From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

Interaction of pigeon cytochrome c-(43–58) peptide analogs with either T cell antigen receptor or I-Ab molecule

We determined that a pigeon cytochrome c-derived peptide, p43–58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43–58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43–58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vβ usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR β chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR β chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.

Ab initio calculation of electronic coupling in the photosynthetic reaction center

We have carried out an ab initio electronic structure calculations of electron transfer couplings between chromophores in the bacterial photosynthetic reaction center. The couplings agree remarkably well with parameters obtained from recent quantum dynamical modeling of experimental data assuming an explicit intermediate mechanism. We also have computed couplings on the M-side of the reaction center and have found that the interaction of the primary donor to the M-side intermediate bacteriochlorophyll is quite small because of destructive interference of the two localized coupling matrix elements. This may explain the slow rate of electron transfer down the M-side of the reaction center.

Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-b]pyridine C8-deoxyguanosine adduct in duplex DNA

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with N-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-N-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is syn, and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.

High copy number I-Ab transgenes induce production of IgE through an interluekin 4-dependent mechanism.

To better understand the role of class II major histocompatibility complex molecules in both normal and autoimmune responses, we have produced a series of I-Ab transgenic mice. One of these transgenic constructs, designated NOD.PD, has the sequence of the NOD beta chain (Abeta(g7)) except at positions 56 and 57, where Pro-Asp replaces His-Ser. Several NOD.PD transgenic lines have been produced. One line of these mice carried a very high number of copies (>50) of the NOD.PD transgene. As has been described in other mice carrying high copy numbers of I-Ab transgenes, B-cell development was abnormal. The steady state numbers of mature B cells (IgM+/IgD(hi)) in the periphery were greatly reduced in transgenic mice compared to nontransgenic littermates. Surprisingly, rather than being accompanied by a generalized hypogammaglobulinemia, this B-cell deficiency was accompanied by elevated concentrations of IgG1 and IgE in the serum. Conversely, the levels of IgG2a were reduced in transgenic mice compared to nontransgenic littermates. Because this isotype pattern was characteristic of interleukin (IL)-4-induced class-switching, we then investigated the role of IL-4 in causing the observed phenotype. We crossed the high copy number transgenic mice with an IL-4-deficient strain of mice. As expected, the elevated levels of IgE in high copy number transgenic mice were eliminated when the IL-4 gene was inactivated. However, the reduction in the number of B cells was not ameliorated. These data indicate that the primary defect caused by the transgene was to reduce the number of B cells in these mice. This reduction was accompanied by a secondary increase in IL-4 production, which drove the remaining B cells toward the production of IgGl and IgE.