The extraordinary specificity of xanthine phosphoribosyltransferase from Bacillus subtilis elucidated by reaction kinetics, ligand binding, and crystallography

Xanthine phosphoribosyltransferase (XPRTase) from Bacillus subtilis is a representative of the highly xanthine specific XPRTases found in Gram-positive bacteria. These XPRTases constitute a distinct subclass of 6-oxopurine PRTases, which deviate strongly from the major class of H(X)GPRTases with respect to sequence, PRPP binding motif, and oligomeric structure. They are more related with the PurR repressor of Gram-positive bacteria, the adenine PRTase, and orotate PRTase. The catalytic function and high specificity for xanthine of B. subtilis XPRTase were investigated by ligand binding studies and reaction kinetics as a function of pH with xanthine, hypoxanthine, and guanine as substrates. The crystal structure of the dimeric XPRTase-GMP complex was determined to 2.05 A resolution. In a sequential reaction mechanism XPRTase binds first PRPP, stabilizing its active dimeric form, and subsequently xanthine. The XPRTase is able also to react with guanine and hypoxanthine albeit at much lower (10(-)(4)-fold) catalytic efficiency. Different pK(a) values for the bases and variations in their electrostatic potential can account for these catalytic differences. The unique base specificity of XPRTase has been related to a few key residues in the active site. Asn27 can in different orientations form hydrogen bonds to an amino group or an oxo group at the 2-position of the purine base, and Lys156 is positioned to make a hydrogen bond with N7. This and the absence of a catalytic carboxylate group near the N7-position require the purine base to dissociate a proton spontaneously in order to undergo catalysis.     

Functional consequences of agonist-mediated state transitions in the cholinergic receptor. Studies in cultured muscle cells.

Parameters associated with activation and desensitization of the nicotinic receptor in the BC3H-1 muscle cell line have been compared with the state transitions that result upon combination with agonist. 125I-labeled cobra alpha-toxin is found to bind to an apparent single class of surface nicotinic receptors on the cells in situ with a rate constant of 1.15 x 10(5) M-1 s-1. The competition between cholinergic ligands and alpha-toxin reveals that agonists, but not classical antagonists, will promote a slow conversion to a receptor state where the affinity for agonists is enhanced. Moreover, agonists such as carbamylcholine elicit a permeability increase to 22Na+ ions that slowly decrements at a rate and to an extent closely paralleled by the conversion of the receptor to the high affinity state. Upon removal of the agonist, both the affinity increase and the diminished permeability change are completely reversible and again exhibit similar kinetics for their return to the original state. A comparison of the capacity of full agonists to compete with alpha-toxin binding and elicit a permeability change suggests that in the absence of agonist, receptor predominates in a low affinity activatable state. Binding of agonists to the low affinity state exhibits little if any cooperativity (n = 0.97 to 1.31), while the corresponding permeability change appears more cooperative (n = 1.31 to 1.52). By contrast, when receptors have been previously equilibrated with agonists, occupation of the receptor occurs over a 3- to 5-fold lower concentration range. Binding following equilibration closely correlates with a concomitant decrease in activatable receptor resulting from equivalent exposure to agonist. Furthermore, under equilibrium conditions, the binding of full agonists is typified by a moderate degree of homotropic cooperativity (1.25 to 1.44), enabling the receptor to desensitize over a narrow range of agonist concentration. Simultaneous measurement of occupation and activation parameters has enabled us to compare a state function for desensitization which is generated from binding parameters with the reduction in permeability seen in the desensitization process. A scheme describing the association of agonist with two functionally distinct receptor states is developed to account for the cooperative relationship between agonist binding and desensitization of the receptor.     

Spontaneous T-cell responses against peptides derived from the Taxol resistance–associated gene-3 (TRAG-3) protein in cancer patients

Expression of the cancer-testis antigen Taxol resistance–associated gene-3 (TRAG-3) protein is associated with acquired paclitaxel (Taxol) resistance, and is expressed in various cancer types; e.g., breast cancer, leukemia, and melanoma. Thus, TRAG-3 represents an attractive target for immunotherapy of cancer. To identify HLA-A*02.01–restricted epitopes from TRAG-3, we screened cancer patients for spontaneous cytotoxic T-cell responses against TRAG-3–derived peptides. The TRAG-3 protein sequence was screened for 9mer and 10mer peptides possessing HLA-A*02.01–binding motifs. Of 12 potential binders, 9 peptides were indeed capable of binding to the HLA-A*02.01 molecule, with binding affinities ranging from strong to weak binders. Subsequently, lymphocytes from cancer patients (9 breast cancer patients, 12 melanoma patients, and 13 patients with hematopoietic malignancies) were analyzed for spontaneous reactivity against the panel of peptides by ELISpot assay. Spontaneous immune responses were detected against 8 epitope candidates in 7 of 9 breast cancer patients, 7 of 12 melanoma patients, and 5 of 13 patients with hematopoietic malignancies. In several cases, TRAG-3–specific CTL responses were scattered over several epitopes. Hence, no immunodominance of any single peptide was observed. Furthermore, single-peptide responses were detected in 2 of 12 healthy HLA-A2⁺ donors, but no responses were detectable in 9 HLA-A2^– healthy donors or 4 HLA-A2^– melanoma patients. The identified HLA-A*02.01–restricted TRAG-3–derived epitopes are targets for spontaneous immune responses in breast cancer, hematopoietic cancer, and melanoma patients. Hence, these epitopes represent potential target structures for future therapeutic vaccinations against cancer, possibly appropriate for strategies that combine vaccination and chemotherapy; i.e., paclitaxel treatment.     

Discovery of a class of calcium sensing receptor positive allosteric modulators; 1-(benzothiazol-2-yl)-1-phenylethanols

1-(Benzothiazol-2-yl)-1-(4-chlorophenyl)ethanol (1) was identified as a positive allosteric modulator (PAM) of the CaSR in a functional cell-based assay. This compound belongs to a class of compounds that is structurally distinct from other known positive allosteric modulators, for example, the phenylalkylamines cinacalcet, a modified analog (13) potently suppressed parathyroid hormone (PTH) release in rats, consistent with its profile as a PAM of CaSRs.     

Ligand-induced conformational change in the alpha7 nicotinic receptor ligand binding domain

Molecular dynamics simulations of a homology model of the ligand binding domain of the alpha7 nicotinic receptor are conducted with a range of bound ligands to induce different conformational states. Four simulations of 15 ns each are run with no ligand, antagonist d-tubocurarine (dTC), agonist acetylcholine (ACh), and agonist ACh with potentiator Ca(2+), to give insight into the conformations of the active and inactive states of the receptor and suggest the mechanism for conformational change. The main structural factor distinguishing the active and inactive states is that a more open, symmetric arrangement of the five subunits arises for the two agonist simulations, whereas a more closed and asymmetric arrangement results for the apo and dTC cases. Most of the difference arises in the lower portion of the ligand binding domain near its connection to the adjacent transmembrane domain. The transfer of the more open state to the transmembrane domain could then promote ion flow through the channel. Variation in how subunits pack together with no ligand bound appears to give rise to asymmetry in the apo case. The presence of dTC expands the receptor but induces rotations in alternate directions in adjacent subunits that lead to an asymmetric arrangement as in the apo case. Ca(2+) appears to promote a slightly greater expansion in the subunits than ACh alone by stabilizing the C-loop and ACh positions. Although the simulations are unlikely to be long enough to view the full conformational changes between open and closed states, a collection of different motions at a range of length scales are observed that are likely to participate in the conformational change.     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.     

The structure and specificity of Escherichia coli maltose acetyltransferase give new insight into the LacA family of acyltransferases

The crystallographic three-dimensional structure of the Escherichia coli maa gene product, previously identified as a maltose O-acetyltransferase (MAT) [Brand, B., and Boos, W. (1991) J. Biol. Chem. 266, 14113-14118] has been determined to 2.15 A resolution by the single anomalous dispersion method using data from a crystal cocrystallized with trimethyllead acetate. It is shown here that MAT acetylates glucose exclusively at the C6 position and maltose at the C6 position of the nonreducing end glucosyl moiety. Furthermore, MAT shows higher affinity toward artificial substrates containing an alkyl or hydrophobic chain as well as a glucosyl unit. The presence of a long hydrophobic patch near the acceptor site provides the structural explanation for this preference. The three-dimensional structure reveals the expected trimeric left-handed parallel beta-helix structure found in all other known hexapeptide repeat enzymes. In particular, the structure shows similarities both overall and at the putative active site to the recently determined structure of galactoside acetyltransferase (GAT), the lacA gene product [Wang, X.-G., Olsen, L. R., and Roderick, S. L. (2002) Structure 10, 581-588]. The structure, together with the new biochemical data, suggests that GAT and MAT are more closely related than previously thought and might have similar cellular functions. However, while GAT is specific for acetylation of galactosyl units, MAT is specific for glucosyl units and is able to acetylate maltooligosaccharides, an important property for biotechnological applications. Structural differences at the acceptor site reflect the differences in substrate specificity.     

Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.     

Asymmetric structural motions of the homomeric alpha7 nicotinic receptor ligand binding domain revealed by molecular dynamics simulation

A homology model of the ligand binding domain of the alpha7 nicotinic receptor is constructed based on the acetylcholine-binding protein crystal structure. This structure is refined in a 10 ns molecular dynamics simulation. The modeled structure proves fairly resilient, with no significant changes at the secondary or tertiary structural levels. The hypothesis that the acetylcholine-binding protein template is in the activated or desensitized state, and the absence of a bound agonist in the simulation suggests that the structure may also be relaxing from this state to the activatable state. Candidate motions that take place involve not only the side chains of residues lining the binding sites, but also the subunit positions that determine the overall shape of the receptor. In particular, two nonadjacent subunits move outward, whereas their partners counterclockwise to them move inward, leading to a marginally wider interface between themselves and an overall asymmetric structure. This in turn affects the binding sites, producing two that are more open and characterized by distinct side-chain conformations of W54 and L118, although motions of the side chains of all residues in every binding site still contribute to a reduction in binding site size, especially the outward motion of W148, which hinders acetylcholine binding. The Cys loop at the membrane interface also displays some flexibility. Although the short simulation timescale is unlikely to sample adequately all the conformational states, the pattern of observed motions suggests how ligand binding may correlate with larger-scale subunit motions that would connect with the transmembrane region that controls the passage of ions. Furthermore, the shape of the asymmetry with binding sites of differing affinity for acetylcholine, characteristic of other nicotinic receptors, may be a natural property of the relaxed, activatable state of alpha7.