229l
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GENERATING LIGAND BINDING SITES IN T4 LYSOZYME USING DEFICIENCY-CREATING SUBSTITUTIONS
OverviewOverview
Several variants of T4 lysozyme have been identified that sequester small, organic ligands in cavities or clefts. To evaluate potential binding sites, for non-polar molecules, we screened a number of hydrophobic, large-to-small mutants for stabilization in the presence of benzene. In, addition to Leu99-->Ala, binding was indicated for at least five other, mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and, thermal denaturation. As predicted from the shape of the cavity in the, benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to, enlarge the cavity of the Met102-->Ala mutant into a deep crevice through, an additional substitution, but the double mutant failed to bind ligands, because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the, protein structure contracted slightly to reduce the volume of the void, created by truncating substitutions and expanded upon binding the, non-polar ligand, with shifts similar to those resulting from the, mutations.A polar molecule binding site was also created by truncating, Arg95 to alanine. This creates a highly complementary buried polar, environment that can be utilized as a specific "receptor" for a, guanidinium ion. Our results suggest that creating a deficiency through, truncating mutations of buried residues generates "binding potential" for, ligands with characteristics similar to the deleted side-chain. Analysis, of complex and apo crystal structures of binding and non-binding mutants, suggests that ligand size and shape as well as protein flexibility and, complementarity are all determinants of binding. Binding at non-polar, sites is governed by hydrophobicity and steric interactions and is, relatively permissive. Binding at a polar site is more restrictive and, requires extensive complementarity between the ligand and the site.
About this StructureAbout this Structure
229L is a Single protein structure of sequence from Bacteriophage t4 with CL, GAI and BME as ligands. Active as Lysozyme, with EC number 3.2.1.17 Full crystallographic information is available from OCA.
ReferenceReference
Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions., Baldwin E, Baase WA, Zhang X, Feher V, Matthews BW, J Mol Biol. 1998 Mar 27;277(2):467-85. PMID:9514755
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