Structural and Thermodynamic Studies on a Salt-bridge Triad in the NADP-binding Domain of Glutamate Dehydrogenase from Thermotoga maritima: Cooperativity and Electrostatic Contribution to Stability ² Joyce H. G. Lebbink, ‡,§ Valerio Consalvi, | Roberta Chiaraluce, | Kurt D. Berndt, ‡,⊥ and Rudolf Ladenstein* ,‡ Center for Structural Biochemistry, Department of Biosciences at NoVum, Karolinska Institutet, 14157 Huddinge, Sweden, Dipartimento di Scienze Biochimiche ‘A Rossi Fanelli’, UniVersita ` ‘La Sapienza’, Piazzale A. Moro 5, 00185 Rome, Italy, and Faculty of Natural Sciences, So ¨ derto ¨ rns Ho ¨ gskola, 141 89 Huddinge, Sweden ReceiVed July 8, 2002; ReVised Manuscript ReceiVed October 16, 2002 ABSTRACT: Cooperative interactions within ion-pair networks of hyperthermostable proteins are thought to be a major determinant for extreme protein stability. While the favorable thermodynamic contributions of optimized electrostatics in general as well as those of pairwise interactions have been documented, cooperativity between pairwise interactions has not yet been studied thermodynamically in proteins from hyperthermophiles. In this study we use the isolated cofactor binding domain of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima to analyze pairwise and cooperative interactions within the salt-bridge triad Arg190-Glu231-Lys193. The X-ray structure of the domain was solved at 1.43 Å and reveals the salt-bridge network with surrounding solvent molecules in detail. All three participating charges in the network were mutated to alanine in all combinations. The X-ray structure of the variant lacking all three charges reveals that the removal of the side chains has no effect on the overall conformation of the protein. Using solvent denaturation and thermodynamic cycles, the interaction energies between each pair of residues in the network were determined in the presence and in the absence of the third residue. Both the Arg190-Glu231 ion pair and the Lys193-Glu231 salt bridge in the absence of the third residue, contribute favorably to the free energy for unfolding of the domain in urea. Using guanidinium chloride as denaturant reveals a strong cooperativity between the two ion-pair interactions, the presence of the second ion pair converts the first interaction from destabilizing into stabilizing by as much as 1.09 kcal/mol. The different energetics of the salt-bridge triad in urea and GdmCl are discussed with reference to the observed anion binding in the crystal structure at high ionic strength and their possible role in a highly charged, high-temperature environment such as the cytoplasm of hyperthermophiles. On the basis of recent progress in comparative structural analysis, molecular dynamics, and biochemical and biophysi- cal characterization of wild-type and mutant proteins from hyperthermophilic organisms, it is now becoming more apparent that charged amino acid residues and their arrange- ment into large networks play an important role in maintain- ing a stable and biologically active protein structure at high temperatures (1-4). Extensive networks of positively and negatively charged residues have been reported for example in glutamate dehydrogenases (GluDH) and lumazine synthase from hyperhermophiles (5-9). Initial mutagenesis studies on proteins from mesophiles have shown that exposed ion- pair interactions contribute only marginally to the stabiliza- tion free energy (10-12). It was argued that the gain in electrostatic energy upon formation of a salt bridge is offset by the entropic cost of desolvation and localization of the flexible side chains. However, at higher temperatures charged interactions become relatively more favorable and contribute more to protein stability. Due to a decreased desolvation penalty at higher temperature, the energy barrier to solvate an ion pair is higher, resulting in an increase in strength of electrostatic interactions. Molecular dynamics calculations have shown that at higher temperatures more energy is required to solvate and thereby disrupt a salt bridge while there is no such barrier for breaking the interaction between hydrophobic isosteres of charged amino acids (13). Using site-directed mutagenesis, ion-pair interactions have been removed from or have been introduced into proteins from hyperthermophiles. In a few cases, these substitutions had no effect on the resistance of the proteins against heat ² This work was partly supported by Marie Curie Individual Fellowship Contract QLK3-CT-1999-51141 to J.L. * Corresponding author. E-mail: rudolf.ladenstein@csb.ki.se. Phone: + 46 8 608 9222. Fax: + 46 8 608 9290. ‡ Karolinska Institutet. § Present address: Department of Molecular Carcinogenesis, Neth- erlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. | Universita ` ‘La Sapienza’. ⊥ So ¨derto ¨rns Ho ¨gskola. 1 Abbreviations: CD, circular dichroism; DTT, dithiotreitol; EDTA, ethylenediaminetetraacetic acid; GdmCl, guanidinium chloride; GluDH, glutamate dehydrogenase; rmsd, root-mean-square deviation. Arg190/ Ala193/Ala231 refers to the protein variant with an arginine at position 190, alanine at position 193, and alanine at position 231. Other protein variants are described accordingly. 15524 Biochemistry 2002, 41, 15524-15535 10.1021/bi020461s CCC: $22.00 © 2002 American Chemical Society Published on Web 12/05/2002