Protein Engineering vol.10 no.8 pp.905–914, 1997 Homology modelling of two subtilisin-like serine proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri Wilfried G.B.Voorhorst1, Angela Warner1, stabilization mechanisms that allow proteins to be functional Willem M.de Vos1,2 and Roland J.Siezen2,3 near the maximum temperature of life. Analysis of three- 1 dimensional structures and modelled structures of proteins Department of Microbiology, Wageningen Agricultural University, Downloaded from https://academic.oup.com/peds/article/10/8/905/1487418 by guest on 24 September 2021 Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands from hyperthermophiles and their comparison with those from and 2Department of Biophysical Chemistry, NIZO, PO Box 20, NL-6710 less stable proteins has resulted in identification of features BA Ede, The Netherlands that may be related to protein stabilization. Mechanisms by 3To whom correspondence should be addressed which proteins from thermophiles are stabilized resemble those identified for mesophilic model proteins and include hydrogen The hyperthermophilic archaeon Pyrococcus furiosus bonds, hydrophobic interactions, ion-pairs, secondary structure produces an extracellular, glycosylated hyperthermo- stabilization, cavity filling, reduction of the length of loops stable subtilisin-like serine protease, termed pyroly- and conformational strain (reviewed by Tomschy et al., 1994; sin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Vo¨ lkl et al., 1994; Muir et al., 1995; Szila´gyi and Za´vodsky, Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. 1995; Korndorfer et al., 1995; Yip et al., 1995; Vieille Chem., 271, 20426–20431). Based on the pyrolysin coding and Zeikus, 1996; Knapp et al., 1997). It seems that each sequence, a pyrolysin-like gene fragment was cloned and thermostable protein is stabilized by a unique combination of characterized from the extreme thermophilic archaeon different mechanisms resulting in a more rigid structure. A Thermococcus stetteri. Like pyrolysin, the deduced sequence common theme that has now emerged from analysis of the of this serine protease, designated stetterlysin, contains a structures identified so far for hyperthermophiles suggests that catalytic domain with high homology with other subtilases, an increased number of ion-pairs, that may be arranged allowing homology modelling starting from known crystal in extensive networks, contributes to the observed extreme structures. Comparison of the predicted three-dimensional thermostability (Korndorfer et al., 1995; Yip et al., 1995; models of the catalytic domain of stetterlysin and pyrolysin Knapp et al., 1997). Moreover, many proteins from hypertherm- with the crystal structure of subtilases from mesophilic ophiles are not only extremely thermostable, but also show a and thermophilic origin, i.e. subtilisin BPN9 and thermitase, higher resistance to chemical denaturation and proteolytic and the homology model of subtilisin S41 from psychro- degradation than their less stable counterparts (Fontana, 1991). philic origin, led to the identification of features that could This can be illustrated with pyrolysin, an extracellular serine be related to protein stabilization. Higher thermostability protease from the archaeon Pyrococcus furiosus that shows was found to be correlated with an increased number of optimal growth at 100°C (Fiala and Stetter, 1986). This residues involved in pairs and networks of charge–charge protease has a half-life value of 4 h at 100°C and is still active and aromatic–aromatic interactions. These highly thermo- in the presence of 6 M urea (Eggen et al., 1990; Voorhorst stable proteases have several extra surface loops and inserts et al., 1996). with a relatively high frequency of aromatic residues and The family of subtilisin-like serine proteases, also referred Asn residues. The latter are often present in putative to as subtilases (Siezen et al., 1991; Siezen and Leunissen, N-glycosylation sites. Results from modelling of known 1997) has been extensively studied from psychrophilic, meso- substrates in the substrate-binding region support the philic and thermophilic origin to gain insight in thermostability, broad substrate range and the autocatalytic activation catalytic activity and substrate specificity (Wells and Estell, previously suggested for pyrolysin. 1988; Davail et al., 1994; Daniel et al., 1995; Feller et al., Keywords: Archaea/Pyrococcus furiosus/serine protease/Ther- 1996). However, knowledge of subtilases from hyperthermo- mococcus stetteri/thermostability philic origin is limited to pyrolysin from P.furiosus (Eggen et al., 1990; Voorhorst et al., 1996) and STABLE from Staphylothermus marinus (Mayr et al., 1996), which have been Introduction extensively characterized on the biochemical level with specific To gain insight in the molecular basis of thermostability attention for thermostability and substrate specificity. More- of proteins, structurally homologous proteins with different over, the subtilase aerolysin from the hyperthermophile thermostability have been compared using known crystal Pyrobaculum aerophilum has been analysed for features that structures or predicted structures from homology modelling could be related to its thermostability using homology model- (Teplyakov et al., 1990; Davail et al., 1994; Heringa et al., ling (Vo¨lkl et al., 1994). 1995; Tomschy et al., 1994; Muir et al., 1995; Szila´gyi and The amino acid sequence homology in the catalytic domain Za´vodszky, 1995). The recent isolation and characterization of pyrolysin with other subtilases is sufficiently high to allow of proteins and their genes from hyperthermophiles, micro- for homology modelling (Voorhorst et al., 1996). However, organisms able to grow optimally at 80°C or higher (Stetter the identification of features as being important for protein et al., 1990), generates the possibility to extend the structural stabilization based on only a single enzyme may lead to analysis to extremely stable proteins to gain insight in the misinterpretation (Querol et al., 1996). Therefore, we have © Oxford University Press 905 W.G.B.Voorhorst et al. now isolated and characterized the gene encoding a homolog sequence analysis showed the presence of a KpnI restriction of pyrolysin, designated stetterlysin, from the extreme thermo- site in the amplified fragment that could be used for cloning philic archaeon Thermococcus stetteri that grows optimally at chromosomal DNA fragments. A Southern blot containing 75°C (Miroshnichenko et al., 1989). The amino acid sequence chromosomal DNA of T.stetteri digested with different com- homology within the core of the catalytic domain between binations of KpnI and other restriction enzymes was probed stetterlysin, pyrolysin and other subtilases allowed homology with the cloned PCR fragment. Positively hybridizing frag- modelling using known three-dimensional protein structures ments were subsequently cloned in appropriately digested of subtilisin BPN9 from the mesophilic bacterium Bacillus pUC18 and characterized by nucleotide sequence analysis. amyloliquefaciens with an optimum growth temperature between 30 and 40°C (Heinz et al., 1991; Gallagher et al., 1995) Sequence analysis and alignment and the more thermostable thermitase from Thermoactinomyces Automated nucleotide sequence analysis of plasmid DNA was vulgaris that grows optimally at 50°C (Gros et al., 1989). The carried out on either an Applied Biosystems 373A sequencer predicted three-dimensional structures of the highly thermo- (Applied Biosystems Inc.) using a PrismTM Ready Reaction stable pyrolysin and stetterlysin were compared with the DyeDeoxyTM Terminator cycle sequencing kit or the Li-Cor Downloaded from https://academic.oup.com/peds/article/10/8/905/1487418 by guest on 24 September 2021 homology model of subtilisin S41 from the psychrophilic 4000L sequencer (Li-Cor Inc.) using the Amersham Thermo Bacillus T41 (optimal growth temperature of 4°C), that has a Sequenase cycle sequencing kit with 7-deaza-dGTP and half-life value of only a few minutes at 50°C (Davail infrared labelled oligonucleotides (MWG-Biotech, Germany). et al.,1994), and with subtilisin BPN9 and thermitase, with Computer analysis of nucleotide and deduced amino acid half-life values of 23 min at 60°C (Braxton and Wells, sequences was carried out with the PC/GENE program version 1992) and 1 h at 85°C (Daniel et al., 1995), respectively. 5.01 (IntelliGenetics Inc.) and the GCG package version 7.0 Conformational characteristics of the catalytic domain of (Devereux et al., 1990) at the CAOS/CAMM Centre of the stetterlysin and pyrolysin were analysed that could be related University of Nijmegen (The Netherlands). to their thermostability regarding overall composition, ion- The amino acid sequences of pyrolysin and stetterlysin were pair formation, aromatic–aromatic interactions, amino acid included in the multiple sequence alignment of the catalytic replacements, helix stabilization and calcium-binding sites. domains of the entire subtilase family (Siezen et al., 1991; Additionally, the substrate binding regions of these archaeal Siezen and Leunissen, 1997). In this way the sites of insertions subtilases were modelled in detail with respect to substrate in the catalytic domain could be identified. The numbering of specificity and interactions between the enzymes and their subtilisin BPN9 indicated by stars (*) was used as reference. known substrates. Modelling of the catalytic domains of pyrolysin