PDBsum entry: 2imz

Hydrolase

PDB-id: 2imz

Contents

Description

Header details

Header records

References

PROCHECK

Protein chains

144 a.a. *

Metal ions

_ZN ×2

Waters ×334

* Residue conservation analysis

Tools

Clefts Calculation

PDB id:

2imz

Name:

Hydrolase

Title:

Crystal structure of mtu reca intein splicing domain

Structure:

Endonuclease pi-mtui. Chain: a, b. Fragment: splicing domain. Engineered: yes. Mutation: yes

Source:

Mycobacterium tuberculosis. Organism_taxid: 1773. Gene: reca. Expressed in: escherichia coli. Expression_system_taxid: 562.

UniProt:

Chains A, B: P0A5U4 (RECA_MYCTU)

Seq:

Struc:

Seq:

Struc:

Seq:

Struc:

Seq:

790 a.a.

Struc:

144 a.a.*

Key:

PfamA domain

Secondary structure

* PDB and UniProt seqs differ at 43 residue positions (black crosses)

Resolution:

1.70Å

R-factor:

0.181

R-free:

0.210

Authors:

P.Van Roey

Key ref:

P.Van Roey et al. (2007). Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue.. J Mol Biol, 367, 162-173. [PubMed id: 17254599] [DOI: 10.1016/j.jmb.2006.12.050]

Date:

05-Oct-06

Release date:

01-May-07

Related entries:

2in0
2in8
2in9

Quick_links

Procheck

Clefts

Surface

Key reference

DOI no: 10.1016/j.jmb.2006.12.050

J Mol Biol 367:162-173 (2007)

PubMed id: 17254599

Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue.

P.Van Roey, B.Pereira, Z.Li, K.Hiraga, M.Belfort, V.Derbyshire.

ABSTRACT

The 440 amino acid Mtu recA intein consists of independent protein-splicing and endonuclease domains. Previously, removal of the central endonuclease domain of the intein, and selection for function, generated a 168 residue mini-intein, DeltaI-SM, that had splicing activity similar to that of the full-length, wild-type protein. A D422G mutation (DeltaI-CM) increased C-terminal cleavage activity. Using the DeltaI-SM mini-intein structure (presented here) as a guide, we previously generated a highly active 139 residue mini-intein, DeltaDeltaI(hh)-SM, by replacing 36 amino acid residues in the residual endonuclease loop with a seven-residue beta-turn from the autoprocessing domain of Hedgehog protein. The three-dimensional structures of DeltaI-SM, DeltaDeltaI(hh)-SM, and two variants, DeltaDeltaI(hh)-CM and DeltaDeltaI(hh), have been determined to evaluate the effects of the minimization on intein integrity and to investigate the structural and functional consequences of the D422G mutation. These structural studies show that Asp422 is capable of interacting with both the N and C termini. These interactions are lacking in the CM variant, but are replaced by contacts with water molecules. Accordingly, additional mutagenesis of residue 422, combined with mutations that isolate N-terminal and C-terminal cleavage, showed that the side-chain of Asp422 plays a role in both N and C-terminal cleavage, thereby suggesting that this highly conserved residue regulates the balance between the two reactions.

Selected figure(s)

Figure 2.
Figure 2. Structure of ΔI-SM. (a) Molecular structure of ΔI-SM. (b) Comparison of the conformation of ΔI-SM (green) with the GyrA intein^12 (red). (c) Dimer observed in the asymmetric unit, with zinc ions shown in cyan. (d) Coordination of the zinc ion by residues Glu424, His429, and the C-terminal aminosucciminide (SUC440) from one molecule (green), and His439 from the second molecule (red) in the asymmetric unit. Figure 2. Structure of ΔI-SM. (a) Molecular structure of ΔI-SM. (b) Comparison of the conformation of ΔI-SM (green) with the GyrA intein[3]^12 (red). (c) Dimer observed in the asymmetric unit, with zinc ions shown in cyan. (d) Coordination of the zinc ion by residues Glu424, His429, and the C-terminal aminosucciminide (SUC440) from one molecule (green), and His439 from the second molecule (red) in the asymmetric unit.

Figure 4.
Figure 4. Effect of the D422G mutation. (a) Superposition of the structures of ΔΔI[hh]-SM (green) and ΔΔI[hh]-CM (red). (b) Detailed comparison of the area of residue 422. Water molecules in this area are shown as spheres, light green for ΔΔI[hh]-SM and rose for ΔΔI[hh]-CM. In ΔΔI[hh]-CM, two water molecules are found bound tightly to the protein in the area of the missing Asp422 side-chain. Figure 4. Effect of the D422G mutation. (a) Superposition of the structures of ΔΔI[hh]-SM (green) and ΔΔI[hh]-CM (red). (b) Detailed comparison of the area of residue 422. Water molecules in this area are shown as spheres, light green for ΔΔI[hh]-SM and rose for ΔΔI[hh]-CM. In ΔΔI[hh]-CM, two water molecules are found bound tightly to the protein in the area of the missing Asp422 side-chain.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 367, 162-173) copyright 2007.

Figures were selected by an automated process.