PDBsum entry: 1jva

Hydrolase

PDB id: 1jva

Contents

Protein chains

427 a.a. *

Waters ×205

* Residue conservation analysis

PDB id:

1jva

Name:

Hydrolase

Title:

Crystal structure of the vma1-derived endonuclease bearing the n and c extein propeptides

Structure:

Vma1-derived homing endonuclease x10sss. Chain: a, b. Fragment: residues 274-747. Synonym: x10sss vde. Engineered: yes. Mutation: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: vma1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PQS)

Resolution:

2.10Å R-factor: 0.198 R-free: 0.240

Authors:

R.Mizutani,Y.Satow

Key ref:

R.Mizutani et al. (2002). Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides. J Mol Biol, 316, 919-929. PubMed id: 11884132 DOI: 10.1006/jmbi.2001.5357

Date:

29-Aug-01 Release date: 29-Aug-02

Protein chains

P17255  (VATA_YEAST) -  V-type proton ATPase catalytic subunit A

Seq: 1071 a.a.

Struc: 427 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.6.3.14 - H(+)-transporting two-sector ATPase.
• Reaction: ATP + H₂O + H⁺(In) = ADP + phosphate + H⁺(Out)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: intein-mediated protein splicing (1 term)
• Biochemical function: endonuclease activity (1 term)

reference

DOI no: 10.1006/jmbi.2001.5357

J Mol Biol 316:919-929 (2002)

PubMed id: 11884132

Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides.

R.Mizutani, S.Nogami, M.Kawasaki, Y.Ohya, Y.Anraku, Y.Satow.

ABSTRACT

Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor model and is then subjected to a 2.1A resolution crystallographic analysis. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation. The S284 O(gamma) atom located at a distance of 3.1A from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S(gamma) atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of Nright arrowS acyl shift at the junction between the N-extein and intein. The S738 O(gamma) atom in the C-terminal junction is placed in close proximity to the S284 O(gamma) atom at a distance of 3.6A, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step. The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site.

Selected figure(s)

Figure 1.
Figure 1. A diagram for the mechanism of the protein-splicing reaction. N and C-extein polypeptides in the N and C-terminal sides of the VDE intein are indicated by the boxes and their N and C labels, respectively. In the N ! S acyl shift step, a thioester intermediate is formed at C284. This step is further examined here as shown in Figure 6. A branched intermediate is formed by the transesterification that follows a nucleophilic attack on the thioester moiety by the side-chain of the conserved C738. In the next step, the intervening region from C284 to N737 is excised by the peptide bond cleavage upon succinimide formation at the conserved N737. The transient ligation product then under- goes the final S ! N acyl rearrangement from a thioester to an amide bond.

Figure 4.
Figure 4. Structure of the X10SSS VDE protein. (a) X10SSS VDE molecules in the asymmetric unit. Molecules A and B are viewed along the non-crystallographic 2-fold axis. The C and N-extein residues are illustrated as stick models. Molecule B is highlighted by colors; red, the N-extein residues; light green, the splicing region of the domain II; green, the DNA-recognition region of domain II; blue, the domain I; yellow, the C-extein residues. This Figure was produced with the program MOLSCRIPT. 27 (b) Stereo view of the electron-density map superposed on the residues located at the splicing site of molecule B. For the calculation of this difference-Fourier map, residues 279-284 and 737- 741 were omitted from the model, and then the resultant model was further refined. The map is contoured at the 3 s level using TURBO-FRODO. 26

The above figures are reprinted by permission from Elsevier: J Mol Biol (2002, 316, 919-929) copyright 2002.

Figures were selected by an automated process.