ShOUL ULD D i S i STAY Y OR SHOULD ULD I S I STAY: : PRO ROTON ON TRANS NSFE FER R REVI VISITED SITED Paul Czodrowski SPPEXA 2016 // Software for Exascale Computing
Proton transfer? standard pKa values 1 protonation state amino acid pKa value pKa value Arg 13.0 0.5 Asp 4.0 Glu 4.4 His 6.3 Lys 10.4 0 Tyr 9.6 1 14 pH 2
pKa calculations? empirical size of studied systems implicit 1 models pK G 2 . 303 a molecular kT dynamics quantum mechanics speed 3
pKa calculations? computed experimentally determined G diss AH (aq) A - (aq) +H + Solvent (aq) G (AH) G (A - ) pK a P-AH P-A - +H + Protein Not to forget: We get pKa values for all titratable residues! 4
Development of peoe_pb : Our partial charge methodology Partial charges solvent ( e =80) Poisson- Implicit solvent model protein Boltzmann ( e =20) equation Electrostatic potential P artial E qualisation of O rbital E lectronegativities (PEOE) : orbital electronegativity distribution of = 0.5*(IP+EA) k k k k B A = a+b*q+c*q 2 +d*q 3 q 5 A
Validation of our partial charge methodology peoe_pb Solvation free energies of small molecules pKa values in proteins calculated G solv [kcal/mol] calculated pKa r 2 e Protein =20 = 0.78 RMSD = 1.57 experimental G solv [kcal/mol] experimental pKa 6
Protonation changes detected by ITC Thrombin Trypsin H H n H meas bind ion 7
Protonation effects for trypsin Thrombin Trypsin 1x 1b 1c 1d 4 5 8
trypsin/1b vs trypsin/1c ITC: n=+0.90 * ITC: n=±0.0 FDPB: n=+0.51 FDPB: n=±0.0 His57: large pKa shift * proton uptake 9
HIV protease – apo state 1HHP 3HVP catalytic dyad Asp25 Asp25 ‘ pK a1 pK a2 Catalytic dyad Experiment 3.1 – 3.7 4.9 – 6.8 Mono-protonated 10 Calculation 3.8 6.8 Mono-protonated
DMP-323 bound to HIVP 1QBS DMP-323 catalytic dyad Asp25‘ Asp25 pK a1 pK a2 Catalytic dyad Experiment > 7.2 > 7.2 Doubly protonated 11 Calculation 5.3 10.7 Doubly protonated
What else can one do with pKa calculations? 12
pKa values & covalent bonds? Addition Reaction of Model Compounds with Glutathione taken from: http://dx.doi.org/10.1021/jm400822z 13
The cysteinome of the kinome taken from: http://dx.doi.org/10.1021/jm101396q 14
Set-up of the calculations • Per target kinase, all public PDB structures are used • The ligand is not considered in the calculation • Processing/Calculation is done by OpenEye‘s protein_pka Amino Acid Dictionary Protein PB pKa pKas Protein PDB Ligand 15
Analysis of the calculations Box plots for all CYS pKa values per protein Box plots for all CYS SASA values per protein CYS pKa value „out of value“ (i.e. >20) SASA pK a CYS model pKa value That‘s my NULL model 16
EGFR: Covalently attacked CYS-797 3w2p SASA: 38,96 Å 2 (w/o covalent Inhibitor) pK a Cys.781 Cys.797 3w2o Cys.775 SASA: 28,49 Å 2 (w/o non-covalent inhibitor) Cys.797 apo structures Cys.818 Covalently attacked Cys.950 Cys.939 Cys.797 17
EGFR: Covalently attacked CYS-797 pK a SASA Cys797 18
EGFR: Covalently attacked CYS-797 Experimental determination of the pKa value Oral communication at the GordonConference ComputerAided Drug Discovery pKa EGFR.CYS797 = 6.53 +/- 0.05 Work done Site point mutation EGFR/CYS at position 796 pKa = 8.43 at Pfizer Site point mutation EGFR/CYS at position 798 pKa = 8.12 19
Summary … because • They tell you something about the protonation effect of ligand-protein binding • The nucleophilicity of the CYS residue pKa seems to be related to the predicted pKa calculations are cool Why not consider protonation changes in long-scale MD simulations? 20
Acknowledgment MERCK • Gerhard Klebe • Carl Deutsch • Anthony Nicholls • Christoph Sotriffer • Christoph Scholz • Mike Word • Frank Dullweber • Jose Batista • Ingo Dramburg • Gunther Stahl 21
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