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PDBsum entry 3ifj

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protein metals Protein-protein interface(s) links
Splicing PDB id
3ifj
Jmol
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    
PROCHECK
Go to PROCHECK summary
 Headers
 References

Protein chains
Pfam  
P9WHJ2  (RECA_MYCTO) -  Protein RecA
Seq:
Struc:
 
Seq:
Struc:
790 a.a.
139 a.a.*
Key:    Secondary structure  CATH domain
* PDB and UniProt seqs differ at 43 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  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.  

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.  
20820635 G.Volkmann, and H.Iwaï (2010).
Protein trans-splicing and its use in structural biology: opportunities and limitations.
  Mol Biosyst, 6, 2110-2121.  
20844013 P.A.Beachy, S.G.Hymowitz, R.A.Lazarus, D.J.Leahy, and C.Siebold (2010).
Interactions between Hedgehog proteins and their binding partners come into view.
  Genes Dev, 24, 2001-2012.  
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.