PDBsum entry 1kmt

Protein binding

PDB id: 1kmt

Contents

Protein chains

138 a.a. *

Waters ×592

* Residue conservation analysis

PDB id:

1kmt

Name:

Protein binding

Title:

Crystal structure of rhogdi glu(154,155)ala mutant

Structure:

Rho gdp-dissociation inhibitor 1. Chain: a, b. Synonym: rho gdi 1, rho-gdi alpha. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.30Å R-factor: 0.160 R-free: 0.195

Authors:

A.Mateja,Y.Devedjiev,D.Krowarsh,K.Longenecker,Z.Dauter,J.Otlewski, Z.S.Derewenda

Key ref:

A.Mateja et al. (2002). The impact of Glu-->Ala and Glu-->Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution. Acta Crystallogr D Biol Crystallogr, 58, 1983-1991. PubMed id: 12454455 DOI: 10.1107/S090744490201394X

Date:

17-Dec-01 Release date: 11-Dec-02

Protein chains

P52565  (GDIR1_HUMAN) -  Rho GDP-dissociation inhibitor 1 from Homo sapiens

Seq: 204 a.a.

Struc: 138 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1107/S090744490201394X

Acta Crystallogr D Biol Crystallogr 58:1983-1991 (2002)

PubMed id: 12454455

The impact of Glu-->Ala and Glu-->Asp mutations on the crystallization properties of RhoGDI: the structure of RhoGDI at 1.3 A resolution.

A.Mateja, Y.Devedjiev, D.Krowarsch, K.Longenecker, Z.Dauter, J.Otlewski, Z.S.Derewenda.

ABSTRACT

It is hypothesized that surface residues with high conformational entropy, specifically lysines and glutamates, impede protein crystallization. In a previous study using a model system of Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI), it was shown that mutating Lys residues to Ala results in enhanced crystallizability, particularly when clusters of lysines are targeted. It was also shown that one of these mutants formed crystals that yielded diffraction to 2.0 A, a significant improvement on the wild-type protein crystals. In the current paper, an analysis of the impact of surface mutations replacing Glu residues with Ala or Asp on the stability and crystallization properties of RhoGDI is presented. The Glu-->Ala (Asp) mutants are generally more likely to produce crystals of the protein than the wild-type and in one case the resulting crystals yielded a diffraction pattern to 1.2 A resolution. This occurs in spite of the fact that mutating surface Glu residues almost invariably affects the protein's stability, as illustrated by the reduced deltaG between folded and unfolded forms measured by isothermal equilibrium denaturation. The present study strongly supports the notion that rational surface mutagenesis can be an effective tool in overcoming problems stemming from the protein's recalcitrance to crystallization and may also yield dramatic improvements in crystal quality.

Selected figure(s)

Figure 1.
Figure 1 A view of the RhoGDI 66 fragment with glutamates shown in full and colored gold. The two sites which were mutated in the highly diffracting crystal form are shown in pink. The sites are numbered according to the amino-acid sequence of the full-length protein. The figure was prepared using RIBBONS (Carson, 1991[Carson, M. (1991). J. Appl. Cryst. 24, 958-961.]).

Figure 3.
Figure 3 Ribbon diagram of the overall arrangement of the molecules in the asymmetric unit, with the location of the point mutations identified by magenta spheres. The two molecules within the asymmetric unit are colored according to the temperature factors. The grey sphere indicates the entrance to the hydrophobic pocket. The figure was prepared using BOBSCRIPT (Esnouf, 1997[Esnouf, R. M. (1997). J. Mol. Graph. 15, 132-143.]).

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2002, 58, 1983-1991) copyright 2002.

Figures were selected by the author.