Yuhui Cheng Jan 7th, 2004

Welcome to Spring Festival Potluck in McCammon’s group on Jan. 21st, 2004

(Next next Wednesday)

Insight into Phosphoryl Transfer Insight into Phosphoryl Transfer Mechanism of PKA with High Mechanism of PKA with High

Level Level ab initioab initio QM/MM QM/MM calculations and MD simulations calculations and MD simulations

((continuedcontinued))Yuhui ChengYuhui Cheng

Jan 7th, 2004Jan 7th, 2004

http://mccammon.ucsd.edu/~ycheng

OutlineOutlineProblems unsolved in our previous QM/MM Problems unsolved in our previous QM/MM simulations.simulations.

Developments to solve these problems.Developments to solve these problems.

@@ High level High level ab initioab initio QM/MM calculations for QM/MM calculations for 1L3R 1L3R crystalcrystal models. models.

@@ MD simulations to prepare different MD simulations to prepare different conformations for QM/MM calculations. conformations for QM/MM calculations.

Future plan.Future plan.

The Structure of C subunitThe Structure of C subunit

How does the C subunit How does the C subunit catalyze ATP to catalyze ATP to phosphorylate the peptide?phosphorylate the peptide?

Joseph A. Adams, Biochemstry, 2003, 42, 601-607

Short Review Short Review

20.0Å

1L3R: 8610 atoms

1ATP: 9652 atoms

Small QM Subgroup: 32 atoms(3 pseudo bond “C-C”)

Large QM Subgroup: 49 atoms(4 pseudo bond “C-C”)

27.0Å

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

ProblemsProblems

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

Higher level QM/MM calculationsHigher level QM/MM calculations

B3LYP(6-31G*)/MMB3LYP(6-31G*)/MM approach was approach was performed on 1L3R crystal structures. performed on 1L3R crystal structures. Both of small and large QM models were Both of small and large QM models were tried and analyzed.tried and analyzed.

Since reaction coordinates Since reaction coordinates R1R1 was proved was proved successfully, here successfully, here R1R1 is used. is used.

ADP

ADP

ADP

Ser

Ser

Ser

D166

D166

D166

0.99[0.99]

1.79[1.76]

3.01[3.15]

1.67[1.64]

1.04

[1.04]

1.54[1.54]

2.10[2.12]

2.40[2.32]

1.05[1.04]

1.48

[1.51]

1.80[1.78] 2.80

[2.77]

0

Kcal/mol

7.38[11.40]

5.23 [9.36]

Reaction progress

B3LYP(6-31+G*)/MM// B3LYP(6-31G*)/MM1L3R small model1L3R large model

Individual MM Residue Electrostatic Individual MM Residue Electrostatic Contributions to TS StabilizationContributions to TS Stabilization

Asymmetric TS Asymmetric TS

1L3R large model 1L3R small model

2.32 2.12

ATP

Ser

D166

K168

ATP

Ser

D166

K168

2.71 1.89

K168

D166

Ser

ATP

2.402.10

Phosphoryl Transfer based on 1L3R Phosphoryl Transfer based on 1L3R crystal structure [crystal structure [B3LYP(6-31G*)/MMB3LYP(6-31G*)/MM]]

ConclusionConclusionB3LYP(6-31G*)/MM obtains consistent and B3LYP(6-31G*)/MM obtains consistent and similar simulation results with HF(3-21G)/MM. similar simulation results with HF(3-21G)/MM. The structural difference of TS between small The structural difference of TS between small and large models is negligible. and large models is negligible. Two possible structural factors influencing Two possible structural factors influencing reaction barrier:reaction barrier:

* H-bond between P-site Ser and Asp166.* H-bond between P-site Ser and Asp166. * The distance between P-site Ser and ATP.* The distance between P-site Ser and ATP.

This phosphoryl transfer model stands late This phosphoryl transfer model stands late proton transfer, and characteristic vibration proton transfer, and characteristic vibration frequency of the TS is frequency of the TS is OOγγ…P…Pγγ……OOserser . .

Parameterization of Phospho Parameterization of Phospho Ser/Thr Ser/Thr

SEP TPO

*

*

MD simulation Result MD simulation Result

Is it right?Is it right?

P-site Ser

ATP

Asp166

Re-parameterization of MgRe-parameterization of Mg22ATPATP

HC

CT

OS(3)P(3)

O2(2)

O2(2)

OS(2)

MgII

P(2) MgI

O2(1)O2(1)

OS(1)

HCP(1)

O3

O3

O3

NEW CHARGES OLD CHARGES MgI(MgII) 1.4532 2.0000

P(1) 1.1534 1.2650 P(2) 0.9691 1.3852 P(3) 0.8642 1.2532 O3 -0.7612 -0.9526

OS(1) -0.5157 -0.5322 OS(2) -0.3043 -0.5689 OS(3) -0.2289 -0.5987 O2(1) -0.6456 -0.8894 O2(2) -0.7370 -0.8799

CT 0.0484 0.6044 HC 0.0774 -0.1372

MD simulation with New ParameterMD simulation with New Parameter

Conformations in the Active SiteConformations in the Active Site

100% 0%OLD

NEW

100% 0%

I II

53% 15%

Frequency of Comformation II in Frequency of Comformation II in new MD simulationnew MD simulation

HC

CT

OS(3)P(3)

O2(2)

O2(2)

OS(2)

MgII

P(2) MgI

O2(1)O2(1)

OS(1)

HCP(1)

O3

O3

O3

Global RMSD in 10ns MD Global RMSD in 10ns MD simulations simulations

The Behavior of Ser53 and Gly-rich LoopThe Behavior of Ser53 and Gly-rich Loop

71%

99%

3%

Open and closing of Lys72Open and closing of Lys72

C Helix

ATP

His87

(P)Thr197Lys72 Glu91

33%

99%

The Behavior of Lys168 and Thr201The Behavior of Lys168 and Thr201

100%100%

63%7%

50%

8%

75%

15%

Open and closing of the “gorge”Open and closing of the “gorge”

Open

Closed

C Helix

185 208

(P)Thr197Glu91

Asp166

Arg165

Phe185 Glu208

Activation Segment

His87

The on-going and future workThe on-going and future work

MD simulation

2ns snapshot 2.2ns snapshot 12ns snapshot2.6ns snapshot2.4ns snapshot

T201A MD

QM/MM calculations

K72A MD K168A MD T197A MD

Specific conformations

Relationship of dynamics and reaction activity

The relationship we try to figure outThe relationship we try to figure out

What are the factors to influence phosphoryl What are the factors to influence phosphoryl transfer mechanism?transfer mechanism?

How do they control the reaction?How do they control the reaction?

The mechanism for them to block or favor the The mechanism for them to block or favor the reaction.reaction.

AcknowledgementAcknowledgement

Prof. J. Andrew. McCammonProf. J. Andrew. McCammon

Yingkai ZhangYingkai Zhang

Jie LiuJie Liu

Other members in McCammon’s groupOther members in McCammon’s group