The role of salt bridges on the temperature adaptation of aqualysin I, a thermostable subtilisin-like proteinase Lilja B. Jónsdóttir a , Brynjar Ö. Ellertsson a , Gaetano Invernizzi c , Manuela Magnúsdóttir a , Sigríður H. Thorbjarnardóttir b , Elena Papaleo c, ⁎, Magnús M. Kristjánsson a, ⁎⁎ a Department of Biochemistry, Science Institute, University of Iceland, Iceland b Institute of Biology, University of Iceland, Iceland c Structural Biology and NMR Laboratory, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen, Denmark abstract article info Article history: Received 20 June 2014 Received in revised form 5 August 2014 Accepted 20 August 2014 Available online 27 August 2014 Keywords: Subtilisin-like Salt bridges Protein stability Thermophilic Protease Molecular dynamics Differences in salt bridges are believed to be a structural hallmark of homologous enzymes from differently temperature-adapted organisms. Nevertheless, the role of salt bridges on structural stability is still controversial. While it is clear that most buried salt bridges can have a functional or structural role, the same cannot be firmly stated for ion pairs that are exposed on the protein surface. Salt bridges, found in X-ray structures, may not be stably formed in solution as a result of high flexibility or high desolvation penalty. More studies are thus needed to clarify the picture on salt bridges and temperature adaptation. We contribute here to this scenario by combin- ing atomistic simulations and experimental mutagenesis of eight mutant variants of aqualysin I, a thermophilic subtilisin-like proteinase, in which the residues involved in salt bridges and not conserved in a psychrophilic homolog were systematically mutated. We evaluated the effects of those mutations on thermal stability and on the kinetic parameters. Overall, we show here that only few key charged residues involved in salt bridges really contribute to the enzyme thermal stability. This is especially true when they are organized in networks, as here attested by the D17N mutation, which has the most remarkable effect on stability. Other mutations had smaller effects on the proper- ties of the enzyme indicating that most of the isolated salt bridges are not a distinctive trait related to the enhanced thermal stability of the thermophilic subtilase. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Aqualysin I (AQUI) is an extracellular subtilisin-like serine protein- ase (subtilase) from the thermophilic bacterium Thermus aquaticus [1–4]. The enzyme is related to subtilases, which have been classified as proteinase K-like on the basis of sequence homology [5]. The subtilase members have been extensively studied structurally and several high-resolution three-dimensional (3D) structures are available in the PDB [6]. These include the structure of proteinase K at atomic resolution [7], but also a structure of the thermophilic aqualysin I [8], as well as that of a close structural homolog from a psychrotrophic Vibrio sp. (VPR) [9]. It has been suggested that one of the main characteristics of thermo- philic enzymes is their highly rigid protein structure compared to their meso- and psychrophilic counterparts. As a result, thermophilic enzymes are highly thermostable, but are characterized by relatively low catalytic efficiency, except at high temperatures [10–13]. Cold- adapted enzymes on the other hand are characterized by high catalytic efficiencies at low temperatures, but their thermal stability is generally compromised, both properties that have been related to higher struc- tural flexibility of the cold-adapted enzymes [12,14–16]. Results of comparative structural studies of homologous enzymes of different temperature origins indicated structural features that may be associated with temperature adaptation of proteins. These involve combination and adjustment of different structural features, e.g. num- ber of salt bridges or their networks, hydrogen bonds, number of proline residues in loops, hydrophobic core packing, as well as hydrophilic vs hydrophobic surface exposure [12,13,16–19]. These studies also sug- gested that an increased number of salt bridges may play a crucial role in thermostabilization, especially for hyperthermophilic enzymes [10, 20–26]. Indeed, in the X-ray structures of hyperthermophilic proteins, ion pairs are often observed in the form of clusters or networks at the protein surfaces or at the domain–domain/protein–protein interfaces Biochimica et Biophysica Acta 1844 (2014) 2174–2181 Abbreviations: PMSF, phenylmethanesulfonyl fluoride; VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species; AQUI, aqualysin I; Suc-AAPF-NH-Np, suc- cinyl-AlaAlaProPhe-p-nitroanilide; MD, molecular dynamics; rmsf, root mean square fluctuation ⁎ Corresponding author. Tel.: +45 35322026. ⁎⁎ Corresponding author. Tel.: +354 525 4800, fax: +354 552 8911. E-mail addresses: elena.papaleo.78@gmail.com, elena.papaleo@bio.ku.dk (E. Papaleo), mmk@hi.is (M.M. Kristjánsson). http://dx.doi.org/10.1016/j.bbapap.2014.08.011 1570-9639/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap