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Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.

The nucleoside diphosphate kinase (NDP kinase) from Myxococcus xanthus has been purified to homogeneity and crystallized (J. Munoz-Dorado, M. Inouye, and S. Inouye, J. Biol. Chem. 265:2702-2706, 1990). In the presence of ATP, the NDP kinase was autophosphorylated. Phosphoamino acid analysis was carried out after acid and base hydrolyses of phosphorylated NDP kinase. It was found that the protein was phosphorylated not only at a histidine residue but also at a serine residue. Replacement of histidine 117 with a glutamine residue completely abolished the autophosphorylation and nucleotide-binding activity of the NDP kinase. Since histidine 117 is the only histidine residue that is conserved in all known NDP kinases so far characterized, the results suggest that the phosphohistidine intermediate is formed at this residue during the transphosphorylation reaction from nucleoside triphosphates to nucleoside diphosphates. Preliminary mutational analysis of putative ATP-binding sites is also presented.

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Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [␥-32 P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-toAla double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides Nucleoside diphosphate (NDP) kinase catalyzes the following transphosphorylation reaction, via the formation of a phosphoenzyme intermediate:The primary role of NDP kinase in the cell was considered the maintenance of a pool of nucleotide triphosphates for the synthesis of DNA and RNA (22). NDP kinase was reported to be the only enzyme responsible for the synthesis of nucleotides other than ATP in Salmonella typhimurium (7). Also, the ndk gene appears to be essential in the gram-negative bacterium Myxococcus xanthus (20). Recently the enzyme has been implicated as a tumor suppressor candidate in studies of highly metastatic human cells (14,26,27) and has been associated with the development of Drosophila larvae (3,25).NDP kinase genes (ndk) from prokaryotic and eukaryotic organisms have been cloned and sequenced (8,19). The deduced amino acid sequence of the enzyme from Escherichia coli indicated that NDP kinases are highly conserved throughout evolution; the E. coli and human (Nm23-H1) enzymes are 43% identical (8). The three-dimensional structures of NDP kinases from Dictyostelium discoideum (4), M. xanthus (33), and Drosophila melanogaster (2) have been determined. These studies again showed that NDP kinases from different sources share highly conserved three-dimensional structures.NDP kinases are known to function by a ping-pong mechanism which involves the formation of a high-energy phosphoenzyme intermediate. Most of the intermediates have been shown to be alkali stable and acid labile, suggesting the formation of phosphohistidine, which has been identified in NDP kinases from various sources (22). Recently, histidine 118 has been identified as the phosphorylation site in one of the human NDP kinases, Nm23 (6). This histidine residue is invariant among all the NDP kinases sequenced so far (8). However phospho-NDP kinases with drastically different stability characteristics have been reported (22). Phosphoserine formation has been reported to occur in NDP kinase from rat mucosal mast cells (11) and in NDP kinase from M. xanthus, in which the formation of phosphohistidine ha...

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