PDBsum entry 1vbf

Transferase

PDB id: 1vbf

Contents

Protein chains

224 a.a. *

Waters ×408

* Residue conservation analysis

References listed in PDB file

Key reference

Title

How oligomerization contributes to the thermostability of an archaeon protein. Protein l-Isoaspartyl-O-Methyltransferase from sulfolobus tokodaii.

Authors

Y.Tanaka, K.Tsumoto, Y.Yasutake, M.Umetsu, M.Yao, H.Fukada, I.Tanaka, I.Kumagai.

Ref.

J Biol Chem, 2004, 279, 32957-32967. [DOI no: 10.1074/jbc.M404405200]

PubMed id

15169774

Abstract

To study how oligomerization may contribute to the thermostability of archaeon proteins, we focused on a hexameric protein, protein L-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii (StoPIMT). The crystal structure shows that StoPIMT has a distinctive hexameric structure composed of monomers consisting of two domains: an S-adenosylmethionine-dependent methyltransferase fold domain and a C-terminal alpha-helical domain. The hexameric structure includes three interfacial contact regions: major, minor, and coiled-coil. Several C-terminal deletion mutants were constructed and characterized. The hexameric structure and thermostability were retained when the C-terminal alpha-helical domain (Tyr(206)-Thr(231)) was deleted, suggesting that oligomerization via coiled-coil association using the C-terminal alpha-helical domains did not contribute critically to hexamerization or to the increased thermostability of the protein. Deletion of three additional residues located in the major contact region, Tyr(203)-Asp(204)-Asp(205), led to a significant decrease in hexamer stability and chemico/thermostability. Although replacement of Thr(146) and Asp(204), which form two hydrogen bonds in the interface in the major contact region, with Ala did not affect hexamer formation, these mutations led to a significant decrease in thermostability, suggesting that two residues in the major contact region make significant contributions to the increase in stability of the protein via hexamerization. These results suggest that cooperative hexamerization occurs via interactions of "hot spot" residues and that a couple of interfacial hot spot residues are responsible for enhancing thermostability via oligomerization.

Figure 2.
FIG. 2. Sequence alignment of PIMT. The residue numbering refers to StoPIMT. Completely conserved residues are highlighted in orange, and conservatively mutated sites are shown in light blue. Cys149 in StoPIMT, which forms intermolecular disulfide linkage, is boxed in red. M. janaschii, Methanococcus janaschii; V. vulnificus, Vibrio vulnificus; P. aeruginosa, Pseudomonas aeruginosa; Mouse, Mus musculus; human, Homo sapiens.

Figure 6.
FIG. 6. Changes in relative CD intensity at 222 nm for StoPIMT and its deletion mutants at various GdnHCl concentrations. Red circles, StoPIMT; blue squares, StoPIMT-d205; yellow squares, StoPIMT-d204; green squares, StoPIMT-d203; purple closed triangles, hexameric StoPIMT-d202; purple open triangles, dashed line, monomeric StoPIMT-d202; blue closed triangles, dimeric StoPIMT-d199; blue open triangles, dashed line, monomeric StoPIMT-d199; green closed triangles, dimeric StoPIMT-d197; green open triangles, dashed line, monomeric StoPIMT-d197.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 32957-32967) copyright 2004.