Hyperthermophile subtilases Homology modelling of two subtilisin-like serine proteases from the hyperthermophile archaea Pyrococcus furiosus and Thermococcus stetteri Protein Engineering 10 (1997) 905-914 Wilfried Voorhorst, Angela Warner, Willem de Vos Wageningen University, Wageningen, the Netherlands Roland J. Siezen NIZO food reseach, Ede, the Netherlands

Temperature-dependent subtilases temp.optimum protease structure psychrophile Bacillus TA41 0 - 5 oC subtilisin TA41 model mesophile Bacillus amyloliquefaciens 40 - 50 oC subtilisin BPN’ X-ray thermophile Thermoactinomyces vulgaris 60 - 70 oC thermitase X-ray hyperthermophile Thermococcus stetteri 80 - 90 oC stetterlysin model Pyrococcus furiosus 95 -100 oC pyrolysin model

Protein stabilization H-bonds S-S bonds hydrophobic interactions aromatic interactions salt bridges (ion pairing) helix dipole, helix capping shorter loops glycosylation In general: REDUCE FLEXIBILITY !!

Protein temperature stabilization Amino acid substitution effect CysH, Met  X reduce oxidation Asn, Gln  X reduce deamination Asp  X reduce peptide cleavage X  Arg, Lys, Glu, Asp more ion pairing/networks Lys  Arg more H-bonds X  Phe, Tyr, Trp more aromatic interactions Gly  Ala helix stabilization add helix caps helix stabilization add Pro in loops less flexible add Asn in loops more glycosylation sites

Homology modelling steps Steps Tools Select protein sequence and family BLAST, Pfam Select known X-ray structures PDB Sequence alignment FASTA, Clustal Create homology model framework Quanta Introduce deletions, insertions Quanta, PDB Transfer new side chains to model Quanta, Charmm (Introduce S-S, ion binding sites) Quanta, Charmm Energy minimization (constraints !) Charmm Evaluate model ProCheck

X-ray structures subtilisin BPN’: thick: red, yellow thermitase: thin: green, blue 3-D alignment superimpose identical residues  conserved core of -helices and -sheet strands

Sequence alignment

Create homology model framework Based on sequence alignment: select the segments of subtilisin BPN’ (green) and thermitase (red) with highest sequence identity best loop length to pyrolysin or stetterlysin

Modelling of insertions, deletions +147 +29 +27 +8 Based on sequence alignment: introduce deletions add insertions (< 7 residues) -2 +6 +5 +4 +2 Pyrolysin model = modelled = not modelled

Completing the model Transfer new side chains to model pyrolysin or stetterlysin sequence regularize molecule (Quanta/Charmm) (Introduce S-S, ion binding sites) no S-S bonds, no known Ca2+ binding sites Energy minimization constrain catalytic residues (Asp, His, Ser, Asn) constrain substrate binding region (-sheet strands) energy minimize (Charmm) Evaluate model parameters Ramachandran plot: main chain phi-psi angles side chain parameters (ProCheck)

Conclusions from models As temperature stability increases: Amino acid composition no CysH, no S-S bonds increase charged residues (mainly acidic) increase aromatic residues (Phe, Trp, Tyr) Helix stabilization no specific trend Side chain interactions increase of surface ion pairs and networks increase of surface aromatics (pairs, clusters)

Pyrolysin: charged residues red: acidic blue: basic yellow: inserts ftp://ftp.cmbi.kun.nl/pub/molbio/siezen97/

Stetterlysin: charged residues red: acidic blue: basic yellow: inserts ftp://ftp.cmbi.kun.nl/pub/molbio/siezen97/

Stetterlysin: aromatic residues purple: aromatic yellow: inserts ftp://ftp.cmbi.kun.nl/pub/molbio/siezen97/

Pyrolysin: aromatic residues purple: aromatic yellow: inserts ftp://ftp.cmbi.kun.nl/pub/molbio/siezen97/

Catalytic domain properties

Conclusions from models Loops in (hyper)thermophile subtilases Loop size many longer loops (inserts) larger loops cannot be modeled !! Amino acid composition rich in aromatic residues (20%) no reduction of thermolabile Asn residues Modification many potential glycosylation motifs Asn-X-(Ser/Thr)

General conclusions Homology model is only a first approximation: several (large) loops cannot be modeled interaction of surface residues difficult to model (no H2O) BUT: model suffices for general predictions Thermostability of hyperthermophilic enzymes may be correlated with: increase of ionic interactions, networks increase of aromatic interactions, clusters