1. A Computational Enzyme Activity Design of HIV-1 Protease Luca De Vico WATOC 2011 Santiago de Compostela, Spain
2. What the project is about Modify a protease in order to cleave another desired sequence New enzymatic activity towards a specific sequence Rational redesign of the enzyme Valuable tool for biological applications Extended to any sequence Experimental and computational collaboration
3. Enzyme redesign: what is needed - A target sequence to cleave - A template protease: HIV-1 protease ✂ Pro His Leu Ser Phe Met Ala Ile Pro Pro
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5. Enzyme redesign: what is needed X-ray structure of wild type protease docked with one of its natural substrates: 1KJ7 The protocol has to produce: - reasonable structures with many different substrate peptides bound - generate mutants with lower binding energy
6. Our PyRosetta optimization protocol Substrate peptide sequence Starting structure x6 f, ψ, χ perturbations Energy minimization (David-Fletcher-Powell) x8 x4 Out of 500 decoy structures the lower in energy is chosen. x6 MC criterion Side-chain packing MC criterion ca. 24 hours on 5 cpus Output decoy
7. The perturbation and minimization are on: Backbone Side chains Substrate peptide Protease cavity Residues inside 5 Å radius from the peptide Any residue reported as active in Chaudhury et al. Their ±1 sequence neighbors Both chains have the same residues as “movable”
11. Used to compute qualitative binding energiesEbinding = Ecomplex – Eapo – Epeptide
12. Qualitative binding energies FMO MP2/PCM/6-31G* (kcal/mol), Wild Type protease Average binding energy Natural Target Cleavable Peptides -60.9 Min value -79.2 Max value -41.1 Standard deviation 11.0 Average binding energy Known NON-CleavablePeptides -13.7 Min value -68.3 Max value 61.0 Standard deviation 28.1 Target peptide sequence binding energy: -24.1
13. Enzyme redesign: mutation protocol Among the cleavable peptides sequences, the closest to the target is mutated into the target sequence, one amino acid at the time Cleavable start ✂ ✂ Val Ser Phe Asn Phe Met Ala Ile Leu Thr Pro His Leu Ser Phe Pro Gln Ile Pro Pro Target
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15. Only the protease 6 specificity determining residues are allowed to mutate (ca. 2000 total rotamers per perturbation)
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18. Evaluation of energy barrier for qualitative differences between wild type and mutated protease
26. Example of optimization convergence MA-CA Each optimization cycle requires ca. 24 hours on 5 cpus
27. FMO MP2/PCM/6-31G* binding energies of cleavable peptides Kcal/mol Differences between the binding energies of WT HIV-1 protease and its natural substrates are expected and will be experimentally checked
28. Enzyme redesign Among the experimentally verified cleavable peptides, the closest to the target sequence is chosen Target sequence TF-PR cleavage sequence, candidate starting sequence Specificity most involved peptide residues The candidate substrate sequence is mutated into the target sequence one amino acid at the time
29. Enzyme redesign The candidate substrate sequence is mutated into the target sequence one amino acid at the time
