PDBsum entry 261l

Hydrolase

PDB id: 261l

Contents

Protein chain

173 a.a. *

Waters ×43

* Residue conservation analysis

PDB id:

261l

Name:

Hydrolase

Title:

Structural characterisation of an engineered tandem repeat contrasts the importance of context and sequence in protein folding

Structure:

Lysozyme. Chain: a. Engineered: yes. Mutation: yes

Source:

Enterobacteria phage t4. Organism_taxid: 10665. Cellular_location: cytoplasm. Gene: gene e from bacteriophage t4. Expressed in: escherichia coli. Expression_system_taxid: 562. Other_details: bacteriophage t4 (mutant gene derived from the m13 plasmid by cloning the t4 ly sozyme gene)

Biol. unit:

Monomer (from PDB file)

Resolution:

2.50Å R-factor: 0.170

Authors:

M.Sagermann,W.A.Baase,B.W.Matthews

Key ref:

M.Sagermann et al. (1999). Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding. Proc Natl Acad Sci U S A, 96, 6078-6083. PubMed id: 10339544 DOI: 10.1073/pnas.96.11.6078

Date:

11-May-99 Release date: 24-May-99

Protein chain

P00720  (ENLYS_BPT4) -  Endolysin from Enterobacteria phage T4

Seq: 164 a.a.

Struc: 173 a.a.*

* PDB and UniProt seqs differ at 16 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: 10.1073/pnas.96.11.6078

Proc Natl Acad Sci U S A 96:6078-6083 (1999)

PubMed id: 10339544

Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding.

M.Sagermann, W.A.Baase, B.W.Matthews.

ABSTRACT

To test a different approach to understanding the relationship between the sequence of part of a protein and its conformation in the overall folded structure, the amino acid sequence corresponding to an alpha-helix of T4 lysozyme was duplicated in tandem. The presence of such a sequence repeat provides the protein with "choices" during folding. The mutant protein folds with almost wild-type stability, is active, and crystallizes in two different space groups, one isomorphous with wild type and the other with two molecules in the asymmetric unit. The fold of the mutant is essentially the same in all cases, showing that the inserted segment has a well-defined structure. More than half of the inserted residues are themselves helical and extend the helix present in the wild-type protein. Participation of additional duplicated residues in this helix would have required major disruption of the parent structure. The results clearly show that the residues within the duplicated sequence tend to maintain a helical conformation even though the packing interactions with the remainder of the protein are different from those of the original helix. It supports the hypothesis that the structures of individual alpha-helices are determined predominantly by the nature of the amino acids within the helix, rather than the structural environment provided by the rest of the protein.

Selected figure(s)

Figure 2.
Fig. 2. (A) Initial electron density showing the overall conformation of the duplicated sequence, as seen in space group P3[2]21. The WT* structure, omitting residues 36-42 (shown as a ribbon drawing) was subject to 10 cycles of rigid-body refinement in the mutant lysozyme cell. The calculated phases and structure factors, F[c], were used to calculate a map with amplitudes (F[mutant] F[c]) at 3.0-Å resolution. The density in the vicinity of the deleted residues, contoured at 2.5 , is shown. (B) Electron density after refinement of the inserted region in space group P3[2]21. Coefficients are (2F[o]-F[c]). The structure factors, F[c], and phases were calculated from the refined model including the inserted region. The resolution is 2.5 Å, and the map is contoured at 1.0 . (C) Superposition of the overall structure of the duplication mutant in space group P3[2]21 (blue bonds) on WT* lysozyme (green bonds). The inserted region in the mutant structure is highlighted in yellow.

Figure 3.
Fig. 3. (A) Map showing the initial electron density for the inserted region of molecule A in space group P2[1]. Amplitudes are (2F[o]-F[c]) weighted by REFMAC (15) where the structure factors, F[c], and phases were calculated from the refined model including the inserted region. The map was calculated at 2.5-Å resolution and contoured at 1.0 . The density in the vicinity of residues 40i-43i is not well defined and could not be fit by a well-defined model. (B) Electron density for molecule B of crystal form P2[1]. This map was calculated with the same coefficients, contouring, and resolution as in A. (C) Superposition of the C trace of the two copies of mutant L20 in crystal form P2[1] (molecule A, blue; molecule B, mauve) and wild-type T4 lysozyme (green). The sequence of the insert is highlighted in yellow for molecule A and in orange for molecule B. The structural rearrangements of loop 18-25 in molecule B are clearly visible. The superpositions were based on the -carbon atoms of residues 51-80 within the amino-terminal domain. Because of slight changes in the hinge-bending angle the C-terminal domains appear out of register although the respective structures within these regions are very similar.

Figures were selected by an automated process.