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

2. Temperature-dependent subtilases
temp.optimum protease structure
psychrophile
Bacillus TA oC subtilisin TA41 model
mesophile
Bacillus amyloliquefaciens oC subtilisin BPN’ X-ray
thermophile
Thermoactinomyces vulgaris oC thermitase X-ray
hyperthermophile
Thermococcus stetteri oC stetterlysin model
Pyrococcus furiosus oC pyrolysin model

3. 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 !!

4. 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

5. 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

6. 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

7. Sequence alignment

8. 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

9. 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

10. 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)

11. 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)

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

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

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

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

16. Catalytic domain properties

17. 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)

18. 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