Protein thermostability above 100°C: A key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability. The discovery of hyperthermophilic microorganisms that grow optimally at or near 100°C has necessitated a radical revision of ideas concerning protein thermostability. Most of the enzymes from these organisms are stable for many hours at or above 100°C (1), suggesting that these enzymes must embody most of the mechanisms of thermostability that occur in extremely thermostable proteins. Thorough study of these proteins may therefore identify key structural determinants of thermal stability at very high temperatures. Trends commonly associated with elevated thermostability in proteins include relatively small solvent-exposed surface area (2), increased packing density that reduces cavities in the hydrophobic core (3, 4, 5, 6), an increase in core hydrophobicity (7, 8) decreased length of surface loops (6), and hydrogen bonds between polar residues (9). A prominent role for ion pairs in stabilization of proteins at or above 100°C, where the hydrophobic effect is minimal (10), has been inferred from the recently solved structures of several proteins from extreme thermophiles (11–14). This is consistent with earlier suggestions made on the role of ion pairs on stability (15). As yet, there is no general rule that governs amino acid composition of thermostable proteins and methods for predicting and designing stabilizing mutations are not reliable (16, 17, 18). Recent work (19–21) has indicated that significant increments of stability may be achieved in proteins from mesophiles by the inclusion of ‘‘rigidifying’’ mutations; however, we believe that the proteins from the hyperthermophiles may have evolved optimal adaptations of this sort. Our work has focused on homology-based modeling and structure comparison of the hexameric glutamate dehydrogenases (GluDHs) from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis, archaea that grow optimally at 100°C (22) and 88°C (23), respectively, and that from a mesophilic organism, Clostridium symbiosum. The two hyperthermophilic enzymes provide a near-ideal comparative experimental system for studying the determinants of exceptional thermostability as they show distinct differences in stability yet are highly homologous. An unusual structural feature of P. furiosus GluDH is a number of extensive networks of buried intersubunit ion pairs (11), which is reduced in extent in homologous, but less thermostable, GluDHs (24, 25). Our strategy has been to identify differences between the ion-pair networks in the P. furiosus and T. litoralis GluDHs and to convert the stability of the T. litoralis GluDH to that of the more thermostable P. furiosus enzyme, to establish a causal relationship between intersubunit ion-pair networks and hyperstability. MATERIALS AND METHODS Site-Directed Mutagenesis. We constructed mutants bearing the single Thr 138 replaced with Glu (T138E) and the double T138EyAsp 167 replaced with Thr (D167T) substitutions. Site-directed mutagenesis was performed by using either a modification of the uracil DNA glycosylase method for generating site-directed mutations or a modification of the Eckstein method (QuickChange mutagenesis system, Stratagene). Mutant T138E was constructed by using the uracil DNA glycosylase system and the following primer pairs: p15J (GGA TGA CCA TGG TTG AGC AAG ACC) and pT138-E Rev (U AUC CUC GUA UGG ACU UAU AAC ATC ATA GAT AGC); p16J (AGT GAG GGA TCC TCA CTT CTT GAT CCA TCC) and pT138-E For (A AGU CCA UAC GAG GAU AUU CCA GCT CCA GAC GT). Primers p15JypT138E Rev and primers p16JypT138E were used to amplify the 59 fragment and the 39 fragment respectively of the gdhA gene. Primers 15J and 16J were designed to anneal to the 59 and 39 ends of the gene, respectively, and contain the NcoI and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9512300-6$2.00y0 PNAS is available online at www.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: GluDH, glutamate dehydrogenase EC 1.4.1.3; T138E, Thr 138 replaced with Glu; D167T, Asp 167 replaced with Thr. Data deposition: The atomic coordinates for Thermococcus litoralis GluDH have been deposited in the Protein Data Bank, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (PDB ID code 1BVU). §To whom reprint requests should be addressed. e-mail: robb@umbi. umd.edu.