PDBsum entry 2in8

Hydrolase

PDB id

2in8

Loading ...

Contents

			Protein chain

					139 a.a. *

			Ligands

					SO4

			Waters ×114

* Residue conservation analysis

PDB id:

2in8

Links

Name:

Hydrolase

Title:

Crystal structure of mtu reca intein, splicing domain

Structure:

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

Source:

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

Resolution:

1.70Å

R-factor:

0.252

R-free:

0.292

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:

06-Oct-06

Release date:

01-May-07

PROCHECK

	Headers

	References

Protein chain

P9WHJ3 (RECA_MYCTU) - Protein RecA from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)

Seq: Struc:

Seq: Struc:					790 a.a. 139 a.a.*

Key:

PfamA domain

Secondary structure

CATH domain

* PDB and UniProt seqs differ at 44 residue positions (black crosses)



		Enzyme reactions

Enzyme class:

E.C.3.1.-.-

[IntEnz] [ExPASy] [KEGG] [BRENDA]

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.

Figures were selected by an automated process.

Literature references that cite this PDB file's key reference

PubMed id

Reference

21539790

P.T.Shemella, N.I.Topilina, I.Soga, B.Pereira, G.Belfort, M.Belfort, and S.K.Nayak (2011).
Electronic structure of neighboring extein residue modulates intein C-terminal cleavage activity.

Biophys J, 100, 2217-2225.

19940146

K.Tori, B.Dassa, M.A.Johnson, M.W.Southworth, L.E.Brace, Y.Ishino, S.Pietrokovski, and F.B.Perler (2010).
Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

J Biol Chem, 285, 2515-2526.

20209535

L.Zhang, N.Xiao, Y.Pan, Y.Zheng, Z.Pan, Z.Luo, X.Xu, and Y.Liu (2010).
Binding and inhibition of copper ions to RecA inteins from Mycobacterium tuberculosis.

Chemistry, 16, 4297-4306.

20449740

S.Elleuche, and S.Pöggeler (2010).
Inteins, valuable genetic elements in molecular biology and biotechnology.

Appl Microbiol Biotechnol, 87, 479-489.

19744499

K.Hiraga, I.Soga, J.T.Dansereau, B.Pereira, V.Derbyshire, Z.Du, C.Wang, P.Van Roey, G.Belfort, and M.Belfort (2009).
Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center.

J Mol Biol, 393, 1106-1117.

PDB codes:

3ifj 3igd

19462022

L.Zhang, Y.Zheng, Z.Xi, Z.Luo, X.Xu, C.Wang, and Y.Liu (2009).
Metal ions binding to recA inteins from Mycobacterium tuberculosis.

Mol Biosyst, 5, 644-650.

19630416

Z.Du, P.T.Shemella, Y.Liu, S.A.McCallum, B.Pereira, S.K.Nayak, G.Belfort, M.Belfort, and C.Wang (2009).
Highly conserved histidine plays a dual catalytic role in protein splicing: a pKa shift mechanism.

J Am Chem Soc, 131, 11581-11589.

19636882

Z.Du, Y.Liu, Y.Zheng, S.McCallum, J.Dansereau, V.Derbyshire, M.Belfort, G.Belfort, P.Van Roey, and C.Wang (2008).
1H, 13C, and 15N NMR assignments of an engineered intein based on Mycobacterium tuberculosis RecA.

Biomol NMR Assign, 2, 111-113.

17586768

M.A.Johnson, M.W.Southworth, T.Herrmann, L.Brace, F.B.Perler, and K.Wüthrich (2007).
NMR structure of a KlbA intein precursor from Methanococcus jannaschii.

Protein Sci, 16, 1316-1328.

PDB codes:

2jmz 2jnq

The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.