His residues). All of the substrates, except caffeic acid, may approach the protein active site. Positively charged Arg372 acts as a gate above the heme distal pocket and seems to address substrate orientation in relation to the side-chain terminal group.

Fourier transform infrared linked analysis of conformational changes in annexin V upon membrane binding.

Annexins are ubiquitous cellular proteins of unknown primary function that bind to anionic phospholipid membranes in a calcium-dependent manner. Correlative studies involving X-ray crystallography and electron microscopy suggest that annexins undergo a structural change upon binding to supported lipid monolayer membranes. In this investigation, novel spectroscopic and analytical techniques have been applied to verify and characterize this change. Soluble annexin V was examined with ordinary transmission infrared spectroscopy, while membrane-bound annexin V was examined with both transmission and internal reflection infrared spectroscopy. Spectra were processed by linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. This approach is shown to enhance the sensitivity and accuracy of the bandfitting procedure. Our results are consistent with the general mode of membrane binding inferred from electron microscopy studies, and they provide independent support for the conclusion that annexin V undergoes a conformational change upon binding to lipid monolayer membranes. Most likely, this change involves the formation of new beta structure in which interstrand hydrogen bonds orient parallel to the membrane surface.

Binding of the anticancer drug ZD1694 to E. coli thymidylate synthase: assessing specificity and affinity

Background Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by 5,10-methylenetetrahydrofolate (CH 2H 4folate) to form deoxythymidine monophosphate (dTMP) and dihydrofolate (H 2folate). The essential role of TS in the cell life cycle makes it an attractive target for the development of substrate and cofactor-based inhibitors that may find efficacy as anticancer and antiproliferative drugs. Antifolates that compete specifically with the binding of CH 2H 4 folate include the cofactor analog CB3717 (10-propargyl-5,8-dideazafolate). However, the development of potent cofactor analog inhibitors of TS, such as CB3717, as drugs has been slowed by their toxicity, which often becomes apparent as hepatic and renal toxicity mediated by the specific chemistry of the compound. Attempts to abolish toxicity in human patients while preserving potency against the target enzyme, have led to the development of ZD1694, which has already shown significant activity against colorectal tumours. Results The three dimensional crystallographic structure of ZD1694 in complex with dUMP and Escherichia coli TS has been determined to a resolution of 2.2 å . This was used to evaluate the specific structural determinants of ZD1694 potency and to correlate structure/activity relationships between it and the closely related ligand, CB3717. ZD1694 binds to TS in the same manner as CB3717 and H 2 folate, but a methyl group on its quinazoline ring, its thiophene ring and the methyl group at N10 are compensated for by plastic accommodation of the enzyme active site coupled with specific rearrangement in the solvent structure. A specific hydrogen bond between the protein and the inhibitor CB3717 is absent in the case of ZD1694 whose monoglutamate tail is reoriented and more well ordered. Conclusions The binding mode of ZD1694 to thymidylate synthase has been determined at atomic resolution. ZD1694 forms a ternary complex with dUMP and participates in the multi-step TS reaction through the covalent bond formation between dUMP and Cys146 thereby competing with CH 2H 4 folate at the active site. Analysis of this inhibitor ternary complex structure and comparison with that of CB3717 reveals that the enzyme accommodates the differences between the two inhibitors with small shifts in the positions of key active site residues and by repositioning an active site water molecule, thereby preserving a general binding mode of these inhibitors.

Thermodynamics of the interaction of inhibitors with the binding site of recombinant human renin

The independent subsite model is widely used for the design of peptide inhibitors of enzymes with extended active sites. This model assumes that the subsites are independent of each other and that the free energies of binding contributed by the several subsites are additive. We questioned the strict application of this model for structure-activity studies, since one can, a priori, conceive of likely deviations from this model. Accordingly, we tested the independent subsite model by measuring the thermodynamic binding parameters of a series of peptide inhibitors of human renin. This enzyme-inhibitor system was chosen as a model by virtue of the high degree of specificity of renin for its natural substrate, angiotensinogen, and the availability of a large number of structurally similar peptide inhibitors. Although we found the general mode of binding of these renin inhibitors to be primarily hydrophobic, serious deviations from additivity and independent subsite model constraints were observed. We conclude that an important determinant of binding is most probably the conformation assumed by the peptide inhibitor in solution. Thus, we suggest that caution be exercised in using affinity constants to assess the interactions of peptide inhibitors with human renin and possibly with other enzymes having extended binding sites. Furthermore, the thermodynamic parameters of a class of compounds provide more information as to the mode of binding of ligands to their respective receptors than do dissociation constants.

Spin-labeled Sulfonyl Fluorides as Active Site Probes of Protease Structure: I. COMPARISON OF THE ACTIVE SITE ENVIRONMENTS IN α-CHYMOTRYPSIN AND TRYPSIN

Abstract α-Chymotrypsin and trypsin were spin-labeled with 14 ortho-, meta-, and para-substituted phenylsulfonyl fluorides. These analogues of tosylfluoride were substituted with either pyrrolidinyl, pyrrolinyl, or piperidinyl nitroxide moieties. A complete description of their synthesis and the enzyme inhibition studies is presented in the following paper (Wong, S. S., Quiggle, K., Triplett, C., and Berliner, L. J. (1974) J. Biol. Chem. 249, 1678–1682). The studies were designed to test for dynamic conformational homologies in these closely related proteins. However, the spectral results at pH 3.5 clearly showed that the active site conformations of trypsin and α-chymotrypsin were different. Model-building studies indicated which spin labels could bind isomorphously to tosyl-α-chymotrypsin. Several of those labels which were shown to be restricted structurally from binding in the chymotrypsin aromatic pocket were found upon exposure to saturated indole to give electron spin resonance spectra closely resembling those for trypsin. Those inhibitors which could theoretically bind in the pocket were affected least or not at all. With trypsin, on the other hand, 1 mm benzamidine produced no spectral shifts. A suggested model for α-chymotrypsin based on steric arguments provides for three general binding modes: (a) one isomorphous to tosylchymotrypsin, (b) one reflecting partial binding at the tosyl hole, and (c) one in a general region of the active site. Indole can displace the partially bound State b toward the general binding Mode c. This general Mode c is probably common to trypsin as well.