PDBsum entry 1d1m

Viral protein

PDB id: 1d1m

Contents

Protein chains

65 a.a. *

Waters ×30

* Residue conservation analysis

PDB id:

1d1m

Name:

Viral protein

Title:

Crystal structure of cro k56-[dgevk]-f58w mutant

Structure:

Lambda cro repressor. Chain: b, a. Fragment: lambda cro repressor. Engineered: yes. Mutation: yes

Source:

Enterobacteria phage lambda. Organism_taxid: 10710. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.05Å R-factor: 0.185 R-free: 0.298

Authors:

P.B.Rupert,A.K.Mollah,M.C.Mossing,B.W.Matthews

Key ref:

P.B.Rupert et al. (2000). The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers. J Mol Biol, 296, 1079-1090. PubMed id: 10686105 DOI: 10.1006/jmbi.1999.3498

Date:

17-Sep-99 Release date: 24-Sep-99

Protein chains

P03040  (RCRO_LAMBD) -  Regulatory protein cro from Escherichia phage lambda

Seq: 66 a.a.

Struc: 65 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1006/jmbi.1999.3498

J Mol Biol 296:1079-1090 (2000)

PubMed id: 10686105

The structural basis for enhanced stability and reduced DNA binding seen in engineered second-generation Cro monomers and dimers.

P.B.Rupert, A.K.Mollah, M.C.Mossing, B.W.Matthews.

ABSTRACT

It was previously shown that the Cro repressor from phage lambda, which is a dimer, can be converted into a stable monomer by a five-amino acid insertion. Phe58 is the key residue involved in this transition, switching from interactions which stabilize the dimer to those which stabilize the monomer. Structural studies, however, suggested that Phe58 did not penetrate into the core of the monomer as well as it did into the native dimer. This was strongly supported by the finding that certain core-repacking mutations, including in particular, Phe58-->Trp, increased the stability of the monomer. Unexpectedly, the same substitution also increased the stability of the native dimer. At the same time it decreased the affinity of the dimer for operator DNA. Here we describe the crystal structures of the Cro F58W mutant, both as the monomer and as the dimer. The F58W monomer crystallized in a form different from that of the original monomer. In contrast to that structure, which resembled the DNA-bound form of Cro, the F58W monomer is closer in structure to wild-type (i.e. non-bound) Cro. The F58W dimer also crystallizes in a form different from the native dimer but has a remarkably similar overall structure which tends to confirm the large changes in conformation of Cro on binding DNA. Introduction of Trp58 perturbs the position occupied by the side-chain of Arg38, a DNA-contact residue, providing a structural explanation for the reduction in DNA-binding affinity.The improved thermal stability is seen to be due to the enhanced solvent transfer free energy of Trp58 relative to Phe58, supplemented in the dimer structure, although not the monomer, by a reduction in volume of internal cavities.

Selected figure(s)

Figure 2.
Figure 2. Superposition of the a-carbon backbone of the original engineered monomer, Cro K56-[DGEVK], in black on the mutant monomer, Cro K56-[DGEVK]-F58W. One of the two crystallographically independent copies of the mutant structure (chain A) is shown in gray and the other with open bonds. This superposition, as well as for all other such Figures, is based on the a-carbon atoms of residues 5-40 which includes the a-helical core region and the helix-turn-helix.

Figure 6.
Figure 6. (a) Superposition of the mutant monomer, Cro-[DGEVK]-F58W (open bonds), on one subunit of the native Cro dimer (filled bonds). (b) Superposition of one subunit of the mutant monomer, Cro-[DGEVK]-F58W (open bonds), on one subunit of the WT Cro dimer (filled bonds) in its conformation when bound to operator DNA. (c) Detailed view of (b) in the vicinity of residue 58 showing the superposition of the native Cro-operator complex (filled bonds) on one subunit of Cro-F58W. The indole ring of Trp58 displaces Arg38' from its position in the Phe58 structure and would tend to disrupt the interaction between Arg38' and the phosphate oxygen atom labeled S12 O2P. This labeling is as used by the Protein Data Bank. In terms of common conventions for the Cro system (e.g. see [Albright and Matthews 1998]), S11 O2P and S12 O2P correspond, respectively, to phosphate groups P[E] and P[D].

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 296, 1079-1090) copyright 2000.

Figures were selected by an automated process.