PDBsum entry 2ijh

Transcription regulator

PDB id: 2ijh

Contents

Protein chains

57 a.a. *

Waters ×152

* Residue conservation analysis

PDB id:

2ijh

Name:

Transcription regulator

Title:

Crystal structure analysis of cole1 rom mutant f14w

Structure:

Regulatory protein rop. Chain: a, b, c. Synonym: RNA one modulator, rom. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: rop. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.80Å R-factor: 0.194 R-free: 0.230

Authors:

J.E.Ladner

Key ref:

E.B.Struble et al. (2008). New crystal structures of ColE1 Rom and variants resulting from mutation of a surface exposed residue: Implications for RNA-recognition. Proteins, 72, 761-768. PubMed id: 18260113 DOI: 10.1002/prot.21965

Date:

29-Sep-06 Release date: 16-Oct-07

Protein chains

P03051  (ROP_ECOLX) -  Regulatory protein rop from Escherichia coli

Seq: 63 a.a.

Struc: 57 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

DOI no: 10.1002/prot.21965

Proteins 72:761-768 (2008)

PubMed id: 18260113

New crystal structures of ColE1 Rom and variants resulting from mutation of a surface exposed residue: Implications for RNA-recognition.

E.B.Struble, J.E.Ladner, D.M.Brabazon, J.P.Marino.

ABSTRACT

In ColE1, the plasmid encoded RNA one modulator (Rom) protein, which is also referred to as Rop, specifically binds and stabilizes an intermediate RNA loop-loop kissing structure formed between the plasmid encoded transcripts RNA I and RNA II and thereby acts as an auxiliary repressor of replication. Rom folds into a homodimeric, cylindrically packed four helix bundle with an exact twofold symmetry axis (Banner et al., J Mol Biol 1987;196:657-675; Eberle et al., J Biomol 1991;1:71-83). Previous studies (Castagnoli et al., EMBO J 1989;8:621-629; Predki et al., Cell 1995;80:41-50) have localized the RNA binding surface to the H1/H1' face of the helical bundle and found Phe14 to be a key determinant of the binding affinity and specificity for RNA kissing complexes. To investigate the role of Phe14 in RNA recognition, we have determined high-resolution crystal structures of two point mutants of Rom (F14Y and F14W), as well as a high-resolution structure of a crystal form of Rom in which the dimer comprises the asymmetric unit. Although the structures of F14Y and F14W share a very high degree of structural identity with that of the wild-type protein and each other, differences are observed between the three polypeptide chains found in the asymmetric unit of each crystal in the packing of the tryptophan and tyrosine side chains at position 14, as well as some of the other surface exposed side chains of key amino acids involved in RNA binding. In both the wild-type Rom and mutant structures, crystal packing forces can break the exact twofold symmetry of the dimer and influence the conformation of the side chains presented on the H1/H1' face of Rom. Since the new structures show such a high degree of structural identity, the disruption in RNA binding observed for the mutant proteins can be attributed specifically to the chemical nature of the side chain at position 14. Moreover, the fact that even subtle changes in the side chain at position 14 cannot be compensated for by the apparent flexibility of this side chain suggests a highly constrained packing of this residue in the RNA-protein complex.

Selected figure(s)

Figure 1.
Figure 1. Superposition of the structures of the three polypeptide chains observed in the crystal of the F14Y mutant variant (A) and the three chains observed in the crystal of the F14W mutant variant (B). The backbone of the protein chains are displayed in stick format. The side chains at position 14 are selectively shown and labeled.

Figure 4.
Figure 4. Comparison of the packing observed for the side chains at and around position 14 in the AB dimer structures (top panels) and CC , twofold symmetry generated dimer structures, (bottom panels) of wild type Rom (A), F14Y Rom variant (B), and F14W Rom variant (C). In the case of the F14Y and F14W mutants, potential H-bonding interactions between the side chain indole or phenol groups and neighboring Gln18 are indicated by dashed lines.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2008, 72, 761-768) copyright 2008.

Figures were selected by the author.