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Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5-sulfamoyl (AdoOSO 2 NH 2 ) or N-ethylsulfamoyl (AdoOSO 2 NHCH 2 CH 3 ) group. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors; titration of each inhibitor into AMP-pNA assays revealed their K i values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8-Å cocrystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp-123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser-107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity.

We have studied quantitatively the effect of the corepressor, S-adenosylmethionine (SAM), on the interaction between the E. coli methionine repressor, MetJ, and an idealised operator fragment, by recording measurements of surface plasmon resonance using a BIAcore instrument. We have recorded kinetic binding data in the presence of SAM, which carries a net positive charge, and two corepressor analogues, adenosylornithine (AO) and aza-SAM, which differ in the location of the atom carrying the positive charge. Our data support the hypothesis that the effect of the corepressor is electrostatic in origin. The difference in electrostatic interaction energy between the SAM- and AO-repressor-operator complexes of approximately 3.5 kJ/mol calculated from the known three-dimensional structure is within the range of our experimentally determined values of 2.8-4.3 kJ/mol. These results illustrate the potential of SPR measurements for studying protein-nucleic acid interactions.

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1 Heteromeric P2X 2/3 receptors are much more sensitive than homomeric P2X 2 receptors to abmethylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells. 2 We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the ab and/or bg oxygen atoms were replaced by other moieties (including -CH 2 -, -CHF-, -CHCl -, -CHBr-, -CF 2 -, -CCl 2 -, -CBr 2 -, -CHSO 3 -, -CHPO 3 -, -CFPO 3 -, -CClPO 3 -, -CH 2 -CH 2 -, -CC -, -NH -, -CHCOOH -). 3 We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X 2 subunits alone (homomeric P2X 2 receptors), or cells expressing both P2X 2 and P2X 3 subunits, in which the current through heteromeric P2X 2/3 receptors was isolated. 4 ADP analogues had no agonist or antagonist effect at either P2X 2 or P2X 2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X 2 receptors, except bgdifluoromethylene-ATP, which was a weak agonist. 5 At P2X 2/3 receptors, bg-imido-ATP, bg-methylene-ATP, and bg-acetylene-ATP were weak agonists, whereas ab,bg-and bg,gd-bismethylene-AP 4 were potent full agonists. bg-Carboxymethylene-ATP and bg-chlorophosphonomethylene-ATP were weak antagonists at P2X 2/3 receptors (IC 50 about 10 mM). 6 The results indicate (a) that the homomeric P2X 2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X 2/3 receptor is much more tolerant of ab and bg substitution; and (c) that a P2X 2/3 -selective antagonist can be obtained by introduction of additional negativity at the bg-methylene.