PDBsum entry: 3ifj

Splicing

PDB id: 3ifj

Contents

Protein chains

139 a.a. *

Metals

_ZN ×2

Waters ×197

* Residue conservation analysis

PDB id:

3ifj

Name:

Splicing

Title:

Crystal structure of mtu reca intein, splicing domain

Structure:

Endonuclease pi-mtui. Chain: a, b. Synonym: protein reca, recombinase a, mtu reca intein. Engineered: yes. Mutation: yes

Source:

Mycobacterium tuberculosis. Organism_taxid: 1773. Gene: mt2806, mtv002.02c, reca, rv2737c. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.90Å R-factor: 0.218 R-free: 0.260

Authors:

P.Van Roey,M.Belfort

Key ref:

K.Hiraga et al. (2009). Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center. J Mol Biol, 393, 1106-1117. PubMed id: 19744499 DOI: 10.1016/j.jmb.2009.08.074

Date:

24-Jul-09 Release date: 06-Oct-09

Protein chains

P0A5U4  (RECA_MYCTU) -  Protein RecA

Seq: 790 a.a.

Struc: 139 a.a.*

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

 Gene Ontology (GO) functional annotation 

• Biological process: intein-mediated protein splicing (1 term)

DOI no: 10.1016/j.jmb.2009.08.074

J Mol Biol 393:1106-1117 (2009)

PubMed id: 19744499

Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center.

K.Hiraga, I.Soga, J.T.Dansereau, B.Pereira, V.Derbyshire, Z.Du, C.Wang, P.Van Roey, G.Belfort, M.Belfort.

ABSTRACT

Inteins are phylogenetically diverse self-splicing proteins that are of great functional, evolutionary, biotechnological, and medical interest. To address the relationship between intein structure and function, particularly with respect to regulating the splicing reaction, and to groom inteins for application, we developed a phage display system to extend current in vivo selection for enhanced intein function to selection in vitro. We thereby isolated inteins that can function under excursions in temperature, pH, and denaturing environment. Remarkably, most mutations mapped to the surface of the intein, remote from the active site. We chose two mutants with enhanced splicing activity for crystallography, one of which was also subjected to NMR analysis. These studies define a "ripple effect", whereby mutations in peripheral non-catalytic residues can cause subtle allosteric changes in the active-site environment in a way that facilitates intein activity. Altered salt-bridge formation and chemical shift changes of the mutant inteins provide a molecular rationale for their phenotypes. These fundamental insights will advance the utility of inteins in chemical biology, biotechnology, and medicine.

Selected figure(s)

Figure 2.
Fig. 2. Intein mutations that enhance phage recovery. (a) Selected mutations (left) and recovery from chitin resin (right). The control phage displaying only CBD, CBD colons ΔΔI[hh] (WT), and CBD colons ΔΔI[hh]-SM (SM) are shown in gray bars. The mutants in the N-terminal extein (− 2 and − 1 positions), the N-terminal intein segment, the loop region, and the C-terminal intein segment are shown in black, turquoise, red, and yellow bars, respectively. (b) Location of mutations on the crystal structure of the Mtu RecA mini-intein ΔΔI[hh]. Residues 1–94 (N-terminal segment) and 403–440 (C-terminal segment) are separated by the seven-amino-acid Hedgehog sequence VRDVETG (the loop residues 95–101). The N-terminal segment and the mutated residues within are turquoise, the loop region is red, the V67L mutations is magenta, the C-terminal segment and F421 residue are yellow, and the active-site residues C1, H439, and N440 are green. (c) Phage recovery of double mutants after in vitro splicing at pH 6.0 (gray bars) and pH 8.6 (black bars). Numbering of mutants corresponds to (a).

Figure 4.
Fig. 4. Structure of selected intein mutants. (a) Structure of parent intein (ΔΔI[hh]). Area of focus in (b)–(d) is boxed. (b) Wild-type intein showing a bifurcated salt bridge between D24, R419, and D50. (c) The D24Y mutant. The bifurcated salt bridge to R419 and the bridge between R419 and D50 are disrupted. (d) The F421Y mutant. The replacement of a Phe residue with Tyr also disrupts the salt bridge between D24 and R419; instead, a salt bridge is formed between D50 and R419. Turquoise versus yellow backbone and residues are colored as in Fig. 2b.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2009, 393, 1106-1117) copyright 2009.

Figures were selected by an automated process.