How Oligomerization Contributes to the Thermostability of an Archaeon Protein

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 ␣-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 ␣-helical domain (Tyr 206 -Thr 231 ) was deleted, suggesting that oligomerization via coiled-coil association using the C-terminal ␣-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.Thermophilic organisms grow optimally at temperatures of Ͼ70°C. The proteins isolated from these organisms are usually more thermostable than homologous proteins from mesophiles, despite the fact that they have similar three-dimensional structures and identical catalytic mechanisms to the mesophilic proteins as well as sequence homologies of 40 -85% (1). Extensive studies have focused on identifying key determinants or common factors responsible for the thermostability of thermophile proteins, and the following structural features have been proposed as responsible for the high thermostability: decreased solvent-exposed surface area (2), increased polar interactions at the molecular surface (3-10), higher packing density within the protein (10 -12), greater core hydrophobicity (10, 13-15), shorter surface loops (11, 16), and more hydrogen bonds (7,10,(17)(18)(19) relative to those of ordinary proteins as well as oligomerization (10, 20 -25).The protein L-isoaspartyl-O-methyltransferase (PIMT 1 ; EC 2.1.1.77) functions as an enzyme in the repair of age-damaged proteins in which asparagines and aspartates have been spontaneously deamidized and isomerized into L-isoaspartyl residues; it catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of the ␣-carboxyl group of the L-isoaspartyl residues to form L-iso-Asp-␣-methyl ester (26,27...

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

“…A multitude of studies showed oligomerization to promote protein stability303132333435. Asking whether octamerization also influences the stability of hUGP, we applied thermostability measurements and compared wt hUGP1 with two mutants (I498D and Δ501–508), previously shown for hUGP2 to cause dissociation into tetramers and dimers, respectively24.…”

Escherichia coli transcription termination factor NusA: heat-induced oligomerization and chaperone activity

“…In this work, we investigated whether NusA can also act as a molecular chaperone because it can be used as a solubility partner in heterologous expression, which is similar to some well-studied chaperones1718. Protein oligomerization has been shown to enhance the thermostability of thermophilic archaeal proteins41. In mesophilic E. coli , a large proportion of heat stable proteins can also form high oligomers20, implying that oligomerization could also contribute to protein stability in mesophiles.…”

Fusion of Self-Assembling Amphipathic Oligopeptides with Cyclodextrin Glycosyltransferase Improves 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis with Soluble Starch as the Glycosyl Donor

“…These high RMSD values suggested a relatively flexible structure in these peptides (35,36). As we knew, reduction of the N-or C-terminal flexibility of enzymes may lead to an increase in their thermostability (37)(38)(39). Therefore, the relatively highly flexible structure at the N termini of SAP5-CGTase and SAP6-CGTase may be responsible for reducing their thermostability compared to that of the wild-type CGTase (see Table 2).…”