Localization and structure of the muscarinic receptor ligand binding site.

A conserved aspartic acid residue in transmembrane helix 3 of the muscarinic acetylcholine receptors is important in binding the headgroup of muscarinic ligands. This acidic amino acid probably points into a relatively hydrophilic cavity whose walls are formed by the amphipathic transmembrane helices of the receptor. Amino acid side chains within this cavity contribute to ligand binding.

An apparently acentric marker chromosome originating from 9p with a functional centromere without detectable alpha and beta satellite sequences.

Recently, we studied a patient with minor abnormalities and an apparently acentric marker chromosome who carried a deleted chromosome 9 and a marker chromosome in addition to a normal chromosome 9. The marker was stable in mitosis but lacked a primary constriction. The origin of the marker was established by fluorescent in situ hybridization (FISH) using a chromosome 9 painting probe. Hybridization of unique sequence 9p probes localized the breakpoint proximal to 9p13. Additional FISH studies with all-human centromere alpha satellite, chromosome 9 classical satellite, and beta satellite probes showed no visible evidence of these sequences on the marker [Curtis et al.: Am J Hum Genet 57:A111, 1995]. Studies using centromere proteins (CENP-B, CENP-C, and CENP-E) were performed and demonstrated the presence of centromere proteins. These studies and the patient's clinical findings are reported here.

The function of a highly-conserved arginine residue in activation of the muscarinic M1 receptor.

Arg123 in the rat muscarinic M1 receptor is part of the highly conserved triad Asp-Arg-Tyr found at the junction of transmembrane helix 3 with the second intracellular loop. Mutation of Arg123 to Lys, Ala, Leu, Glu and Gln had no effect on levels of receptor expression in COS-7 cells, or on affinities for the antagonist N-methylscopolamine. Acetylcholine stimulation of the Lys123 receptor evoked the same maximum phosphoinositide response as the wild type, although the potency was reduced six-fold, but mutation to other residues strongly disrupted receptor function. Mutation of Arg123 always decreased the ratio of the high affinity to the low affinity agonist binding constant, but the absolute effect on the latter varied from a 4-fold increase for the Lys123 to a small decrease for the Leu123 mutation. These results suggest that a positive charge at position 123 is central to the activation of G-proteins by the muscarinic M1 receptor.

The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis.

Mutation of the Asp in transmembrane domain three of the muscarinic receptors to Asn (M1) or Glu (M1 and M2) strongly reduced the high-affinity component of agonist binding, and the M1 phosphoinositide response. Formation of the acetylcholine-receptor binary complex was also strongly inhibited. In contrast, binary complex formation by other agonists, as well as the antagonist (-)-N-methylscopolamine, was less affected. Ionic bonding between the carboxylate oxyanion and the positively-charged headgroup probably anchors acetylcholine when it is bound in its active conformation, but alternative, non-productive, binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands.

The conformational switch in 7-transmembrane receptors: the muscarinic receptor paradigm.

The rhodopsin-like superfamily of 7-transmembrane receptors is the largest class of signalling molecules in the mammalian genome. Recently, a combination of mutagenesis, biophysical and modelling studies have suggested a credible model for the alpha-carbon backbone in the transmembrane region of the 7-transmembrane receptors, and have started to reveal the structural basis of the conformational switch from the inactive to the active state. A key feature may be the replacement of a network of radial constraints, centred on transmembrane helix three, which stabilise the inactive ground state of the receptor by a new set of axial interactions which help to stabilise the activated state. Transmembrane helix three may act as a rotary switch in the activation mechanism.

The role of charge interactions in muscarinic agonist binding, and receptor-response coupling.

Site-directed mutagenesis has been used to evaluate the roles of the key aspartate and arginine residues in transmembrane domain three of the muscarinic receptors. The results suggest that the formation of an ionic bond between the Asp carboxylate group and the onium headgroup is essential to anchor acetylcholine in its active, bound conformation in both binary agonist-receptor and ternary agonist-receptor-G-protein complexes, but that secondary, non-productive binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands. The positive charge of the arginyl side-chain is central to the recognition, and subsequent activation of G-proteins by the agonist-M1 mAChR complex.

The conformational switch in muscarinic acetylcholine receptors.

The recently-determined structure of rhodopsin has provided a suitable basis for modeling the three-dimensional structure of the M1 muscarinic acetylcholine receptor. Using this as a framework for interpreting mutagenesis studies, we have been able to suggest most of the contacts which the receptor makes with acetylcholine and many of the intramolecular contacts which are important for the ground-state structure of the receptor. It is possible to outline a mechanism of G-protein interaction.

Genetic linkage between C-bands and storage protein genes in chromosome 1B of tetraploid wheat.

Genetic mapping of polymorphic C-bands allows direct comparisons between genetic and physical maps. Eleven C-bands and two seed storage protein genes on chromosome 1B, polymorphic between Langdon durum and four accessions of T. dicoccoides, were used to study the distribution of recombination along the entire length of the chromosome. Recombination in the short arm was almost completely restricted to the satellite, two-thirds of the arm's length from the centromere; the Gli-B1 gene was found to be tightly linked to the telomeric C-band. In the long arm, the distal 51.4% of the arm accounted for 88% of recombination; the proximal half of the arm accounted for the remaining 12%. While the amount of crossing-over differed significantly between the four T. dicoccoides 1B chromosomes, there were no significant differences in the relative distributions of crossing-over along the chromosome. Consequently, the genetic maps obtained from the four individual T. dicoccoides chromosomes were combined to yield a consensus map of 14 markers (including the centromere) for the chromosome.

Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions.

The control of expression of the Bacillus subtilis spoIIA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIIA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglII, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by beta-galactosidase activity. The results indicate that spoIIA was under positive control by a moiety present at about five copies per chromosome. Spore formation was not affected by amplification, so spoIIA-lacZ reiteration did not sequester a molecule required elsewhere for sporulation.

Physical distribution of recombination in B-genome chromosomes of tetraploid wheat.

Several studies have indicated a noncorrespondence between genetic and physical distances in wheat chromosomes. To study the physical distribution of recombination, polymorphism for C-banding patterns was used to monitor recombination in 67 segments in 11 B-genome chromosome arms of Triticum turgidum. Recombination was absent in proximal regions of all chromosome arms; its frequency increased exponentially with distance from the centromere. A significant difference was observed between the distribution of recombination in physically short and physically long arms. In physically short arms, recombination was almost exclusively concentrated in distal segments and only those regions were represented in their genetic maps. In physically long arms, while a majority of the genetic distance was again based upon recombination in distal chromosome segments, some interstitial recombination was observed. Consequently, these regions also contributed to the genetic maps. Such a pattern of recombination, skewed toward terminal segments of chromosomes, is probably a result of telomeric pairing initiation and strong positive chiasma interference. Interference averaged 0.81 in 35 pairs of adjacent segments and 0.57 across the entire recombining portions of chromosome arms. The total genetic map lengths of the arms corresponded closely to those expected on the basis of their metaphase-I chiasma frequencies. As a consequence of this uneven distribution of recombination there can be a 153-fold difference (or more) in the number of DNA base pairs per unit (centiMorgan) of genetic length.

The biosynthesis and linkage of teichuronic acid to peptidoglycan in Bacillus licheniformis.

Membrane and wall-membrane preparations of Bacillus licheniformis 94 will, if supplied with the appropriate precursors, synthesize teichuronic acid and link it to peptidoglycan although teichuronic acid is absent from walls of this organism. B. licheniformis 94 lacks phosphoglucomutase activity and therefore cannot synthesize the precursor UDPglucuronic acid. The initial reaction of teichuronic acid biosynthesis is catalysed by a translocase and results in the formation of polyprenyl-diphospho-N-acetylgalactosamine and the release of UMP. This reaction is not inhibited by tunicamycin. The disaccharide repeating unit of the polymer is then formed by the transfer of glucuronic acid from UDPglucuronic acid with the release of UDP. Polymerization of the repeating units occurs by incorporation of new units at the reducing terminus of the growing teichuronic acid chain and the release of polyprenyl diphosphate. The subsequent dephosphorylation of the lipid diphosphate for reuse in the biosynthesis cycle is inhibited by bacitracin. Linkage to peptidoglycan occurs by the formation of a phosphodiester bond between the reducing N-acetylgalactosamine terminus of the teichuronic acid chain and a 6-hydroxyl group of a muramic acid residue in the glycan of peptidoglycan. Wall-membrane preparations synthesizing teichuronic acid, poly(glycerol phosphate) teichoic acid and peptidoglycan link the teichuronic and teichoic acids to different glycan chains.

Dye sensitivity correlated with envelope protein changes in dye (sfrA) mutants of Escherichia coli K12 defective in the expression of the sex factor F.

Mutations of the dye gene on the E. coli chromosome result in sensitivity to the dye toluidine blue and, in male cells, cause loss of F pili, producing sterility in conjugation. Compared with its dye+ parent, a strain deleted for dye (delta dye) showed an altered sensitivity to a wide range of dyes and antibiotics which affect different intracellular processes, and hence it appeared likely that the barrier properties of the cell envelope were impaired. Unlike mutants known to be defective in LPS structure, there appeared to be no correlation between the hydrophobicity of the compounds and the sensitivity of the delta dye strain. Moreover there was no difference between dye+ and delta dye strains in their sensitivity to LPS-specific phages, and chemical and GLC analysis of LPS components revealed no difference between the two strains. Examination of outer and inner membrane proteins from isogenic strains having the delta dye deletion and the dye+ gene cloned into the plasmid pACYC184, with or without insertional inactivation of dye by the transposon gamma delta, was performed by SDS-PAGE. This revealed a number of differences in the profile of proteins from both inner and outer membranes, correlated with mutation in the dye gene. The dye gene appears to be identical to the sfrA gene, which has been shown to be required for efficient transcription of the sex factor F. It is therefore proposed that the dye (sfrA) gene product may also control the expression of chromosomal genes coding for envelope proteins.

Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis.

Plasmids carrying different portions of the polycistronic spoIIA locus, and unable to replicate autonomously in Bacillus subtilis, were able to transform a Spo+ B. subtilis strain, BR151, for the plasmid-determined chloramphenicol resistance by Campbell-like insertion into the region of homology on the chromosome. Two such plasmids, pPP35 and pPP36, yielded Spo- transformants, indicating that the cloned regions of these plasmids were entirely within the chromosomal spoIIA transcriptional unit. The cloned regions overlapped the end of a known spoIIA cistron, so that the transcriptional unit was larger than this cistron, and was polycistronic. This is the first demonstration of such a polycistronic sporulation transcriptional unit. The DNA sequence of the region has now been determined (given in an accompanying paper) and suggests a transcriptional unit with three open reading frames. Two other plasmids yielded Spo+ transformants of BR151, and these define the outer limits of the transcriptional unit. The adjacent sporulation locus identified by the spoV A89 mutation was not part of the same transcriptional unit.

Alanine-scanning mutagenesis of transmembrane domain 6 of the M(1) muscarinic acetylcholine receptor suggests that Tyr381 plays key roles in receptor function.

Transmembrane domain 6 of the muscarinic acetylcholine (ACh) receptors is important in ligand binding and in the conformational transitions of the receptor but the roles of individual residues are poorly understood. We have carried out a systematic alanine-scanning mutagenesis study on residues Tyr381 to Val387 within the binding domain of the M(1) muscarinic ACh receptor. The seven mutations were then analyzed to define the effects on receptor expression, agonist and antagonist binding, and signaling efficacy. Tyr381Ala produced a 40-fold reduction in ACh affinity and a 50-fold reduction in ACh-signaling efficacy. Leu386Ala had similar but smaller effects. Asn382Ala caused the largest inhibition of antagonist binding. The roles of the hydroxyl group and benzene ring of Tyr381 were probed further by comparative analysis of the Tyr381Phe and Tyr381Ala mutants using three series of ligands: ACh analogs, azanorbornane- and quinuclidine-based ligands, and atropine analogs. These data suggested that the hydroxyl group of Tyr381 is primarily involved in forming hydrogen bond interactions with the oxygen atoms present in the side chain of ACh. We propose that this interaction is established in the ground state and preserved in the activated state of the receptor. In contrast, the Tyr381 benzene ring may form a cation-pi interaction with the positively charged head group of ACh that contributes to the activated state of the receptor but not the ground state. However, the hydroxyl group and benzene ring of Tyr381 both participate in interactions with azanorbornane- and quinuclidine-based ligands and atropine analogs in the ground state as well as the activated state of the receptor.

The role of the aspartate-arginine-tyrosine triad in the m1 muscarinic receptor: mutations of aspartate 122 and tyrosine 124 decrease receptor expression but do not abolish signaling.

An Asp-Arg-Tyr triad occurs in a majority of rhodopsin-like G protein-coupled receptors. The fully conserved Arg is critical for G protein activation, but the function of the flanking residues is not well understood. We expressed in COS-7 cells m1 muscarinic receptors that were mutated at Asp122 and Tyr124. Most mutations at either position strongly attenuated or prevented the expression of binding sites for the antagonist [3H]N-methylscopolamine. However, sites that were expressed displayed unaltered affinity for the antagonist. Receptor protein, visualized with a carboxyl-terminally directed antibody, was reduced but never completely abolished. The effects of these mutations were partially reversed by the deletion of 129 amino acids from the third intracellular loop of the receptor. In several cases, comparison of immunocytochemistry with binding measurements suggested the presence of substantial amounts of inactive, presumably misfolded, receptor protein. Some of the variants that bound [3H]N-methylscopolamine underwent small changes in their affinities for acetylcholine. All retained nearly normal abilities to mediate an acetylcholine-induced phosphoinositide response. We propose that Asp122 and Tyr124 make intramolecular contacts whose integrity is important for efficient receptor folding but that they do not participate directly in signaling. The role of these residues is completely distinct from that of Arg123, whose mutation abolishes signaling without diminishing receptor expression.

Purification and characterization of two phage PBSX-induced lytic enzymes of Bacillus subtilis 168: an N-acetylmuramoyl-L-alanine amidase and an N-acetylmuramidase.

After heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-L-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.

Scanning mutagenesis identifies amino acid side chains in transmembrane domain 5 of the M(1) muscarinic receptor that participate in binding the acetyl methyl group of acetylcholine.

The exofacial part of transmembrane domain 5 (TMD 5) of the cationic amine-binding subclass of 7-transmembrane receptors is thought to be important in binding the side chain of the agonist. Residues Ile-188 through Ala-196 in TMD 5 of the M(1) muscarinic acetylcholine receptor (mAChR) have been studied by Cys- and Ala-scanning mutagenesis. The results are consistent with a helical conformation for this sequence. The positively charged sulfhydryl reagent N-trimethyl-2-aminoethyl methanethiosulfonate reacted selectively with Phe-190 --> Cys, Thr-192 --> Cys, and Ala-193 --> Cys, indicating that the face of TMD 5 accessible from the binding site crevice is consistent with a recent model by Baldwin and colleagues of the transmembrane domain of the 7-transmembrane receptors. In contrast, the acetylcholine derivative bromoacetylcholine reacted selectively with Thr-192 --> Cys, which forms the focus of a group of amino acids (Ile-188, Thr-189, Thr-192, Ala-196) whose mutation decreased the binding affinity of the transmitter ACh itself. The center of this patch of residues is offset to one side of the binding pocket, suggesting that a rotation of TMD 5, relative to that implied by the Baldwin model, may be necessary to optimize the anchoring of acetylcholine within the binding site of the M(1) mAChR. An induced rotation of TMD 5 could contribute to the formation of the activated state of the receptor.

Acetylcholine mustard labels the binding site aspartate in muscarinic acetylcholine receptors.

Acetylcholine mustard (AChM) is an analogue of acetylcholine (ACh) in which the onium headgroup is replaced by a chemically reactive aziridinium moiety. AChM aziridinium has agonist activity, but, having bound, reacts with and blocks the muscarinic receptor (mAChR) binding site. Purified mAChRs from rat forebrain have been specifically labeled with [3H]AChM. The linkage formed is cleaved by hydroxylamine, is found within cyanogen bromide (CNBr) peptides with molecular masses of approximately 2.4 and 3.9 kDa, and is close to a disulfide-bonded cysteine. Edman degradation reveals a site of label attachment 26 residues C-terminal to a CNBr cleavage site. As in the case of the alkylating antagonist analogue [3H]propylbenzilylcholine mustard, these findings indicate that a conserved aspartic acid residue in transmembrane helix 3 of the mAChRs, corresponding to Asp-105 (m1 sequence), is the site of label attachment.