Yuhui Cheng Jan 7th, 2004

1. Welcome to Spring Festival Potluck in McCammon’s group on Jan. 21st, 2004 (Next next Wednesday)

2. Insight into Phosphoryl Transfer Mechanism of PKA with High Level ab initioQM/MM calculations and MD simulations (continued) Yuhui Cheng Jan 7th, 2004 http://mccammon.ucsd.edu/~ycheng

3. Outline • Problems unsolved in our previous QM/MM simulations. • Developments to solve these problems. @ High level ab initio QM/MM calculations for 1L3R crystal models. @ MD simulations to prepare different conformations for QM/MM calculations. • Future plan.

4. The Structure of C subunit How does the C subunit catalyze ATP to phosphorylate the peptide? Joseph A. Adams, Biochemstry, 2003, 42, 601-607

5. Short Review Small QM Subgroup: 32 atoms(3 pseudo bond “C-C”) Large QM Subgroup: 49 atoms(4 pseudo bond “C-C”) 20.0Å 27.0Å 1L3R: 8610 atoms 1ATP: 9652 atoms

6. Phosphoryl Transfer based on 1L3R crystal structure [HF(3-21G)/MM]

7. Problems • The calculations were accomplished under HF(3-21G)/MM, is it accurate enough to describe phosphoryl transfer in this enzyme? • The models were successful in 1L3R and 1ATP crystal structures, how about the relationship between the reaction activity, protein dynamics and different conformations?

8. Higher level QM/MM calculations • B3LYP(6-31G*)/MM approach was performed on 1L3R crystal structures. Both of small and large QM models were tried and analyzed. • Since reaction coordinates R1 was proved successfully, here R1 is used.

9. B3LYP(6-31+G*)/MM// B3LYP(6-31G*)/MM Ser 1L3R small model Kcal/mol 2.10 [2.12] 2.40 [2.32] 1L3R large model 1.04 [1.04] ADP 1.54 [1.54] Ser 1.80 [1.78] 2.80 [2.77] 1.48 [1.51] ADP D166 7.38 [11.40] 1.05 [1.04] D166 5.23 [9.36] 1.79 [1.76] Ser 3.01 [3.15] ADP 0.99 [0.99] 1.67 [1.64] D166 0 Reaction progress

10. Individual MM Residue Electrostatic Contributions to TS Stabilization

11. Asymmetric TS Ser Ser Ser 2.32 2.12 2.10 1.89 2.71 2.40 ATP D166 D166 ATP D166 ATP K168 K168 K168 1L3R small model 1L3R large model

12. Phosphoryl Transfer based on 1L3R crystal structure [B3LYP(6-31G*)/MM]

13. Conclusion • B3LYP(6-31G*)/MM obtains consistent and similar simulation results with HF(3-21G)/MM. • The structural difference of TS between small and large models is negligible. • Two possible structural factors influencing reaction barrier: * H-bond between P-site Ser and Asp166. * The distance between P-site Ser and ATP. • This phosphoryl transfer model stands late proton transfer, and characteristic vibration frequency of the TS is Oγ…Pγ…Oser .

14. Parameterization of Phospho Ser/Thr * * TPO SEP

15. MD simulation Result

16. Is it right? P-site Ser ATP Asp166

17. Re-parameterization of Mg2ATP O2(1) O2(1) O2(2) P(2) MgI OS(3) OS(2) P(3) HC P(1) O3 CT OS(1) HC O2(2) O3 MgII O3

18. MD simulation with New Parameter

19. Conformations in the Active Site II I 0% 100% 0% 100% OLD 15% NEW 53%

20. Frequency of Comformation II in new MD simulation O2(1) O2(1) O2(2) P(2) MgI OS(3) OS(2) P(3) HC P(1) O3 CT OS(1) HC O2(2) O3 MgII O3

21. Global RMSD in 10ns MD simulations

22. The Behavior of Ser53 and Gly-rich Loop 71% 99% 3%

23. Open and closing of Lys72 C Helix His87 (P)Thr197 Glu91 Lys72 99% 33% ATP

24. The Behavior of Lys168 and Thr201 15% 100% 100% 7% 8% 63% 50% 75%

25. Open and closing of the “gorge” Open Closed C Helix His87 Glu91 (P)Thr197 Arg165 208 185 Asp166 Glu208 Phe185 Activation Segment

26. The on-going and future work Relationship of dynamics and reaction activity QM/MM calculations Specific conformations K168A MD T197A MD K72A MD

27. The relationship we try to figure out • What are the factors to influence phosphoryl transfer mechanism? • How do they control the reaction? • The mechanism for them to block or favor the reaction.

28. Acknowledgement • Prof. J. Andrew. McCammon • Yingkai Zhang • Jie Liu • Other members in McCammon’s group