Nuclear magnetic resonance (NMR) analysis of ligand receptor interactions: the cholinergic system--a model.

Elucidation of the molecular mechanisms that govern ligand-receptor recognition is essential to the rational design of specific pharmacological reagents. Whereas often the receptor and its binding site are the target of investigation, study of the ligand in its free and bound state can also reveal important information regarding this recognition process. Nuclear magnetic resonance (NMR) spectroscopy can be extremely useful for such studies. In this review, we discuss the attributes of NMR in the study of ligand receptor interactions. The cholinergic receptor and its binding to the neurotransmitter, acetylcholine, and cholinergic antagonists serve as a model system, illustrating the power of ligand analysis by NMR. The results discussed prove that the region of residues alpha 180-205 of the nicotinic acetylcholine receptor are an essential component of the cholinergic binding site and that ligand binding involves a positively charged hydrophobic motif.

Differences in cognitions during chest pain of patients with panic disorder and ischemic heart disease.

BACKGROUND: A significant number of patients with chest pains who undergo coronary angiography (20-30%) have normal coronary arteries. Up to 50% of this group are eventually diagnosed as Panic Disorder and most continue to complain of their symptoms, in spite of the normal coronary angiogram. We hypothesized that the cognitions of panic disorder subjects on presentation with chest pain would differ from those of patients suffering from true angina pectoris. METHODS: We investigated the cognitions associated with chest pain of three patient groups: proven symptomatic coronary artery disease (CAD+), subjects with chest pain and a normal coronary angiogram (CAD-), and patients with panic disorder (PD). All patients were classified according to whether the symptomatology was, firstly, associated with frightening cognitions (during the episode), and, secondly, whether either these cognitions (cognitive predominance), or the physical symptom (physical predominance), dominated the clinical picture. RESULTS: We observed that in the CAD+ group, 18% experienced frightening cognitions but in only 4% (2 of 66 patients) were the cognitions the dominant experience during the chest pain. In contrast, all the PD patients experienced frightening cognitions and in 83% of this group, the cognitions were the predominant experience. In the CAD- group, 48% were found to be PD compatible. CONCLUSIONS: This study indicates that the cognitions of patients during episodes of chest pain, evaluated by three questions, help to differentiate between PD and true coronary symptoms. Consequently, the presence of frightening cognitions in the presence of chest pain, particularly at the onset of the clinical problem, makes necessary the need for psychiatric evaluation with the objective of excluding PD.

Identification of common motifs in unaligned DNA sequences: application to Escherichia coli Lrp regulon.

We describe a relatively simple method for the identification of common motifs in DNA sequences that are known to share a common function. The input sequences are unaligned and there is no information regarding the position or orientation of the motif. Often such data exists for protein-binding regions, where genetic or molecular information that defines the binding region is available, but the specific recognition site within it is unknown. The method is based on the principle of 'divide and conquer'; we first search for dominant submotifs and then build full-length motifs around them. This method has several useful features: (i) it screens all submotifs so that the results are independent of the sequence order in the data; (ii) it allows the submotifs to contain spacers; (iii) it identifies an existing motif even if the data contains 'noise'; (iv) its running time depends linearly on the total length of the input. The method is demonstrated on two groups of protein-binding sequences: a well-studied group of known CRP-binding sequences, and a relatively newly identified group of genes known to be regulated by Lrp. The Lrp motif that we identify, based on 23 gene sequences, is similar to a previously identified motif based on a smaller data set, and to a consensus sequence of experimentally defined binding sites. Individual Lrp sites are evaluated and compared in regard to their regulation mode.

NMR analysis reveals a positively charged hydrophobic domain as a common motif to bound acetylcholine and d-tubocurarine.

A complete 1H assignment of d-tubocurarine was carried out using 1D and 2D NMR techniques. Geometries of free acetylcholine (ACh) and d-tubocurarine were compared with those of the ligands bound to a recombinant cholinergic binding site (T alpha 184-200 expressed as a fusion protein in Escherichia coli). The conformations of the free ligands were determined by NOESY experiments while those of the bound molecules were obtained by transferred NOESY. The complete relaxation matrix was solved yielding distance constraints which were further refined by a sigma back-calculation. ACh bound to recombinant T alpha 184-200 closely resembled the conformation previously reported for ACh bound to the intact receptor. d-Tubocurarine in the bound state undergoes extensive induced conformational rearrangements generating a "cup"-shaped structure. A unique positively charged hydrophobic domain is identified as characteristic of both bound cholinergic ligands.

Platelet dysfunction associated with clozapine therapy.

Clozapine, an atypical antipsychotic agent used in cases of treatment-resistant schizophrenia, is known for its relative absence of extrapyramidal side effects and its potential hazardous effect on white blood cell function. We have described a case of clozapine-associated epistaxis and reduction of the platelet count. Discontinuance of clozapine therapy resulted in cessation of epistaxis followed by normalization of the platelet count. We suggest routine monitoring of platelet count and function in patients treated with clozapine.

The criminal liability of the mental patient in Jewish law (Halacha).

The legal status of the mentally impaired has been dealt with in Halachic literature since the third century. This article presents four Halachic viewpoints regarding the exemption of the mentally ill patient from legal responsibility in tort and criminal law: lack of mental awareness; lack of free will; lack of judgment; categorial personal status. Halachic legislations with regard to the liability of the insane predate the criteria as applied by modern psychiatry. The similarities between the Halachic and contemporary medicolegal approaches are presented.

Acetylcholine interactions with tryptophan-184 of the alpha-subunit of the nicotinic acetylcholine receptor revealed by transferred nuclear Overhauser effect.

Acetylcholine interactions with three genetically engineered fusion proteins containing peptides from the nicotinic acetylcholine receptor were studied by 1D and 2D nuclear magnetic resonance methods. The three proteins were Torpedo alpha 184-200, Torpedo alpha 186-198, and human alpha 183-204 of the acetylcholine receptor fused to the first 323 residues of the E. coli protein trpE. Nuclear Overhauser effect studies revealed interactions of bound acetylcholine with tryptophan-184 present in the Torpedo alpha 184-200, and the human alpha 183-204 sequences. These interactions are between the N(CH3)3+ and CH3 groups of acetylcholine with the aromatic protons of tryptophan. The appearance of these cross-peaks indicates a distance of less than 5 A between tryptophan and the bound ligand; however, direct contact has yet to be proven.

Molecular dissection of cholinergic binding sites: how do snakes escape the effect of their own toxins?

Snakes have evolved a novel binding site demonstrating selective biorecognition. The snake nicotinic acetylcholine receptor is sensitive to acetylcholine while resistant to the effect of the lethal neurotoxins secreted in their own venom. By subjecting recombinant binding sites to point mutagenesis, biochemical analyses and NMR spectroscopy the binding characteristics of three cholinergic ligands have been measured. The amino acid residue at position 189 has been found to be of particular importance to toxin binding.

NMR studies of recombinant active site peptides of the nicotinic acetylcholine receptor.

Interactions of ligands with recombinant cholinergic binding sites have been monitored by NMR. Monitoring the selective T1 relaxation of the protons of acetylcholine, nicotine, d-tubocurarine, and gallamine reveals specific binding to peptide constructs containing the alpha 183-204 or shorter sequences of the nicotinic acetylcholine receptor of Torpedo, Human, Chicken, Xenopus, Mouse, Calf, and Drosophila. The trend of the KD values of the different ligands shows that the binding of the low molecular weight agonists and antagonists is very weak to the Drosophila sequence which is different from the vertebrate sequences in the N and C terminals. Within the vertebrates, the antagonists d-tubocurarine and gallamine display a KD trend different from that of acetylcholine and alpha-bungarotoxin. Specificity of binding is proven by the fact that atropine, a muscarinic inhibitor, binds non-specifically. Temperature dependence indicates a fast exchange limit (T1 bound greater than tau bound) for gallamine bound to the Torpedo alpha 184-200 sequence. This limit should apply also for the other ligands which have weaker binding constants.

Direct measurement of agonist binding to genetically engineered peptides of the acetylcholine receptor by selective T1 NMR relaxation.

Interactions of four ligands of the nicotinic acetylcholine receptor with genetically engineered peptides have been studied by NMR. A recombinant cholinergic binding site was prepared as a fusion protein between a truncated form of the bacterial protein trpE and a peptide corresponding to the sequence alpha 184-200 from the Torpedo californica receptor. This construct binds alpha-bungarotoxin while the trpE protein alone does not, and thus serves as a negative control [Aronheim, A., Eshel, Y., Mosckovitz, R., & Gershoni, J. M. (1988) J. Biol. Chem. 263, 9933-9937]. In this study agonist binding to alpha 184-200 is demonstrated by monitoring the T1 relaxation of the ligand's protons in the presence and absence of the recombinant binding site. This binding is specific as it can be competed with alpha-bungarotoxin. Quantitative analyses of such competitions yielded the concentration of binding sites, which corresponded to 3.3% and 16.5% of the total protein, for partially purified and affinity-purified alpha 184-200 constructs, respectively. The KD values for the binding of acetylcholine, nicotine, d-tubocurarine, and gallamine to the affinity-purified construct were 1.4, 1.4, 0.20, and 0.21 mM, respectively, while KD's with the nontoxin binding protein were all above 10 mM. Thus, this is a direct demonstration that the toxin binding domain alpha 184-200 may comprise a major component of the cholinergic agonist site.