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Predicting ligand-binding function in families of bacterial receptors.

ABSTRACT: The three-dimensional fold of a new protein sequence can often be inferred directly from sequence homology to a protein of known structure. The function of a new protein sequence is more difficult to predict, however, since homologues can have different molecular and cellular functions. To develop and automate computational methods for determining molecular function, we have analyzed ligand-binding specificity in two related families of binding proteins. One of these families includes Escherichia coli lactose repressor and ribose-binding protein, and the other includes E. coli sulfate- and phosphate-binding proteins. These proteins have similar folds but varying specificity, binding many different small molecules, including mono- and disaccharides, purines, oxyanions, ferric iron, and polyamines. Starting from template structural alignments, alignments of over 90 sequences per family were generated by iterative database searches with hidden Markov models. Phylogenetic trees were made of full-length sequences and of subsets of residues lining the binding cleft, to determine whether subbranches of the trees correlate with ligand-binding preference. Automated analyses of residues in the binding pocket were also used to predict ligand-binding function for many uncharacterized database sequences and to identify specific side chain-ligand contacts in proteins without solved structures. Our results demonstrate the utility of anchoring functional annotation within a protein family context.

SUBMITTER: Johnson JM

PROVIDER: S-EPMC18125 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Predicting ligand-binding function in families of bacterial receptors.

Johnson J M JM Church G M GM

Proceedings of the National Academy of Sciences of the United States of America 20000401 8

The three-dimensional fold of a new protein sequence can often be inferred directly from sequence homology to a protein of known structure. The function of a new protein sequence is more difficult to predict, however, since homologues can have different molecular and cellular functions. To develop and automate computational methods for determining molecular function, we have analyzed ligand-binding specificity in two related families of binding proteins. One of these families includes Escherichi ...[more]

PMID: 10737762

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Project description:ATP-sensitive K(+) channels (K(ATP) channels) are complexes of inwardly rectifying K(+) channels (Kir6.x) and sulphonylurea receptors (SURs). Kir6.2-containing channels are closed by ATP binding to Kir6.2, and opened by MgADP binding to SUR. Channel activity is modulated by synthetic compounds such as the channel-blocking sulphonylureas and the K(ATP) channel openers, which both act by binding to SUR. By interacting with Kir6.2, phosphatidylinositol-4,5-bisphosphate (PIP(2)) and oleoyl-coenzyme A (OCoA) decrease the ATP-sensitivity of the channel and abolish the effect of the synthetic channel modulators. Here, we have investigated whether lipids and related compounds interfered with the binding of the sulphonylurea, glibenclamide (GBC) and of the opener, N-cyano-N'-(1,1-dimethylpropyl)-N''-3-pyridylguanidine (P1075), to the SUR subtypes. Lipids (100-300 microM) inhibited binding of [(3)H]GBC and [(3)H]P1075 to SUR subtypes in the rank order OCoA>dioleylglycerol-succinyl-nitriloacetic acid (DOGS-NTA)>oleate>malonyl-CoA>PIP(2.)OCoA inhibited radioligand binding to SUR completely, with IC(50) values ranging from 6 to 44 microM. Inhibition was reversed by increasing the concentration of the radioligands in agreement with an essentially competitive mechanism. MgATP and coexpression with Kir6.2 decreased the potency of OCoA. DOGS-NTA inhibited radioligand binding to SUR by 40-88%, with IC(50) values ranging from 38 to 120 microM. Poly-lysine increased radioligand binding to SUR by up to 30% but did not affect much the inhibition of ligand binding by OCoA and DOGS-NTA. Radioligand binding to SUR2A but not to the other SUR subtypes was slightly (10-20%) stimulated by lipids at concentrations approximately 10 x lower than required for inhibition. The data show that certain lipids, at high concentrations, interact with SUR and inhibit the binding of GBC and P1075; with SUR2A, a modest stimulation of ligand binding precedes inhibition. Regarding K(ATP) channel activity, these effects will be overruled by the interaction of the lipids with Kir6.2 at lower (physiological) concentrations. They are, however, of pharmacological importance and must be taken into account if high concentrations of these compounds (e.g. OCoA>10 microM) are used to interfere with the action of sulphonylureas and openers on K(ATP) channel activity.

Dataset Information

Predicting ligand-binding function in families of bacterial receptors.

Publications

Predicting ligand-binding function in families of bacterial receptors.

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