PDBsum entry 252l

Hydrolase

PDB id: 252l

Contents

Protein chain

164 a.a. *

Ligands

HED

Metals

_CL

Waters ×136

* Residue conservation analysis

PDB id:

252l

Name:

Hydrolase

Title:

Generating ligand binding sites in t4 lysozyme using deficiency- creating substitutions

Structure:

T4 lysozyme. Chain: a. Engineered: yes. Mutation: yes

Source:

Enterobacteria phage t4. Organism_taxid: 10665. Cellular_location: cytoplasm. Gene: gene e. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Monomer (from PDB file)

Resolution:

2.10Å R-factor: 0.163

Authors:

E.P.Baldwin,W.A.Baase,X.-J.Zhang,V.Feher,B.W.Matthews

Key ref:

E.Baldwin et al. (1998). Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions. J Mol Biol, 277, 467-485. PubMed id: 9514755 DOI: 10.1006/jmbi.1997.1606

Date:

28-Oct-97 Release date: 18-Mar-98

Protein chain

P00720  (ENLYS_BPT4) -  Endolysin from Enterobacteria phage T4

Seq: 164 a.a.

Struc: 164 a.a.*

* PDB and UniProt seqs differ at 6 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.17 - lysozyme.
• Reaction: Hydrolysis of the 1,4-beta-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid in peptidoglycan heteropolymers of the prokaryotes cell walls.

DOI no: 10.1006/jmbi.1997.1606

J Mol Biol 277:467-485 (1998)

PubMed id: 9514755

Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions.

E.Baldwin, W.A.Baase, X.j.Zhang, V.Feher, B.W.Matthews.

ABSTRACT

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.

Selected figure(s)

Figure 7.
Figure 7. Changes in solvent structure near residue 104. Solvents are denoted by starred atoms and the broken lines connect solvent atom pairs that are within hydrogen bonding distance. (a) WT* lysozyme; (b) mutant F104A; (c) benzene complex F104A/BZ.

Figure 10.
Figure 10. (a) Superposition of M102A/M106A (filled bonds) on WT* (open bonds) showing the "alternative" conformation with hydrogen bonds indicated by broken lines. (b) Superposition of residues 106 to 114 of the "alternative" conformation of M102A/M106A (filled atoms and bonds), together with the "wild-type-like" conformation of M102A/M106A (thin open bonds and starred atoms), on WT* (open thick bonds and atoms), based on atoms 81 to 161.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1998, 277, 467-485) copyright 1998.

Figures were selected by an automated process.