Theoretical Investigation of the Structural Characteristics in Active

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Theoretical Investigation of the Structural Characteristics in Active State of Akt1 Kinase Lanxuan Liu, Shuobing FAN, Wenjuan Li, Wentao Tao, Ting Shi, and Yi-Lei Zhao J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00506 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 20, 2018

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1

Theoretical Investigation of the Structural Characteristics in

2

Active State of Akt1 Kinase

3 4

Lanxuan Liu1, Shuobing Fan1, Wenjuan Li1, Wentao Tao1, Ting Shi1,*, Yi-Lei Zhao1,*

5 6 7 8 9

1State

Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

10 11 12 13 14 15

*To whom correspondence should be addressed:

16 17

Dr. Ting Shi

18

School of Life Sciences and Biotechnology

19

Shanghai Jiao Tong University

20

800 Dongchuan Road, Shanghai 200240, China

21

Tel/Fax: +86-21-34207347;

22

Email: [email protected]

23 24

Prof. Yi-Lei Zhao

25

School of Life Sciences and Biotechnology

26

Shanghai Jiao Tong University

27

800 Dongchuan Road, Shanghai 200240, China

28

Tel/Fax: +86-21-34207190;

29

Email: [email protected] 1


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1

TABLE OF CONTENTS (TOC) GRAPHIC

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2



1

ABSTRACT

2

Akt (known as protein kinase B, or PKB) is a serine/threonine kinase that regulates

3

multiple biological processes, including cell growth, survival and differentiation. Akt

4

plays a critical role in intracellular signaling network through conformational changes

5

responsive to diverse signal inputs, and dysregulation of Akt activity could give rise to

6

a number of diseases. However, understanding of Akt’s dynamic structures and

7

conformational transitions between active and inactive states remains unclear. Herein,

8

classical MD simulations and QM/MM calculations were carried out to unveil the

9

structural characteristics of Akt1, especially its active state. The doubly protonated

10

H194 was investigated and both ATP-Akt1 and ADP-Akt1 complexes were constructed

11

to demonstrate the significance of ATP in maintaining ATP-K179-E198 salt bridge and

12

the corresponding allosteric pathway. Besides, conformational transitions from inactive

13

to active state showed different permeation patterns of water molecules in the ATP

14

pocket. The coordination modes of Mg2+ in the dominant representative conformation

15

(I and I′) were presented. Unlike the water-free conformation I′, three water molecules

16

were appeared around Mg2+ in the water-occupied conformation I, which would finally

17

exert an influence on the catalytic mechanism of Akt1. Furthermore, QM/MM

18

calculations were performed to study the phosphoryl-transfer reaction of Akt1. The

19

transfer of ATP γ-phosphate was achieved through a reversible conformational change

20

from reactant to a critical prereaction state, with a water molecule moving into the

21

reaction center to coordinate with Mg2+. After that γ-phosphate group transferred from

22

ATP to the substrate. Taken together, we elucidated the structural characteristics of

23

Akt1 active state and shed a new light on the catalytic mechanism of Akt kinases.

24 25

KEYWORDS

26

Akt1, structural characteristics, active conformation, molecular dynamics (MD)

27

simulation, QM/MM

28 3


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1

INTRODUCTION

2

As a serine/threonine kinase, Akt (also called protein kinase B, or PKB) regulates

3

multiple biological processes by post-translational phosphorylation of serine or

4

threonine residue.1 Akt is encoded by a proto-oncogene, and malfunction of its

5

regulatory machinery often leads to various diseases and disorders, including cancers,

6

diabetes, cardiovascular diseases, insulin resistance and type 2 diabetes, inflammatory

7

and autoimmune disorders, and neuronal disorders.2 Frequent activation of Akt has

8

been reported in various human cancers, such as glioblastoma3, ovarian carcinoma4,

9

breast carcinoma5, non-small cell lung carcinoma6 and hematological malignancies7,

10

etc. Akt activation has been shown to correlate with advanced disease and/or poor

11

prognosis in some of these cancer types3, 8 and inhibitors developed to suppress Akt

12

activation have showed significant clinical potentials to improve therapeutic strategies9.

13

Design of ATP competitive inhibitors in the active site of Akt kinases as anticancer

14

agents has yielded great research interests,10-12 and thorough explorations of Akt active

15

state on atomic level for further drug developments are still desiderated nowadays.

16

Activation of Akt is triggered by the production of phosphatidylinositol 3,4,5

17

triphosphate (PIP3) through upstream phosphoinositide 3 kinase (PI3K) activation.13

18

Three types of Akt kinases are identified, Akt1, Akt2 and Akt3, sharing above 80%

19

sequence identity.14 All three Akt kinases consist of a N-terminal PH-linking domain,

20

a central kinase domain and a C-terminal regulatory domain.15 Akt kinase domain is

21

divided into two parts, the N-terminal small lobe and C-terminal large lobe which

22

together constitute a nucleotide-binding pocket (Figure 1A). The N-lobe consists of five

23

antiparallel β sheets (β1-5 strand) and two α-helices (αB and αC helix). A glycine-rich

24

loop (G-loop), located between the β1 and β2 strand, shadows the phosphate groups of

25

ATP and contributes to phosphoryl transfer. C-lobe is mainly composed of α-helices

26

and the dynamics of significant structural elements of both N-lobe and C-lobe are

27

addressed.16 The conserved Asp-Phe-Gly (DFG) sequence, which directly binds to

28

active site Mg2+ ions, is extremely important for catalysis.17 Conformational changes 4



1

of Akt include αC-in/αC-out, DFG-in/DFG-out, and structural transformation in G-loop,

2

activation loop, pT308, and hydrophobic motif. There are two phosphorylation sites in

3

Akt1, T308 in the A-loop and S473 in the hydrophobic motif.18 Although only 10% of

4

reactive ability is remained in monophosphorylation pT308 Akt compared to the fully

5

activated diphosphorylation state, phosphorylation of T308 is essential in Akt

6

catalysis.19 Besides, pT308 can also resist dephosphorylation by interacting with H194

7

in αC helix and R273 (YRD motif) in the catalytic loop.20 Over 100 Akt substrates have

8

been reported since the first substrate, glycogen synthase kinase 3 (GSK 3).21 Akt1

9

tends to recognize substrate peptide sequence like RXXRXX(S/T) Hyd and conduct

10

phosphorylation on site (S/T) at P(0) position, arginine (R) at P(-3) and P(-5), and a

11

hydrophobic residue (phenylalanine) at P+1 (Figure 1B).19

12 13

Figure 1. (A) Crystal structure of Akt1 (PDB code: 4EKK26, where AMP-PNP was replaced by

14

ATP). N-lobe and C-lobe are shown as surface in green and yellow, respectively. ATP and pT308

15

are shown as sticks. Key structural elements are indicated by different colors in cartoon. (B) The

16

substrate peptide is shown as stick.

17 5


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1

To date, numerous crystal structures have provided static information for studying

2

protein dynamics of general kinases in both active and inactive states.22-25 ATP-

3

dependent allosteric regulation of Akt phosphorylation has already been studied

4

extensively using both computational and experimental methods.20,26-28 Cheng et al.

5

studied both phosphorylated and dephophorylated forms of Akt to reveal the

6

differentiated motion patterns and the crucial role of the protonation state of H196 in

7

the active conformation.16 Chan et al. reported that targeting Akt kinase to the cell

8

membrane could protect it from phosphatase, which was further amplified by

9

occupancy of the ATP binding pocket with ATP or ATP-competitive inhibitors.20

10

Besides, Lu et al. and Mou et al. also demonstrated similar protection effects, where

11

ATP or ATP-competitive inhibitors could be utilized to stabilize the active state

12

conformation, and the corresponding allosteric pathway in Akt1 (ATP/GDC-0068 →

13

T160→F161→E191→H194→pT308) was proposed.27-28

14

Herein,

classical

molecular

dynamic

(MD)

simulations

and

quantum

15

mechanics/molecular mechanics (QM/MM) calculations were carried out to explore the

16

conformational characteristics of the active state of Akt1 kinase. Functions of polar

17

hydrogen atoms in imidazole of H194 with different protonation states of Akt1 were

18

studied. ADP-Akt1 system was compared with ATP-Akt1 to investigate the influence

19

of γ-phosphate group on ATP-K179-E198 salt bridge and the allosteric pathway.

20

Conformational transitions from inactive to active states were captured and three

21

conformations were employed to present the dynamical variations. Structural

22

characteristics of representative conformations (I and I′) were analyzed and the

23

mechanism of Akt1 phosphoryl-transfer reaction were explored by QM/MM

24

calculation.

25 26

MATERIALS AND METHODS

27

System preparation and MD simulations. Initial protein structure of ATP-bound

28

Akt1 complex was obtained from X-ray crystal data (with AMP-PNP, Mg2+, and 6



1

substrate peptide, PDB entries: 4EKK), and AMP-PNP was replaced by ATP.26 Three

2

systems with different His194 protonation states (HID, HIE and HIP) were constructed.

3

The ADP-bonded structure was generated through removal of the γ-phosphate of ATP

4

from the HIP complex. Parameters preparation for Akt1 was carried out using ff12SB

5

force field in AMBER 12 package.29 Force field parameters for ATP, ADP, pT308 and

6

Mg2+ ion, are available in the AMBER parameter database. The protonation states of

7

residues at pH 7.0 were determined by the PDB2PQR web server,30 and pKa

8

calculations were performed by PROPKA.31-33 The protein was externally solvated in

9

an octahedral box of TIP3P water molecules with a 10 Å buffering distance. Sodium

10

ions were then added as counterions to maintain system neutrality.

11

The solvated system was subjected to 10000 steps of steepest descent energy

12

minimization followed by 1000 cycles of conjugate gradient minimization. Then,

13

gradual heating of the whole system from 0 K to 300 K in 50 ps controlled by Langevin

14

dynamics was carried out with a 2-ps temperature collision constant. Positional

15

harmonic restraints were imposed on the protein and ligand using a force constant of 5

16

kcal*mol-1*Å-2. Thereafter, 50 ps equilibration was carried out at constant pressure and

17

temperature (NPT ensemble) to adjust the density of the system to the experiment level.

18

Following equilibration, simulations were performed in the NPT ensemble at 300 K

19

and 1 atm.34-35 During simulations, the time step was set to 2 fs, and lengths of all

20

chemical bonds involving hydrogen atoms were constrained with the SHAKE

21

algorithm.36 The cutoff of non-bonded interactions was set to 10.0 Å and the Particle

22

Mesh Ewald (PME) algorithm was employed to handle long range electrostatic

23

interactions.37 The aforementioned protocol was applied to all of the simulation systems

24

used in this study.

25

Trajectories were analyzed with cpptraj module implanted in Ambertools14.28

26

Root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) of Cα,

27

C and N atoms in the whole system were calculated for all MD trajectory frames.

28

Stability of a hydrogen bond was measured by the frequency of its appearance in the 7


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1

entire trajectory (the acceptor-donor cutoff distance was set to 3.0 Å and the acceptor-

2

hydrogen-donor cutoff angle was set to 135°). Clustering calculations using the

3

average-linkage algorithms were performed to assign frames from the trajectory into

4

different categories according to their structural similarity, and representative structures

5

of each cluster were further studied.38

6

QM/MM calculation. The ONIOM39 calculations were performed with the Gaussian

7

09 software.40 Representative conformations of the dominant cluster in MD trajectories

8

were extracted as reactants (denoted as R and R′) and subjected to geometry

9

optimization and energy calculations. Structural superimpose between crystal structure

10

(inactive state) and the representative conformation (active state) was shown in Figure

11

S1. The quantum mechanical (QM) region consisted of the sugar ribose and

12

triphosphate of ATP, two Mg2+ ions, six residues (T160, K179, D274, K276, N279,

13

D292), the substrate serine P(0)), and four water molecules in close interaction with the

14

active site (Figure S2). The total system had -3 net charges and 10257 atoms, with 1698

15

solvent water molecules. The 114-atom QM model, bearing zero net charge, was treated

16

with hybrid functional M06-2X41 and basis set 6-31G(d). The MM region was treated

17

with Amber-type force field.42 Link atom scheme was used to partition QM and MM

18

region.43 To generate the conformation of intermediate (prereaction state), distance

19

between O3γ atom of ATP and MgII atom was scanned from 2.00 Å to 4.00 Å with 0.1

20

Å as the interval step. During this process, one water molecule gradually approached to

21

MgII ion, and finally the O3γ atom was liberated from the constraint of MgII ion. The

22

intermediate structure was obtained through full optimization of the last scanned

23

structure and the final distance between O3γ atom of ATP and MgII atom was 4.03 Å.

24

Besides, the transition state (TS1) was located at the distance of 2.70 Å. Similarly, Pγ

25

atom was drew near to the Osub atom of substrate to form P-O bond, accompanied with

26

the transfer of phosphoryl by breaking the Oβ-Pγ bond. The scanned distances ranged

27

from 3.90 Å to 2.00 Å, and in the optimized product structure, the P-O bond was 1.66

8



1

Å. Also, TS2 was located at 2.40 Å. All the optimizations were performed at M06-

2

2X/6-31G(d) level (Figure S3).

3 4

RESULTS AND DISCUSSION

5

Protonated States of His194

6

In previous studies, the essential role of H194 in Akt1 has been uncovered through

7

experimental mutagenesis, i.e., H194A mutant revealed markedly reduced T308

8

phosphorylation levels.20 Structural analyses indicated H194A mutation caused pT308

9

to expose from the catalytic cleft and become accessible to phosphatase. On the other

10

hand, different pKa values of H194 in Akt1 kinase (PDB ID: 4EKK) at physiological

11

condition (pH=7.0) were calculated to be 9.0, 7.7 and 6.8 by H++,44 Accelrys,45 and

12

PDB2PQR program,30 respectively. The first two values correspond to doubly

13

protonated H194 in physiological condition and these results are in accord with that

14

reported by Cheng.16 However, it still remains unknown how and to what extent could

15

protonation states of H194 affect the active conformation of Akt1.

16

To understand the role of H194 in Akt1 at the atomic level, we carried out 3×3×50

17

ns MD simulations with all possible protonation states of H194, including (1)

18

monoprotonated state on the δ nitrogen (HID) of imidazole, (2) monoprotonated state

19

on the ε nitrogen (HIE) and (3) diprotonated (HIP) state on both δ and ε nitrogen of

20

H194 (Figure 2A-C). Ligands such as ATP, two magnesium ions and substrate were

21

also considered in these systems. Calculation of root-mean-square deviation (RMSD)

22

values demonstrated that all nine trajectories rapidly reached equilibrium (Figure S4).

23

In HID system, a hydrogen bond was observed between NδH of H194 and E191. Since

24

E191 was located at the junction of helix αB and αC, we speculated that the hydrogen

25

bond would position αB and αC in the right orientation and facilitate the stability of

26

active conformation of Akt1. Our proposal was validated by HIE system, where

27

absence of hydrogen on Nδ of H194 resulted in partial uncoiling of αB and αC helix.

28

Through the root-mean-square fluctuation (RMSF) calculation, bigger fluctuations of 9


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1

αB and αC helix were observed in HIE systems than in HID system (Figure S5), and

2

the frequency of residues (184-187) adopting helix conformation dramatically

3

decreased (Figure 2D), suggesting the key role of hydrogen bond between HID194 and

4

E191 in maintaining the structural stability of αB helix. On the other side, NεH of H194

5

was found to interact with phosphate moiety of pT308 through a hydrogen bond in both

6

HIE and HIP systems. This hydrogen bond may play a significant role in keeping the

7

kinase in a closed conformation and protecting pT308 from being dephosphorylated.

8 9

Figure 2. Representative conformations of Akt1 in three protonation states of His194: HIE (A),

10

HID (B), and HIP (C). αB helix is shown in marine and αC helix in blue. Proportion of hydrogen

11

bond formation between H194-E191 and H194-pT308, as well as residues (184-187 and 193-202)

12

adopting alpha helix conformation in three systems are shown in (D).

13 14

Taken together, it was proposed that doubly protonated H194 undertook critical

15

functions in Akt1 active state: NεH of H194 was associated with the protection of pT308,

16

while NδH was conducive to the maintenance of helix/structural stability by interacting

17

with E191. Therefore, in our subsequent studies, diprotonated His194 model was

18

utilized to investigate the structural characteristics of active Akt1 states.

19 10



1

ATP Binding and ADP Binding

2

Experimental results have reported that ATP could exert a protective effect on

3

phosphorylated sites of Akt against phosphatases; ADP binding, on the other hand, may

4

lead to a transformation of Akt from the phosphorylated (active) state into the

5

unphosphorylated (inactive) state.20 To investigate the role of ATP/ADP as an “on-off”

6

switch in catalytic cycle, ATP-Akt1 and ADP-Akt1 complexes were constructed and

7

3×100 ns MD simulations were performed on both systems. RMSD analysis suggested

8

all simulations rapidly reached their respective equilibrium (Figure S6).

9 10

Figure 3. Representative conformations of ATP-Akt1 (A) and ADP-Akt1 (B) system. (C) Distances

11

between ATP-K179 and K179-E198 salt bridge in three separate simulations for ATP-Akt1 (left)

12

and ADP-Akt1 (right) systems.

13 14

A conserved K179 on rigid β3 strand was thought to interact with both ATP α/β

15

phosphate and E198 in the αC helix, and mediated Akt1 reactivity by positioning ATP

16

at a proper location thus facilitating phosphorylation. Therefore, it is commonly

17

believed that ATP-K179-E198 salt bridge constitutes one of the main characteristics of

18

Akt1 activated state.46 In accordance with this opinion, our simulations proved that the

19

salt bridge could be well maintained in ATP-Akt1 system, while it became structurally 11


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1

unstable and ended up cracking in ADP-Akt1 system. To be precise, the average

2

distances between ADP and K179 distributed in the range from 7.09 Å to 8.69 Å (Figure

3

3), while the average distance in ATP-Akt1 systems was 7.04 Å. Meanwhile, in another

4

ADP-Akt1 trajectory, K179 stayed 8.45 Å away from E198, 1.14 Å longer than the

5

average distance in ATP-Akt1 system (7.31 Å), signifying the fracture of the salt-bridge

6

interaction. It should be noted that the distances presented here are not the direct atom-

7

to-atom distances. Actually, the distances between the terminal heavy atoms of ATP

8

(Oβ), K179 (Nζ) and E198 (Oδ) are near to 3 Å (Table S1). These results suggested the

9

importance of ATP γ-phosphate group in holding together the ATP-K179-E198 salt

10

bridge and keeping Akt1 in an activated catalytic conformation.

11 12

Figure 4. (A) The allosteric regulatory pathway in ATP-Akt1 system (ATP→T160→F161→

13

E191→H194→pT308); (B) The allosteric pathway was interrupted in ADP-Akt1 system; (C) The

14

distribution of distances between the residues involved in the allosteric pathway of Akt1.

15 16

As

illustrated

by

Lu

et

al.,27

the

allosteric

regulatory

pathway

17

(ATP→T160→F161→E191→H194→pT308) played a decisive role in protecting

18

pT308 in a phosphorylated state. Residues involved in the pathway scattered on

19

different parts of Akt1P: T160 and F161 were located in G-loop, E191 at the junction 12



1

of αB and αC helix, H194 in αC helix. RMSF values indicated that in comparison to

2

ATP-Akt1 system, these residues presented larger fluctuations in ADP-Akt1 systems

3

(Figure S7), which might lead to bigger conformational change, and disturb the

4

interaction in allosteric pathway. In our study, distances between the centroids of these

5

key residues were calculated to reveal their interactions. As shown in Figure 4, in

6

comparison to the relative concentration of distances among these residues in ATP-

7

Akt1 system, distances in ADP-Akt1 system were distributed in a wider range,

8

demonstrating more structural transformations. More importantly, distances between

9

certain residue pairs (for example, ADP/T160, F161/E191, and H194/pT308) increased

10

significantly, suggesting interrupted communications in the allosteric pathway. Taken

11

together, the larger the distances, the weaker the interactions, and finally the less

12

protection effect, which was validated by experimental observations.20 These results

13

demonstrated the critical role of ATP γ-phosphate group in protecting the allosteric

14

pathway of Akt1 phosphorylation and its catalytic bioactivity.

15 16

Flexibility of G-loop

17

As we all know, Akt1 is a very dynamic and malleable kinase with large structural

18

disparity in its active and inactive states. Previous studies have reported that

19

communication between ATP and pT308 was orchestrated by interaction between the

20

G-loop and helix αC.27-28 G-loop region (GKGTFG, residues 157-162), which was

21

located between β1 and β2 strands in N-lobe, served as a lid for the phosphates of the

22

ATP, and was suggested to play an important role in both phosphoryl transfer and

23

ATP/ADP exchange during the catalytic cycle.

24

Herein, seven separate 50-ns MD simulations were conducted with Akt1 crystal

25

structure (4EKK) as initial structure, in which no interaction was present between H194

26

and pT308. It was found that all seven systems rapidly reached equilibrium after 10-ns

27

simulations (Figure S8). Fortunately, hydrogen bond formation between diprotonated

28

H194 and pT308 was observed in two “active” trajectories (denoted as md1 and md2, 13


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1

Figure 5A), which, according to our previous knowledge, was indispensable for

2

maintenance of active state. Next, the representative structures from seven trajectories’

3

dominant cluster were extracted and compared with the crystal structure, and the major

4

difference was found in the G-loops (Figure 5B). In comparison to the crystal structure,

5

the largest shift was found to be 4.32 Å, as designated by the Cα of T160, which was

6

located at the center of G-loop.

7 8

Figure 5. (A) Crystal structure of Akt1 (yellow) and representative conformations of md1 and md2

9

trajectories (salmon and cyan, respectively). Hydrogen bonds between H194 and pT308 are shown

10

in black. (B) The position of G-loop before and after simulations (yellow and gray) and the trajectory

11

(cyan) with the largest shift from the crystal structure.

12 13

Conformational transition with permeation of water

14

Next, we prolonged the two “active” trajectories (md1 and md2) to 100 ns (Figure

15

S9) to investigate the structural characteristics of active state of Akt1, and three

16

conformations (a, b and c) in terms of T160’s binding mode were summarized. They

17

could be applied to present the dynamical variations of G-loop from inactive to active

18

state of Akt1. Specially, conformation a was applied to mimics the active state of Akt1,

19

whereas conformation b and c were more likely to emphasize the ‘transition’ process. 14



1

In conformation a (Figure 6), β-phosphate of ATP (Oβ) clutched the hydroxyl (OT160)

2

of T160 by a hydrogen bond, exposing the γ-phosphate, and further facilitating its

3

transfer onto the substrate. In conformation b, the hydroxyl of T160 (OT160) in G-loop

4

was anchored to γ-phosphate of ATP (Oγ), and as a result, ATP was fixed and shielded

5

by the G-loop. In conformation c, however, the hydroxyl of T160 (OT160) distanced

6

itself away from ATP and instead formed a hydrogen bond with hydroxyl oxygen atom

7

(Osub) of serine in substrate peptide. In order to illustrate the three distinctive

8

conformations more clearly, we used distances, d1(OT160-Oβ), d2(OT160-Oγ) and

9

d3(OT160-Osub), to describe conformation a, b and c, respectively. Either conformation

10

a (active state) or c (inactive state) is quite similar between the md1 and md2 trajectories

11

(Figure S10).

12

13

15


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1

Figure 6. Analysis of conformational transition from inactive to active state of Akt1 kinase. (A)

2

Representative conformations (a, b, and c) of different stages. (B) The fluctuation of distances

3

d1(OT160-Oβ), d2(OT160-Oγ) and d3(OT160-Osub) along md1 and md2 simulations. The number of water

4

molecules within 4 Å around the ATP oxygen atom bridging Pβ and Pγ was also displayed. Stages

5

of conformational change from inactive (c, b) to active (a) are marked with blue, green, and red

6

colors along the trajectories, respectively.

7 8

As shown in Figure 6, conformation of Akt1 active site was transformed from c to

9

b at 10.5 ns in md1 trajectory, as indicated by the increase of d1 (from 2.80 Å to 4.21

10

Å) and the decrease of d2 (from 4.45 Å to 2.68 Å). The second conformational transition

11

b to a occurred at 33.5 ns, when d3 declined from 5.04 Å to 2.97 Å, and d2 rose from

12

2.68 Å to 4.12 Å. On the other hand, during the first 52.0 ns of md2 simulation, the

13

average of d1 is 2.93 Å, indicating the domination of conformation c. Then, d1 started

14

fluctuating violently and increased to 5.32 Å, when the distance d3 decreased from 4.95

15

Å to 2.71 Å, which implied that conformation c collapsed and was substituted by

16

conformation a. Moreover, we calculated the number of water molecules in a sphere

17

with a radius of 4 Å centering on the oxygen atom of ATP bridging Pβ and Pγ. It’s

18

worthy of note that, along with the conformational transition, there was a massive

19

change in the number of water molecules in ATP pocket. Take trajectory md1 for

20

example, when conformation b turned to conformation a, the average number of water

21

molecules increased from 5.55 to 9.92, giving us a hint that water diffusion may play a

22

crucial role in both phosphoryl transfer and ATP/ADP exchange during the catalytic

23

cycle.

24

To sum up, there are three typical conformations obtained from two "active"

25

trajectories, in which the hydrogen bond between H194 and pT308 was readily formed

26

to ensure the sustenance of Akt1 active state. Compared with conformation a,

27

conformation b and c differed in the shape of G-loop and binding mode with ATP, but

28

both of them could spontaneously transform to conformation a, where the hydroxyl of 16



1

T160 formed a hydrogen bond with the β-phosphate of ATP. Moreover, following the

2

conformational transition, water molecules flooded into the ATP pocket, which might

3

influence the binding mode of Akt1 with ATP of Mg2+.

4 5

Binding mode of Mg2+ ion

6

Next, we carefully examined the equilibrium structures (i.e. the representative

7

structure of the dominant cluster) obtained from md1 and md2 trajectories

8

(conformation I and conformation I′) (Figure 7). Although hydrogen bond between

9

OT160 of T160 and Oβ of ATP could maintain in both conformations, the structure of

10

their ATP pocket differed from each other. Besides the number of water molecules as

11

mentioned above, the primary dissimilarity between conformation I′ and conformation

12

I lay in the relative location between ATP and Mg2+. The ribose of ATP was forced

13

closer to the Mg2+ ion (MgII) in conformation I′ (2.12 Å) than conformation I (4.31 Å),

14

which resulted in different ATP-Mg2+ binding modes.

15 16

Figure 7. Conformations of active sites obtained from md1 (conformation I, salmon) and md2

17

(conformation I′, cyan) simulations.

18 19

To be more detailed, in conformation I (Figure 8), MgI ion hexahedrally

20

coordinated with Oδ2 of Asp274 in catalytic loop, Oδ1 and Oδ2 Asp292 of DFG motif,

21

O2β and O3γ of ATP, and an oxygen atom from a water molecule; MgII ion was

22

coordinated with Oδ1 of Asn279, Oδ2 of Asp292, O2α and O2γ of ATP, and two oxygen 17


Page 18 of 40

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1

atoms from two water molecules. On the other hand, in comparison to I′, both carboxyl

2

groups of Asp292 and γ-phosphate group of ATP were drawn closer to the MgII ion in

3

conformation I′ and as a result, Asp292 served as a bidentate ligand to MgII and the

4

hydroxyl oxygen on ATP-ribose occupied the remaining coordination space of MgII.

5

In sharp contrast, three water molecules were present around Mg2+ in conformation I

6

while no water was involved in Mg2+ coordination for conformation I′. These structural

7

difference would finally exert an influence on the catalytic mechanism of Akt1

8

phosphoryl-transfer reaction.

9 10

Figure 8. Coordination mode of two Mg2+ ions with ATP and key residues in conformation I

11

(salmon) and conformation I′ (cyan).

12 13

Catalytic mechanism

14

Our previous work indicated that auto phosphoryl-transfer reaction of CheA

15

histidine kinase started with a rapid and reversible conformational change from ground

16

state to prechemistry state (i.e. prereaction state),47 where Oγ atom of ATP in

17

coordination with catalytic Mg2+ ion was replaced by an O atom from a water molecule,

18

subsequently liberating γ-phosphate group from the constraint of Mg2+ and facilitating

19

phosphorylation of its substrate. Herein, the γ-phosphate group of ATP in either water-

20

occupied conformation I or water-free conformation I′ was coordinated with MgII ion 18



1

as well, which was unfavorable for the phosphorylation reaction. Thus, it was

2

postulated that a similar conformational change was required in our systems. For this

3

purpose, key structures including, transition states (TS1 and TS2), ground states (R and

4

R′), prereaction structures (IM and IM′) and phosphate group-accepting polypeptide

5

products (P and P′) corresponding to conformation I and conformation I′ were

6

optimized at ONIOM (M06-2X/6-31G(d):Amber) level. Our calculations focused on

7

potential energies, free energies were also suggested for further explorations.48, 49

8

After the optimizations of ground states, we found the water-occupied

9

conformation I was more stable than the water-free conformation I′, therefore, we

10

carefully studied the phosphoryl-transfer reaction with the former system. As shown in

11

Figure 9, distance r(MgII-Owat) was found to be 3.90 Å in the optimized reactant R, and

12

reduced to 2.04 Å in the optimized intermediate IM, while distance r(MgII-Oγ) was

13

initially 1.91 Å in R and enlarged to 4.03Å in IM. These indicated the formation of a

14

new bond between MgII and a water molecule and breakage of the coordination

15

interaction between MgII and the oxygen of ATP γ-phosphate group. The transition

16

state was obtained during the escape of Oγ atom from the constraint of MgII, and the

17

energy barrier was calculated to be 9.1 kcal/mol. Since R was less stable than IM by

18

only 2.6 kcal/mol, we proposed the conformational transition between R and IM might

19

be reversible. Then, the phosphoryl-transfer reaction proceeded when γ-phosphate

20

group of ATP conducted a nucleophilic attack, accompanied by deprotonation of

21

hydroxyl group of substrate. It was found that distance r(Pγ-Osub), which represented

22

the nucleophilic attack process, was 3.98 Å in IM and decreased to 1.66 Å in the

23

optimized product P, indicating the formation of the Pγ-Osub bond and the completing

24

of the phosphoryl-transfer reaction. Also, the transition state of this step was located by

25

scanning the formation of Pγ-Osub bond. The transfer of ATP γ-phosphate group to

26

substrate was considered to be a rate-determining step with an energy barrier of 19.8

27

kcal/mol. It should be noted that P was more stable in energy than R by 17.0 kcal/mol

19


Page 20 of 40

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1

(corrected to approximately 10.0 kcal/mol on larger basis set, Table S2), suggesting the

2

favorability of phosphor-accepting polypeptide products in thermodynamics.

3 4

Figure 9. Potential energy profile of proposed phosphoryl-transfer mechanism with optimized key

5

structures of Akt1 kinase.

6 7

Next, we conducted optimization on conformation I′ (Figure S11). In IM′, a water

8

molecule was also found to squeeze its way into the coordination site of MgII. Although

9

γ-phosphate group of ATP was liberated by MgII, it got caught by MgI instead, which

10

posed another hindrance to its transfer onto the substrate. In terms of energy, IM′ was

11

calculated to be 19.2 kcal/mol less stable than R′, indicating an unfavorable

12

conformational change. We think the energy discrepancy might be overestimated and

13

it could be reduced when different computational methods were considered. Besides,

14

the energy of P′ was found to be approximately 17.0 kcal/mol more stable than R′ (Table

15

S2), consistent with the energy gap between P and R.

16

Moreover, energetic corrections of all obtained structures at different levels of

17

precision were performed (Table S2) and the results suggested that conformation I was 20



1

energetically more favorable than conformation I′, since both the reactant and the

2

product bore lower energy than R′ and P′. Conformational transformation between R

3

and IM was easily achieved with a small energy gap of less than 3 kcal/mol, while

4

transition from R′ to IM′ confronted with a relative large barrier. To sum up, when the

5

water molecules diffused into the ATP pocket, a critical prereaction state was formed

6

through a reversible conformational change. After ATP γ-phosphate group escaped

7

from MgII-bound state to approach the substrate, the phosphoryl-transfer reaction

8

completed.

9 10

CONCLUSIONS

11

Nowadays numerous experimental and computational studies have been

12

conducted to decipher the catalytic mechanism of Akt1. However, the detailed

13

structure-function interactions in active and inactive states of Akt1 still remain unsolved.

14

We utilized an integration of classical MD simulations and QM/MM calculations to

15

investigate the structural characteristics of Akt1’s active state. MD simulations on three

16

protonated states of H194 revealed NεH of H194 could exert a protection effect on

17

pT308 by locking pT308 in the catalytic cleft with a hydrogen bond, and NδH of H194

18

promoted protein helix/structural stability of Akt1 by interacting with E191. In a word,

19

the doubly protonated H194 was superior to singly protonated state in sustaining Akt1

20

in its active conformation. Next, comparison between ADP-Akt1 and ATP-Akt1

21

systems was performed, and ADP-Akt1 system was validated to be less stabilized, since

22

both an ATP-K179-E198 salt bridge and a previously proposed allosteric pathway

23

(ATP→T160→F161→E191→H194→pT308) tended to crack in ADP-Akt1 system.

24

Further, seven 50-ns simulations initialized from Akt1 crystal structure revealed the

25

inherent flexibility of G-loop, and two favorable trajectories were prolonged to 100-ns

26

simulations. Three conformations (a, b and c) regarding interactions between G-loop

27

and ATP were extracted from the trajectories. Analysis revealed that conformation a

28

was the most stable and their conformation change was always accompanied by ingress 21


Page 22 of 40

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1

of water molecules. What’s more, as a crucial part of Akt1 activation, the coordination

2

mode of Mg2+ in representative conformation I and I′ was presented. Unlike the water-

3

free conformation I′, three water molecules were observed around Mg2+ in

4

conformation I. Besides, the key structures R/R′, IM/IM′ and P/P′ in phosphoryl-

5

transfer reaction were optimized by QM/MM calculations. We proposed an Akt1

6

catalytic mechanism: a prereaction state was attained through reversible conformation

7

change from R, with a water molecule moving into the reaction center to coordinate

8

with Mg2+, while γ-phosphate group seized the chance and transferred from ATP to the

9

substrate. In summary, our study revealed the structural characteristics of Akt1 active

10

state and the catalytic mechanism of γ-phosphate transfer reaction, which could help

11

improving the understanding of structure-function relationships of Akt kinases.

12 13

ASSOCIATED CONTENT

14

Supporting Information

15

The Supporting Information is available free of charge on the ACS Publications website:

16

Structural comparison between inactive crystal structure and active conformation, root-

17

mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) values of

18

MD simulations of HIE, HID, and HIP systems, as well as ATP-Akt1 & ADP-Akt1

19

systems, distances between the terminal heavy atoms of ATP, K179 and E198, atoms

20

of key residues included in the QM region in QM/MM calculations, potential energy

21

paths from reactant to product, optimized key structures R′, IM′ and P′ in phosphoryl-

22

transfer reaction obtained from md2 and the energetic corrections to structures of

23

conformation I and conformation I′ at different level with M06-2X method.

24 25

AUTHOR INFORMATION

26

Corresponding Author

27

*Email: [email protected], Tel/Fax: +86-21-34207190; [email protected], +86-

28

21-34207347 22



1

Author Contributions

2

TS and YLZ conceived and designed the investigation. LLX, WJL, WTT and TS

3

performed calculations and analyses. LLX, SBF, WJL, TS and YLZ wrote up the paper.

4 5

Notes

6

The authors declare no competing financial interest.

7 8

ACKNOWLEDGMENTS

9

The authors thank the National Basic Research Program of China “973”

10

(2012CB721005) and the National High-tech R&D Program of China “863”

11

(2012AA020403), National Science Foundation of China (21377085 and 31770070),

12

SJTU-YG2016MS42, and the SJTU-HPC computing facility award for financial

13

supports and computational hours.

14 15

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Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo,

17

C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi,

18

R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;

19

Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas,

20

O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09; Gaussian,

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Inc. Wallingford, CT, 2013.

22

41. Zhao, Y.; Truhlar, D. G., The M06 suite of density functionals for main group

23

thermochemistry, thermochemical kinetics, noncovalent interactions, excited states,

24

and transition elements: two new functionals and systematic testing of four M06-

25

class functionals and 12 other functionals. Theor. Chem. Acc. 2008, 120, 215-241.

26

42. Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M.; Ferguson, D.

27

M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A., A Second

28

Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic 28



1

Molecules. J. Am. Chem. Soc. 1995, 117, 5179-5197.

2

43. Field, M. J.; Bash, P. A.; Karplus, M., A combined quantum mechanical and

3

molecular mechanical potential for molecular dynamics simulations. J. Comput.

4

Chem. 1990, 11, 700-733.

5

44. Gordon, J. C.; Myers, J. B.; Folta, T.; Shoja, V.; Heath, L. S.; Onufriev, A., H++: a

6

server for estimating pK(a)s and adding missing hydrogens to macromolecules.

7

Nucleic Acids Res. 2005, 33, W368-W371.

8 9

45. Spassov, V. Z.; Yan, L., A fast and accurate computational approach to protein ionization. Protein Sci. 2008, 17, 1955-1970.

10

46. Jura, N.; Zhang, X.; Endres, N. F.; Seeliger, M. A.; Schindler, T.; Kuriyan, J.,

11

Catalytic Control in the EGF Receptor and Its Connection to General Kinase

12

Regulatory Mechanisms. Mol. Cell 2011, 42, 9-22.

13

47. Shi, T.; Lu, Y.; Liu, X.; Chen, Y.; Jiang, H.; Zhang, J., Mechanism for the

14

Autophosphorylation of CheA Histidine Kinase: QM/MM Calculations. J. Phys.

15

Chem. B 2011, 115, 11895-11901.

16

48. Smith, G. K.; Ke, Z.; Guo, H.; Hengge, A. C., Insights into the Phosphoryl Transfer

17

Mechanism of Cyclin-Dependent Protein Kinases from ab Initio QM/MM Free-

18

Energy Studies. J. Phys. Chem. B 2011, 115, 13713-13722.

19

49. Valiev, M.; Yang, J.; Adams, J. A.; Taylor, S. S.; Weare, J. H., Phosphorylation

20

reaction in cAPK protein kinase-free energy quantum mechanical/molecular

21

mechanics simulations. J. Phys. Chem. B 2007, 111, 13455-13464.

22 23

FIGURE LEGEND

24

Figure 1. (A) Crystal structure of Akt1 (PDB code: 4EKK26, where AMP-PNP was replaced by

25

ATP). N-lobe and C-lobe are shown as surface in green and yellow, respectively. ATP and pT308

26

are shown as sticks. Key structural elements are indicated by different colors in cartoon. (B) The

27

substrate peptide is shown as stick.

29


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1

Figure 2. Representative conformations of Akt1 in three protonation states of His194: HIE (A),

2

HID (B), and HIP (C). αB helix is shown in marine and αC helix in blue. Proportion of hydrogen

3

bond formation between H194-E191 and H194-pT308, as well as residues (184-187 and 193-202)

4

adopting alpha helix conformation in three systems are shown in (D).

5

Figure 3. Representative conformations of ATP-Akt1 (A) and ADP-Akt1 (B) system. (C) Distances

6

between ATP-K179 and K179-E198 salt bridge in three separate simulations for ATP-Akt1 (left)

7

and ADP-Akt1 (right) systems.

8

Figure 4. (A) The allosteric regulatory pathway in ATP-Akt1 system (ATP→T160→F161→

9

E191→H194→pT308); (B) The allosteric pathway was interrupted in ADP-Akt1 system; (C) The

10

distribution of distances between the residues involved in the allosteric pathway of Akt1.

11

Figure 5. (A) Crystal structure of Akt1 (yellow) and representative conformations of md1 and md2

12

trajectories (salmon and cyan, respectively). Hydrogen bonds between H194 and pT308 are shown

13

in black. (B) The position of G-loop before and after simulations (yellow and gray) and the trajectory

14

(cyan) with the largest shift from the crystal structure.

15

Figure 6. Analysis of conformational transition from inactive to active state of Akt1 kinase. (A)

16

Representative conformations (a, b, and c) of different stages. (B) The fluctuation of distances

17

d1(OT160-Oβ), d2(OT160-Oγ) and d3(OT160-Osub) along md1 and md2 simulations. The number of water

18

molecules within 4 Å around the ATP oxygen atom bridging Pβ and Pγ was also displayed. Stages

19

of conformational change from inactive (c, b) to active (a) are marked with blue, green, and red

20

colors along the trajectories, respectively.

21

Figure 7. Conformations of active sites obtained from md1 (conformation I, salmon) and md2

22

(conformation I′, cyan) simulations.

23

Figure 8. Coordination mode of two Mg2+ ions with ATP and key residues in conformation I

24

(salmon) and conformation I′ (cyan).

25

Figure 9. Potential energy profile of proposed phosphoryl-transfer mechanism with optimized key

26

structures of Akt1 kinase.

30




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Theoretical Investigation of the Structural Characteristics in Active

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