proteinsSTRUCTURE O FUNCTION O BIOINFORMATICS

Structural basis of conformational variancein phosphorylated and non-phosphorylatedstates of PKCbIIBaljinder K. Grewal, R. Venkata Krishnan, and M. Elizabeth Sobhia*

Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research, Mohali, Punjab 160 062, India

ABSTRACT

PKCbII activation is achieved by primary phosphorylation at three phosphorylation sites, followed by the addition of sec-

ondary messengers for full activation. Phosphorylation is essential for enzyme maturation, and the associated conforma-

tional changes are known to modulate the enzyme activation. To probe into the structural basis of conformational changes

on phosphorylation of PKCbII, a comprehensive study of the changes in its complexes with ATP and ruboxistaurin was per-

formed. ATP is a phosphorylating agent in its phosphorylation reaction, and ruboxistaurin is its specific inhibitor. This

study provides insight into the differences in the important structural features in phosphorylated and non-phosphorylated

states of PKCbII. Less conformational changes when PKCbII is bound to inhibitor in comparison to when it is bound to its

phosphorylating agent in both states were observed. The interactions of ruboxistaurin significant in restricting PKCbII to

attain the conformational state competent for full activation are reported.

Proteins 2014; 00:000–000.VC 2013 Wiley Periodicals, Inc.

Key words: activation loop; conformations; glycine loop; molecular dynamics; phosphorylation; PKCbII.

INTRODUCTION

Protein kinase C (PKC) is a super family of structur-

ally and functionally related 12 isoforms which are

widely distributed in the intracellular environment with

significant roles in signal regulation of important physio-

logical cellular processes.1 PKCbII, a conventional PKC

isoform, plays a prominent role in cell signaling path-

ways, but elevation of its expression levels is associated

with various diabetic complications viz. diabetic cardio-

myopathy, diabetic retinopathy, and so forth.2,3 Various

clinical trial studies addressing the role of PKCbII in

these complications are performed and ruboxistaurin

(RBX), a specific PKCbII inhibitor, has been reported to

be highly beneficial in ameliorating these complica-

tions.4–6 Targeting PKCbII is a main challenge owing to

the high structural similarities among the PKC iso-

forms.7 The structural similarities in PKCs are observed

only in their active states while a markedly distinct con-

formation is seen in the inactive states.8 Activation,

deactivation, and auto inhibition processes of kinases are

coupled with various conformational changes in activa-

tion loop, glycine loop, and other structural features. The

ability of the activation loop of different kinases to adopt

distinct conformations has been recently exploited suc-

cessfully in medicinal field.9 The potent anticancer drug

Gleevec is one such drug which selectively binds to the

inactive state of Abl kinase.10,11

PKC catalyses post-translational phosphorylation of

various substrates, and they themselves are regulated by

phosphorylation at their phosphorylation sites. Phospho-

rylation is one of the vital steps in modulating various

kinases, including PKCbII’s activity. PKCbII is under

acute structural and spatial regulation. Its phosphoryla-

tion state, conformation, and subcellular location must be

precisely defined for exhibiting its physiological function.

Mutations of the Thr641 were reported to destabilize the

kinase domain, thus signifying the importance of phos-

phorylation at this turn motif Thr641 in stabilization of

Additional Supporting Information may be found in the online version of this

article.

Grant sponsors: Council of Scientific and Industrial Research (CSIR) and Depart-

ment of Science and Technology (DST), New Delhi.

*Correspondence to: M. Elizabeth Sobhia, Department of Pharmacoinformatics,

National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S.

Nagar, Mohali, Punjab 160 062, India. E-mail: mesophia@niper.ac.in

Received 31 May 2013; Revised 9 November 2013; Accepted 9 December 2013

Published online 19 December 2013 in Wiley Online Library (wileyonlinelibrary.

com). DOI: 10.1002/prot.24500

VVC 2013 WILEY PERIODICALS, INC. PROTEINS 1

catalytic domain. Phosphorylated Thr500 at activation

loop seems to hold the activation loop in the extended

conformation and modulate the PKCbII activity by vari-

ous conformational changes.12 This conformation favors

the phosphate group transfer from ATP, bound in

PKCbII’s ATP binding site, to the substrate (Fig. S1 of

the Supporting Information).

PKCbII activation is achieved by primary phosphoryla-

tion at Thr500, Thr641, and Ser660 followed by the addi-

tion of secondary messengers for full activation.13,14 The

ATP competitive specific PKC inhibitors demonstrate dis-

tinct state-dependent inhibition. They bind to the phos-

phorylated protein, and inhibit further conformational

changes favorable for binding of second messenger and

thereby the full activation of PKCbII. Similar actions can

be expected from PKCbII-specific inhibitor RBX, as it is

also an ATP competitive inhibitor. Hence, a deeper under-

standing of the principles governing conformational adap-

tations in PKCbII would be of utmost practical

importance to enhance the affinity of existing PKCbII

inhibitors. The study of the structural basis of conforma-

tional changes on phosphorylation of PKCbII in complex

with the phosphorylating agent (ATP) and the specific

inhibitor (RBX) will provide insights in this area. The

objective of this study is to probe into the structural basis

of conformational changes during PKCbII activation asso-

ciated with its phosphorylation.

ATP is a phosphorylating agent in the phosphorylation

reaction performed by PKCbII, and RBX is a potent and

specific inhibitor of PKCbII. Hence, binding of ATP or

RBX is expected to modulate or inhibit the PKCbII activ-

ity, respectively, by inducing various conformational

changes. It can be presumed that if ATP binds to an

“intermediate” conformation of phosphorylated PKCbII it

would drive the equilibrium toward the active state. Con-

versely, if RBX binds to this state it may inhibit the con-

formational changes toward the active state. We studied

this phenomenon in PKCbII using its four complexes,

wherein ATP and RBX are bound to two different states

of kinase domain crystal structure of PKCbII. One state is

the reported form of kinase domain crystal with all its

three phosphorylating sites viz. Thr500, Thr641, and

Ser660 being phosphorylated,15 and the other where these

residues are mutated to non-phosphorylated form using

in silico process. Molecular dynamics (MD) approach was

then used to perform comparative analysis of phosphoryl-

ated and non-phosphorylated PKCbII complexes with

ATP and RBX. Exhaustive analysis of the conformational

changes during the simulations, with an emphasis on the

conformational changes of important structural compo-

nents and their surroundings are reported.

Two extreme conformational states, viz. open and

close are known for protein kinases. But the conforma-

tion of both the reported PKCbII crystal structures is in

between these two conformations.15,16 Nevertheless,

fully active and inactive crystal structures, including the

complexed structure with inhibitors, ATP, and apoform

crystal structures for other kinases, have been reported in

literature. Primarily, to characterize the exact active and

inactive states of PKCbII, a comparative analysis of

reported crystal structures of protein kinases in different

conformations against that of PKCbII was performed.

The work-plan to study the phosphorylation induced

changes is shown in Figure 1.

Figure 1The work-plan designed to study phosphorylation induced conformational changes in PKCbII.

B.K. Grewal et al.

2 PROTEINS

MATERIALS AND METHODS

Comparative crystal structure analysis

For the comparative crystal structure analysis, the crys-

tal structures of various protein kinases reported in dif-

ferent conformations, viz. open, close, and intermediate

were downloaded from the Protein Data Bank. Of the

two crystal structures of PKCbII,15,16 the kinase domain

crystal structure studied by Grodsky et al. was used for

this study.15 It was co-crystallized with an ATP binding

site inhibitor, 2-methyl-bisindolylmaleimide (2MB), and

had a better resolution. PyMOL was used for the com-

parative crystal structure analysis.17 The residue numbers

in all the crystal structures under consideration were dif-

ferent, although their nature was similar. In this discus-

sion, all the residue numbers are mentioned as per

PKCbII kinase domain crystal structure.15

Molecular docking

The co-crystallized complexes of ATP and RBX with

PKCbII crystal structure were not available. Hence, both

the ligands were docked in the ATP binding site of the

kinase domain crystal structure of PKCbII using Glide

module of Schr€odinger Maestro 9.3.18 Initially, the miss-

ing residues of PKCbII crystal structure were added using

Modeller 9v8. Subsequently, in silico mutation of the three

phosphorylated residues viz. Thr500, Thr641, and Ser660,

to the corresponding non-phosphorylated form was per-

formed. The two protein structures, one fully phosphoryl-

ated and the other non-phosphorylated, were prepared

using the protein preparation wizard of Schr€odinger Mae-

stro 9.3. Glide uses a series of hierarchical filters to search

for possible locations of the ligand to be docked in the

active site region of the receptor. In Glide, the properties

of an active site region are represented by a grid that has

different sets of fields which provide progressively precise

scoring of the ligand pose. Glide uses Emodel for

pose selection, and GlideScore (GScore) to rank these

poses. The grid for both phosphorylated and non-

phosphorylated forms of PKCbII was prepared around the

co-crystallized ligand (2MB) with 9 A inner-box and 19 A

outer-box. The 3D structures of the ligands were sketched

and prepared using ligprep as per the steps of docking

methodology of Glide. The prepared ligands were docked

into both phosphorylated and non-phosphorylated struc-

tures. The Extra Precision (XP) mode was used and the

output was set to produce 10 docking poses per ligand.

The four PKCbII complexes are named as:

i. PKCbIIATP-p: PKCbII-ATP complex; Thr500, Thr641,

Ser660 being phosphorylated.

ii. PKCbIIATP-np: PKCbII-ATP complex; Thr500, Thr641,

Ser660 being non-phosphorylated.

iii. PKCbIIRBX-p: PKCbII-RBX complex; Thr500, Thr641,

Ser660 being phosphorylated.

iv. PKCbIIRBX-np: PKCbII-RBX complex; Thr500,

Thr641, Ser660 being non-phosphorylated.

Molecular dynamics simulation

The above docked complexes were subjected to MD

simulation for a time-scale of 150 ns using the PMEMD

module of AMBER11 in CUDA enabled GPU with

amber force fields (GAFF and ff99SB).19

The ligands were geometrically optimized using HF/6-

31G (*) basis set in Gaussian03. The antechamber module

was used to calculate atom centered restrained electrostatic

surface potential (RESP) by fitting ESP estimated from

quantum electronic structure calculation.20 Missing param-

eters of the ligands were generated using the general amber

force field (GAFF) and the parmchk module of antecham-

ber.21,22 The parameters of the three phosphorylated resi-

dues viz. Thr500, Thr641, and Ser660 of protein structure

were obtained from the AMBER parameter database. After

applying the force field, all missing parts of residues and

hydrogen atoms were automatically added by tleap module

of AMBER11. The molecular systems were neutralized by

addition of counter-ions followed by its solvation using

TIP3P water model by creating an isometric water box.

The distance of the box was 9 A from the periphery of the

protein. The systems were energy minimized in two stages.

In the first stage, the protein and the ligand were kept fixed

and only the water molecules were allowed to move to

eliminate close contacts and steric clashes among newly

added atoms. In the second stage, all atoms were allowed

to move. Also, in the first stage, the energy minimization

was performed in 500 and 2000 steps of steepest descent

and conjugate gradient methods, respectively. In the second

stage, the energy minimization was performed in 1000 and

2500 steps of steepest descent and conjugant gradient

methods, respectively. The system was virtually heated for

50 ps, where the protein–ligand complex was restrained

with a force constant of 10 kcal/mol/A2. Density equilibra-

tion was then achieved with restraint force of 2 kcal/mol/A2

on the protein-ligand complex. The system was further

equilibrated under constant pressure at 300 K for a period

of 2 ns. The production phase was performed for 150 ns on

an NPT ensemble at 300 K temperature and 1 atm pressure

with a step size of 2 fs during the entire simulation. Lange-

vin thermostat and Berendsen barostat were used for tem-

perature and pressure coupling, respectively. SHAKE

algorithm was used to constrain all bonds containing

hydrogen atoms. The non-bonded cutoff was kept at 10 A

and long range electrostatic interactions were treated by

Particle Mesh Ewald (PME) method with fast Fourier

transform grid with approximately 0.1 nm space. Trajec-

tory snapshots were taken every 10 ps. PyMOL, VMD,23

and ptraj module of AMBER tools were used for visualiza-

tion and analyses. The B factor of all the complexes was

calculated from the mean square fluctuations (MSF) using

the following equation:

Conformational Variance in PKCbII

PROTEINS 3

B factor 58p2

3

� �� �ðmsf Þ: (1)

Clustering

Clustering helps to reduce the complexity of data

resulting from long simulation runs while retaining the

relevant information of the whole trajectory. This is well

suited to objectively organize trajectory data and analyze

the stabilizing interactions. The conformational transi-

tions are grouped based on the self-similar conformations

and then each group is represented by representative

structure. Clustering analysis of the stabilized trajectories

for 150 ns molecular simulations run of the four PKCbII

complexes was performed using ptraj module of

AMBER11. Ptraj is the AMBER utility for analyzing the

AMBER trajectory files generated during molecular simu-

lations. It uses average link algorithm, and generates a

distance matrix with the RMSD comparisons of each con-

formation with all the others present in the trajectory.

Following this, it counts the number of neighbors for

each conformation using RMSD cut-off, and selects a

structure with largest number of neighbors as the repre-

sentative structure. It considers all the neighbors as a

cluster and eliminates them from the pool of conforma-

tions.24 This procedure is repeated for the remaining

structures in the pool to find new clusters.

RESULTS AND DISCUSSION

Comparative analysis of crystal structures

To characterize the active and inactive conformational

features of PKCbII, comparative crystal structure analysis

of its kinase domain crystal structure with the crystal

structures of other protein kinases reported in different

conformations, including fully active and inactive forms,

was performed (Table I). The two available PKCbII crys-

tal structures are in different conformations; the kinase

domain structure (PDB Code: 2I0E)15 is in intermediate

conformation and full length crystal structure (PDB

Code: 3PFQ)16 is in partially open conformation where

C1B domain clamps NFD helix of kinase domain in an

inactive conformation. The crystal structures considered

for the present study along with their PDB codes in

parenthesis are as:

1. Insulin receptor kinase (1IRK, a tyrosine kinase): phos-

phorylated structure in active form, complexed with a

peptide substrate and an ATP analog (1IR3)25; non-

phosphorylated structure (1IRK).26

2. Cyclic-AMP-dependent protein kinase (PKA): com-

plexed with a 20-amino acid substrate analog inhibitor

(2CPK)27; apoenzyme (1J3H)28; complexed with

potent natural product inhibitor Balanol (1BX6)29;

complexed with substrate (1JLU)30; myristylated cata-

lytic subunit complexed with substrate (1CMK).31

3. PKCi: apoform (3A8X)32; ATP bound state (3A8W)32; complexed with the bisindolylmaleimide inhibitor

BIM1 (1ZRZ).33

The superimposition of PKCbII with various kinases

showed that the orientation of glycine loop (349–354),

activation loop (484–511), Thr500, conformation of sub-

strate binding site near Thr500, aB-helix (375–381), aC-

helix (384–395), the connecting loop of aB and aC helix

(382–383), and NFD helix (628–630) differ significantly

among the above kinases. DFG motif (484–486) also

showed relatively significant structural changes between

the active and inactive states. As mentioned above,

PKCbII’s full length structure is more close to active

form. The NFD loop was bent toward ATP binding site

of PKCbII to form an “in” conformation in its full

length structure with respect to its kinase domain struc-

ture (Fig. S2 of the Supporting Information). The NFD

helical structure was not present in IRK crystal struc-

tures. The “in” conformation of NFD loop with respect

to intermediate PKCbII structure was also observed in

other active protein kinases. The aB, aC, and connecting

loop between them are oriented toward ATP binding site

in active form of IRK, and away from ATP binding site

in its inactive form with respect to their conformations

in PKCbII. In inactive IRK, the activation loop was non-

phosphorylated and collapsed onto the ATP binding site

while in active kinases it was phosphorylated at three

sites and was in extended conformation making surface

for substrate binding and catalysis. The distance between

its Ser1006:CA and Gly1152:CA of glycine and activation

loops, respectively, in 1IRK crystal structure was 11.3 A

in active form and 4.7 A in inactive form. The corre-

sponding residues in PKCbII crystal structure are

Ser352:CA and Gly486:CA. The glycine loop of all the

above kinases in active form was above and in inactive

form was below with respect to PKCbII glycine loop

conformation. The distances of 13–15 A, 4–5 A, and

8.7 A between Ser352:CA of the glycine-rich loop and

Gly486:CA of the activation loop specifies open, close,

and intermediate states, respectively.15

Analysis of active conformation of fully phosphorylated

three crystal structures of PKCi, one in apo form and

other two co-crystallized with ATP and BIM1 showed

that the overall conformations of all these structures are

similar except for minor differences in certain structural

features (Table S1 of Supporting Information). Hence,

irrespective of the bound ligand, non-phosphorylated

structures resemble inactive state and fully phosphoryl-

ated structures closely resemble the active state. A strictly

conserved ion-pair (Lys371 and Glu390) was observed in

conformations closer to active form and was found to be

responsible for proper alignment of aC helix for substrate

binding and catalysis. However, this ion-pair was also

B.K. Grewal et al.

4 PROTEINS

Tabl

eI

Co

mp

arat

ive

Cry

stal

Str

uct

ure

An

alys

iso

fP

rote

inK

inas

esin

Dif

fere

nt

Co

nfo

rmat

ion

sw

ith

the

Cry

stal

Str

uct

ure

of

Kin

ase

Do

mai

no

fP

KC

bII

PDB

ID3P

FQ1I

R31I

RK2C

PK1J

3H1B

X61J

LU1C

MK

3A8X

3A8W

1ZRZ

Gly

cine

loop

Abo

veA

bove

Clos

eIn

term

edia

teO

pen

Ope

nIn

term

edia

teIn

term

edia

teIn

term

edia

teO

pen

Ope

n

Dis

tanc

ebe

twee

nSe

r352

:CA

and

Gly

486:

CA(�

)

13.5

11.3

4.7

10.4

13.5

11.5

10.8

10.0

10.0

13.5

13.8

aB

aTo

war

dTo

war

dAw

ayAw

ayTo

war

dTo

war

dTo

war

dD

efor

med

Ove

rlap

Ove

rlap

Tow

ard

Link

betw

een

aB

and

aCa

Tow

ard

Tow

ard

Away

Tow

ard

Tow

ard

Tow

ard

Tow

ard

Def

orm

edAw

ayAw

ayTo

war

d

aC-

helix

:383

–395

aTo

war

dTo

war

dAw

ayTo

war

dTo

war

dTo

war

dTo

war

dO

verla

pO

verla

pO

verla

pTo

war

dLy

s371

orie

ntat

ion

Sim

ilar

Dis

sim

ilar

Sim

ilar

Sim

ilar

Dis

sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Dis

sim

ilar

Sim

ilar

Sim

ilar

Lys3

71-G

lu39

0io

n-pa

irO

neN

one

Non

eO

neN

one

One

Two

One

Non

eTw

oN

one

Hin

gere

gion

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

DFG

mot

ifbO

verla

pO

verla

pO

ppos

iteor

ient

atio

nO

verla

pO

verla

pO

verla

pO

verla

pO

verla

pO

verla

pO

verla

pO

verla

p

Cata

lytic

resi

dues

Ove

rlap

Sim

ilar

Not

sim

ilar

Ove

rlap

exce

ptLy

s371

Ove

rlap

Ove

rlap

Ove

rlap

exce

ptLy

s371

Ove

rlap

Ove

rlap

Ove

rlap

Ove

rlap

Act

ivat

ion

loop

Nom

inal

lybe

ntup

war

dN

omin

ally

bent

upw

ard

Colla

psed

insi

deSh

ort,

colla

psed

Shor

t,co

llaps

edSh

ort,

colla

psed

Shor

t,co

llaps

edN

omin

alco

llaps

eA

lmos

tsi

mila

rSi

mila

rne

glig

ible

colla

pse

Asp

466

orie

ntat

ion

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Thr5

00(N

-CA

-CB

-OG

1)

orie

ntat

ion

Sim

ilar

Abs

ent

Abs

ent

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

Sim

ilar

aE

ith

erb

ent

tow

ard

glyc

ine

loo

p(t

ow

ard

),o

ro

pp

osi

ted

irec

tio

n(a

way

)o

rsi

mil

arco

nfo

rmat

ion

(ove

rlap

)w

ith

resp

ect

toa

Bo

f2

I0E

.bD

FG

mo

tif

resi

du

esar

eA

sp4

84

,P

he4

85

,an

dG

ly4

86

.

All

mea

sure

men

tsan

do

bse

rvat

ion

sar

ew

ith

resp

ect

toP

KC

bII

crys

tal

stru

ctu

re(P

DB

ID:

2I0

E).

Cat

alyt

icre

sid

ues

are

Lys

37

1,

Asp

46

6,

Asn

47

1,

and

Asp

48

4.

Th

ese

are

also

spin

ere

sid

ues

.In

2C

PK

,1

BX

6,

and

1JL

Ua

C-h

elix

was

mo

ved

tow

ard

glyc

ine

loo

p,

bu

tit

was

less

than

the

oth

erp

rote

ins.

In1

CM

K,

3A

8X

,an

d3

A8

W

tyro

sin

e(Y

)w

asp

rese

nt

inst

ead

of

ph

enyl

alan

ine

(F)

inD

FG

mo

tif.

InD

FG

mo

tif

of

3A

8W

,th

eam

ino

acid

“F”

isre

pla

cew

ith

“Y.”

Conformational Variance in PKCbII

PROTEINS 5

observed in PKCbII kinase domain crystal structure.

These identified conformational changes were studied in

PKCbII complexes with ATP and RBX viz. PKCbIIATP-np,

PKCbIIATP-p, PKCbIIRBX-np, and PKCbIIRBX-p throughout

the simulation period.

Molecular docking

PKCbII crystal structure is bilobal with b sheet rich

N-terminal lobe being connected to a helix rich C-

terminal lobe by a hinge region.15 Both, the ATP and

substrate-binding sites are located in the cleft between

these lobes. ATP binding site (XGXGX2GX16KX) is in

highly conserved C3 domain, and substrate binding site

which takes part in the phosphoryl transfer is in C4

domain. It starts at the end of the ATP-binding site and

continues till the beginning of the phosphate transfer

group (Fig. S3 of the Supporting Information). Both

ATP and RBX were docked in ATP binding site of

PKCbII. The top five docked poses of each of ATP and

RBX are shown in Figure 2. To verify the docking result

and select the best docked pose for further study, the top

five docked poses of both ATP and RBX were compared

with the co-crystallized poses of their structurally similar

molecule. In the ATP binding site of the crystal struc-

tures of kinase domain and full length PKCbII;

2-methyl-1H-indol-3-yl-BIM-1 (2MB) and adenylyl-

imidodiphosphate (ANP) are co-crystallized, respec-

tively.15,16 The docked ATP was compared with

co-crystallized ANP in full length PKCbII crystal struc-

ture,16 and docked RBX was compared with co-

crystallized 2MB in its kinase domain crystal structure.15

RMSD of docked ATP and RBX with co-crystallized

ANP and 2MB was 0.476 A and 0.155 A, respectively.

ATP in PKCbII’s ATP binding site showed interactions

with Lys350, Ser352, Phe353, Gly354, Lys371, Glu421,

Val423, and Asp470. Docked RBX showed interactions

with Thr404, Glu421, Val423, and Asp470. The interact-

ing residues of their respective co-crystallized ligands are

shown in Table II. It was observed that docked ATP

reproduced all the interactions of ANP, and it also

showed additional interactions. In RBX, all the interac-

tions of 2MB were reproduced though the docked poses

of 2MB and RBX differed in their indole ring

Figure 2Docked poses of (A1) ATP, and (B1) RBX in PKCbII; and superimposition of docked pose of (A2) ATP with co-crystallized ANP (full length

PKCbII, PDB Code: 3PFQ), (B2) RBX with co-crystallized 2MB (kinase domain PKCbII, PDB Code: 2I0E). [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]

B.K. Grewal et al.

6 PROTEINS

orientation. This small difference between the docked

poses of ATP and RBX with ANP and 2MB may be

attributed to their structural differences.

Stability of MD trajectories

The docked complexes of ATP and RBX in both phos-

phorylated and non-phosphorylated forms of PKCbII viz.

PKCbIIATP-p, PKCbIIATP-np, PKCbIIRBX-p, and PKCbIIRBX-

np were used for subsequent MD studies. The RMSD of

these four complexes was stabilized during the simulation

period (Fig. S4 of Supporting Information). In addition,

the Ca RMSD of N-terminal (339–421), hinge (422–424),

and C-terminal (426–620) regions with reference to the

initial conformation of docked complexes indicate that

the lobes move rigidly as a whole (Table S2 of Supporting

Information).

Glycine loop

Among the two reported PKCbII crystal structures, the

glycine loop conformation in the full length structure is

above in comparison to the glycine loop conformation in

its kinase domain structure. The full length structure rep-

resents the active state conformation more closely than

that of the kinase domain structure. Consequently, the

glycine loop is expected to move above or below with

respect to its intermediate conformation in PKCbII kinase

domain crystal structure while the structure attains active

or inactive form, respectively. The stabilized trajectories

of the simulation run for the above complexes were ana-

lyzed by clustering the 4000 conformations generated

during 150 ns run into 10 clusters using ptraj module of

AMBER11 (Table III, Fig. 3). The representative structure

from each cluster was superimposed with kinase domain

crystal structure of PKCbII. In the PKCbIIATP-p complex,

all the representative structures sampled were above the

glycine loop conformation of PKCbII crystal structure.

Here, cluster5 (29.5%, magenta) was highly populated

and its glycine loop conformation was above the confor-

mation observed in the crystal structure. The clusters of

PKCbIIATP-np sampled covered a wide conformational

space of glycine loop involving both the above and below

conformations with respect to crystal structure conforma-

tion of glycine loop. Here, cluster10 (36.20%, light-blue)

was highly populated and its glycine loop conformation

was below the crystal structure conformation. The confor-

mation of moderately populated cluster1 (27.40%) was

above the glycine loop in the crystal structure conforma-

tion. It seems that in PKCbIIATP-np, open-state was exhib-

ited for a prolonged period, but close-state was also

present for considerable amount of time. This indicates

that the non-phosphorylated PKCbII is not completely

mature to exhibit its kinase activity. Thus, PKCbIIATP-p

can be considered achieving active state while PKCbIIATP-

np being relatively unstable during simulation.

In both, PKCbIIRBX-p and PKCbIIRBX-np, the confor-

mational space covered by glycine loops of the represen-

tative structures from 10 clusters was less than that in

PKCbIIATP-p and PKCbIIATP-np. Also, in both the com-

plexes, conformation of representative structure from

highly populated clusters viz. cluster1 (23.70%; red) in

PKCbIIRBX-p and cluster1 (32.50%; red) in PKCbIIRBX-np

were below the crystal structure conformation of glycine

loop. But, movement of glycine loop in PKCbIIRBX-p

cluster was less than that in PKCbIIRBX-np. In

PKCbIIRBX-p, one moderately populated cluster, cluster3

(11.10%), and one low populated cluster, cluster5

(0.20%), showed the glycine loop above relative to its

crystal structure conformation. Hence, in PKCbIIRBX-p,

11.3% of the total conformations represent near active

Table IIParameter Comparison of Docked ATP and RBX with Co-crystallized

ANP and 2MB, Respectively, in ATP Binding Site of PKCbII

S. No. Ligands Interacting residues RMSDa

1. ATP Glu421, Val423, Lys350, Ser352, Phe353,Gly354, Lys371, Asp470

0.476�

2. ANP Glu421, Val423, Lys3713. RBX Glu421, Val423, Thr404, Asp470 0.155 �4. 2MB Thr404, Glu421, Val423, Asp470

aOf ATP with respect to ANP; RBX with respect to 2MB.

Table IIIQuantitative Distribution of Conformations from Molecular Simulation Study for the Four PKCbII Complexes in 10 Clusters

Cluster no. Color

Percentage occurrence of conformations in each cluster

PKCbIIATP-p PKCbIIATP-np PKCbIIRBX-p PKCbIIRBX-np

1 Red 2.20 27.40 23.70 32.502 Green 0.20 2.20 2.00 2.503 Blue 10.60 3.40 11.10 3.104 Yellow 9.10 1.70 21.50 2.005 Magenta 29.50 8.20 0.20 6.206 Orange 8.60 6.30 0.10 5.107 Olive green 8.50 1.70 16.00 26.808 Light pink 1.00 11.70 12.00 3.309 Black 19.30 1.20 7.00 8.7010 Light blue 10.90 36.20 6.40 9.70

Conformational Variance in PKCbII

PROTEINS 7

state in comparison to the crystal structure conforma-

tion, whereas remaining are toward inactive state. In

PKCbIIRBX-np all the states of the glycine loop are below

the crystal structure conformation of glycine loop. Con-

clusively, the conformational space of PKCbII complexes

with RBX includes most of the states toward inactive

form along with a small number of active states in

PKCbIIRBX-p. Also, a change in glycine loop conformation

was comparatively less in PKCbII complexes with RBX in

comparison to its complexes with ATP.

Distance between Ser352:CA andGly486:CA

In PKCbII crystal structure, the distance between the

residue Ser352:CA of glycine loop and Gly486:CA of acti-

vation loop is 8.7 A, signifying an intermediate conforma-

tion.11 The distance in its full-length structure, which is

closer to active state in comparison to kinase domain, is

13.5 A. The comparative analysis of distance between

Ser352:CA and Gly486:CA was performed for all the four

complexes (Fig. 4). In PKCbIIATP-p, the distance was

around 13–14 A till 100 ns of simulation period, and

from 100 to 150 ns the distance was around 14–16 A. In

PKCbIIATP-np, the distance was quite variable in initial

simulation period of 0–25 ns. However, after this time

period, stabilized distance of 4–5 A was observed through-

out simulation period of 150 ns. In PKCbIIRBX-p, the

Ser352:CA and Gly486:CA distance was quite variable

from 10 to 15 A during initial time period of �0–50 ns.

After this time period, the variation in distance compara-

tively reduced to 8–12 A for maximum simulation period.

In PKCbIIRBX-np, �8–9 A distance, a conformational

arrangement similar to PKCbII crystal structure was

maintained during the whole simulation period. These

observations also point that the inhibitor RBX tries to

retain the intermediate state, but phosphorylated residues

move the structural conformations toward active form.

RBX is an ATP competitive inhibitor and similar types of

observations for ATP-competitive inhibitors were also

reported by Smith and Hoshi for other protein kinases.9

Conformation of activation loop

Based on comparative crystal structure analysis and

reported studies for other protein kinases, we can say in

PKCbII, the activation loop (484–511) forms part of

Figure 3Superimposition of PKCbII crystal structure (cyan) with representative structures of the 10 clusters for the trajectories of: (A) PKCbIIATP-p, (B)

PKCbIIATP-np, (C) PKCbIIRBX-p, and (D) PKCbIIRBX-np. Above row is enlarged view of glycine loop; below row is superimposition of crystal struc-ture with structure of representative structure from highly populated cluster.

B.K. Grewal et al.

8 PROTEINS

substrate binding site and provides a surface for substrate

binding. When PKCbII is active, the activation loops are

in a fully extended conformation and facilitates the phos-

phorylation transfer from ATP to substrate. Conversely,

in the non-phosphorylated state, the substrate binding

region is expected to collapse thereby hindering the sub-

strate binding. The conformational changes in activation

loop were less in comparison to glycine loop conforma-

tions during 150 ns simulation period. Figure S2 of Sup-

porting Information shows the activation loop of both

full length PKCbII crystal structure, and active form of

insulin receptor (IR3) to be nominally up in comparison

to activation loop conformation in PKCbII kinase

domain. However, clear distinction is observed between

superimposition of the open and extended conformation

of fully phosphorylated activation loop of an active pro-

tein kinase, IR3 with collapsed non-phosphorylated acti-

vation loop of inactive IRK (Fig. S5 of Supporting

Information). In PKCbII only one residue viz. Thr500 is

phosphorylated unlike IRK, where three phosphorylating

sites are present. Also, in crystal structure of two different

states a more clear distinction between extended and col-

lapsed forms will be observable in comparison to simulat-

ing the same conditions for 150 ns. So, to study the

conformational changes in activation loop, it was pre-

sumed that flexible residues of the activation loop, other

important residues, and phosphorylated Thr500 play a key

role in its collapse. The structural basis of activation loop

movement in phosphorylated and non-phosphorylated

PKCbII was studied in terms of the movement of these

flexible residues.

Flexible residues

As per B-factor graphs, residues Met487, Asn491,

Asp494, Thr497, and Cys502 of activation loop were

showing high fluctuation (Fig. S6 of Supporting Informa-

tion). So in all the four complexes, the distances between

the glycine loop (Ser352:CA) and CA of these residues

were calculated throughout the simulation period of 150

ns (Fig. 5). In PKCbIIATP-p, the results show a sudden

increase in all the distances after initial simulation period.

This can be correlated to the extension of the activation

loop. This indicates that the conformational changes in

enzyme are progressing to allow the substrate binding. In

PKCbIIATP-np also frequent changes were observed in the

initial phase signifying the continually changing confor-

mation of the activation loop. This might affect phospho-

rylation activity of kinases on their substrates by not

allowing substrate binding; thus suggesting an inactive

state of the kinase. In PKCbIIRBX-p complex, these distan-

ces show an overall slight increase, besides the intermedi-

ate variations of increase and decrease. In PKCbIIRBX-np,

all these measured distances were found to be stable

throughout the simulation. This can be attributed to the

effect of the bound inhibitor, RBX, which tries to stabilize

the enzyme in its quiescent state but its fully phosphoryl-

ated residues try to bring the protein to an active state.

Arg465 and Asp466 (RD) residues

PKCbII belongs to the RD kinases category and these

kinases are characterized by the presence of Arg465 (R)

residue preceding Asp466 (D).8 The RD kinases are

known to be activated by phosphorylation of the activa-

tion loop. The R residue of RD kinases shows a few ionic

interactions with phosphate or carboxylate group of the

neighboring residues. In PKCbII, Arg465 shows three

ionic contacts with phosphate of phosphorylated Thr500

(TPO500), and additional interactions with Met487,

Tyr518, and Asp523 (Table IV). The D residue, Asp466 is

essential for substrate binding. It acts as a base in the

catalytic mechanism of PKCbII and activates the incom-

ing substrate hydroxyl, interacting with the nucleophilic

hydroxyl side chain of the substrate.

The interactions between Thr500 and Arg465 in all the

four complexes were analyzed. It was observed that distance

between the centers of mass of these residues is relatively

Figure 4Distance between Ser352:CA of glycine loop and Gly486:CA of activation loop during simulation period in (A) PKCbIIATP-p (red), PKCbIIATP-np

(blue); (B) PKCbIIRBX-p (red), PKCbIIRBX-np (blue). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Conformational Variance in PKCbII

PROTEINS 9

smaller and stable in PKCbIIATP-p and PKCbIIRBX-p in

comparison to PKCbIIATP-np and PKCbIIRBX-np [Fig.

6(A,B)]. In phosphorylated form, TPO500 holds together

Arg465 and Lys489 by making ionic interactions with both

of them [Fig. 6(C)]. Removal of phosphate from TPO500

disrupts its interactions with Arg465 and Lys489. As a

result, the amine groups of Arg465 and Lys489 repel each

other which may affect the substrate binding region of

PKCbII. This provides a probable explanation of the role

of phosphorylated form of Thr500 in substrate binding.

The Thr500 adopts the central position in activation loop

and the ion-pair formed by its phosphorylated form

(TPO500) is important in rotating DFG motif into proper

orientation for catalysis.The dihedral angle analysis of

Thr500 (N-CA-CB-OG1) throughout the simulation

showed that its orientation was stable in phosphorylated

complexes, but flipped by �180� in non-phosphorylated

state (Fig. 7). This also reflects the loss of interaction

between Thr500 and Arg465 in the latter case.

Gating residues

Comparative analysis of PKCbII with other kinases

suggests that Met487 and Gly503 act as gate keeper resi-

dues for substrate binding site. The distance between

these residues is indicative of the opening and closing of

the gate thereby, giving the substrate access to Asp466 of

PKCbII active site to get phosphorylated. The distance

Figure 5Distance between glycine loop and activation loop in (A) PKCbIIATP-p, (B) PKCbIIATP-np, (C) PKCbIIRBX-p, (D) PKCbIIRBX-np during the simula-tion period. The distances are between Ser352:CA of glycine loop and CA of the activation loop residues Met487:CA (light blue), Asn491:CA

(brown), Asp494:CA (gray), Thr497:CA (yellow), and Cys502:CA(dark blue). [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

Table IVIonic Interactions of Arg465 and Asp466 with Other Residues as Observed in PKCbII Crystal Structure, PKCbIIATP-p, PKCbIIATP-np, PKCbIIRBX-p,PKCbIIRBX-np

Residue name

Ionic interactions with other residues

PKCbII crystal structure PKCbIIRBX PKCbIIATP

Asp466 Asn471, HOH101 Lys468 (in p and np)a Asn471(in p), Asn471 and Lys468 (in np)Arg465 Phosphate of TPO500 (3),

Met487 (2), Tyr518 (1), Asp523Similar interactions as in crystal structure

(in p) Only two contacts with Met487 (in np)Similar interactions as in crystal structure

(in p) Thr500(1), Met487(2), Asp523(1) (in np)

ap signifies the phosphorylated complex, and np signifies the non-phosphorylated complex.

B.K. Grewal et al.

10 PROTEINS

between Met487 and Gly503, and each of these residues

from Asp466, was measured in all the four complexes

(Fig. 8). The distance between the gating residues is

highest in the case of PKCbIIATP-p (orange) indicating

the easy access of the substrate to Asp466 to get phos-

phorylated by transfer of the gamma phosphate of bound

ATP to the phosphorylation site of the substrate. The

distance fluctuates in the case of PKCbIIATP-np (blue)

and the access gate is not as wide open as in PKCbIIATP-p

to permit phosphorylation of the substrate. In the case

Figure 6Distance between Arg465 and Thr500 residues during simulation period in (A) PKCbIIATP-p (red), PKCbIIATP-np (blue); (B) PKCbIIRBX-p (red),PKCbIIRBX-np (blue). (C) Ionic interactions shown by TPO500 in PKCbII crystal structure. [Color figure can be viewed in the online issue, which

is available at wileyonlinelibrary.com.]

Figure 7Dihedral angle of Thr500 (N-CA-CB-OG1) for (A) PKCbIIATP-p (red), PKCbIIATP-np (blue); (B) PKCbIIRBX-p (red), PKCbIIRBX-np (blue) duringsimulation period. (C1) Pictorial representation of Thr500 (N-CA-CB-OG1); and (C2) fixed and flipped position of Thr500 (N-CA-CB-OG1)

(cyan and deep blue) in phosphorylated and non-phosphorylated complexes, respectively. [Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

Conformational Variance in PKCbII

PROTEINS 11

of PKCbIIRBX-np (gray), the distance remains compara-

tively stable owing to the nature of the inhibitor to freeze

the conformation of the protein, while in PKCbIIRBX-p

(yellow), the window in which the distance fluctuates is

wide which can be attributed to the conflicting behavior

of the phosphorylated state and the inhibitor on the pro-

tein. Considering these observations, it can be stated that

PKCbIIATP-p complex is close to active state of PKCbII

for exhibiting its kinase activity while the remaining

three complexes exhibit a state unsuitable for the kinase

activity.

A sudden decrease in distance between two gating resi-

dues Met487 and Gly503; and between substrate binding

residue Asp466 and gating residue Gly503 was observed

after �130 ns time period in PKCbIIATP-p and

PKCbIIRBX-np complexes. Also the distance between

Asp466 and Gly503 was less stable in PKCbIIRBX-p. How-

ever, distance between the substrate binding residue

Asp466 and gating residue Met487 was unchanged. This

indicates the movement of Gly503 in PKCbIIATP-p,

PKCbIIRBX-p, and PKCbIIRBX-np. The position of Gly503

is expected to be effected by mutation of phosphorylated

Thr500 to non-phosphorylated state, as Gly503 is only

three positions away from Thr500. Longer simulation

runs for these complexes can be expected to provide a

better picture for behavior of Gly503 in these complexes.

aC-helix

In active form of the kinases, conserved Glu390 of

aC-helix forms ion-pair with Lys371, whereas in inactive

state the aC-helix is rotated outward about its long

axis.8 This swings the conserved Glu390 out of the active

site and disrupts its interaction with Lys371 that coordi-

nates ATP. Activation induces reverse rotation that

restores the Lys371–Glu390 ion pair. Movement of cru-

cial Glu390 is correlated with an inhibitory conformation

of the activation loop. In PKCbII crystal structure, Nz of

Lys371 showed interactions with OE1 and OE2 of

Glu390. The distances from Lys371:Nz to Glu390:OE1

and Glu390:OE2 were calculated in all the four com-

plexes (Fig. 9). The average distances from Lys371:Nz to

Glu390:OE1 in PKCbIIATP-p, PKCbIIATP-np, PKCbIIRBX-p,

and PKCbIIRBX-np were 3.01, 3.44, 3.34, and 3.29 A,

Figure 8Distance between (A) gating residues Met487 and Gly503 of activation loop, (B) gating residue Met487, and substrate binding siteAsp466, (C)

gating residue Gly503 and substrate binding site Asp466 for the complexes PKCbIIATP-p (orange), PKCbIIATP-np (blue), PKCbIIRBX-p (yellow),PKCbIIRBX-np (gray). All the distances are measured between CA of respective residues and the linear trend line is drawn with reference to

PKCbIIATP-p (orange). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

B.K. Grewal et al.

12 PROTEINS

respectively. This distance got reduced to 3.25 A after 40

ns in the case of PKCbIIATP-np. Additionally, the average

distances from Lys371:Nz to Glu390:OE2 in PKCbIIATP-p,

PKCbIIATP-np, PKCbIIRBX-p, and PKCbIIRBX-np were 3.03,

3.04, 3.45, and 3.44 A, respectively. Collectively analyzing

these average distances indicated that the Lys371–Glu390

ion pair was more prominent in PKCbIIATP-p than that

in PKCbIIATP-np, while the same was much less promi-

nent in both the inhibitor bound complexes, PKCbIIRBX-p

and PKCbIIRBX-np. These average distances support the

statement that the ion pair is formed in the active kinases

(PKCbIIATP-p) and not in the inactive ones (PKCbIIATP-

np). However, from �100 to 150 ns, both in PKCbIIATP-p

and PKCbIIATP-np the Lys371-Glu390 seems to be inter-

acting. Longer simulation run in this case also will make

the picture clearer. The superimposition of PKCbII crystal

structure with representative structures of PKCbIIATP-p,

PKCbIIATP-np, PKCbIIRBX-p, and PKCbIIRBX-np is shown

in Figure 10.

RBX interaction with ATP binding siteresidues of PKCbII

Smith and Hoshi reported that PKC inhibitors are

state dependent, and the ATP competitive inhibitors tar-

get quiescent PKC and stabilize PKC in the quiescent

conformation itself.9 It generates slower activation and

suppressed translocation upon activation of PKC. The

reported kinase domain crystal structure of PKCbII is in

fully phosphorylated intermediate state with ATP com-

petitive inhibitor, 2MB being co-crystallized in it. The

study of both glycine loop conformation and Ser352:CA

and Gly486:CA distance shows that PKCbIIRBX-p and

PKCbIIRBX-np exhibited less conformational changes dur-

ing molecular simulation period of 150 ns in comparison

to PKCbIIATP-p and PKCbIIATP-np. All these observations

indicate that RBX seems to bind in intermediate state

and inhibit further conformational changes which are

favorable for binding of second messenger and full acti-

vation of PKCbII. To elucidate the RBX interaction with

PKCbII, hydrogen bond interactions formed by the rep-

resentative structures of 10 clusters obtained from the

molecular simulation of 150 ns time-period are shown in

Table V.

In PKCbIIRBX-p residues Glu421, Val423 formed H-

bond with N1 and O25 throughout the simulation,

whereas O24 showed interaction with Thr404 for partial

period of 150 ns (Fig. 11). The a and b indole of RBX

are surrounded by hydrophobic residues. In comparison

to 2MB, the a and b indole rings of RBX were not tilted

with respect to each other [Fig. 2(B2)]. This ensures bet-

ter hydrophobic interactions in RBX than 2MB. The

Lys371, Met473, Ala483, and Asp484 are near the a-

indole and Phe353, Asp470, and Asn471 are near its side

chain substituents. The residues Leu348, Val356, and

Asp427 are near the b-indole of RBX. The a-indole ring

formed hydrophobic interactions with Phe353, Ala483,

and Met473. The RBX atoms C12, C13, C14 are close to

Figure 9Distance from Lys371:Nz toGlu390:OE1 and Glu390:OE2 interacting atoms during simulation period in (A and C) PKCbIIATP-p (red), PKCbIIATP-np

(blue); (B and D) PKCbIIRBX-p (red), PKCbIIRBX-np (blue). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.

com.]

Conformational Variance in PKCbII

PROTEINS 13

Ala483; C30, C31, C32 are close to Phe353; and C8, C14

are near Met473. The b-indole showed hydrophobic

interactions with Leu348 and Val 356; C22 and C23

closer to Leu348; C15, C19 close to Val356. The N9 and

N18 bridge of RBX span the ATP binding site, and N33

atom of this bridge seems to be in proximity to two

polar amino acids; Asp470 and Asn471. Among them,

the Asp470 formed the H-bond with N33 of RBX. In

PKCbIIRBX-np, the hydrogen bonding interaction with

Glu421 and Val423 was observed. But, RBX interactions

with Thr404, Phe353, and Asp470 were missing. The resi-

dues Asp470 and Asn471 are near activation loop.

Removal of phosphate from Thr500 affects the activation

loop conformation, which may have resulted in the disrup-

tion of Asp470 H-bond in non-phosphorylated residues.

Conclusively, RBX showed interactions with Glu421

and Val423 both in PKCbIIRBX-p and PKCbIIRBX-np. In

addition, RBX showed interactions with Thr404 for

smaller simulation period; and with Phe353 and Asp470

during 150 ns molecular simulations in PKCbIIRBX-p.

However, these interactions were not observed in

PKCbIIRBX-np. Phe353 is part of flexible glycine loop, and

Asp470 is present near activation loop. According to the

study of conformational changes in glycine and activation

loop discussed above, in silico mutation of phosphorylated

residues to non-phosphorylated increases their flexibility.

The interactions of RBX with residue Phe353 of glycine

loop and Asp470 near activation loop are the difference

between RBX’s interaction patterns in PKCbIIRBX-p and

PKCbIIRBX-np. We can conclude that better interaction

ability of PKCbII inhibitors with Phe353 and Asp470 will

Figure 10Superimposition of PKCbII crystal structure (cyan) with representative structures of the highly populated cluster from PKCbIIATP-p (magenta),

PKCbIIATP-np (light-blue), PKCbIIRBX-p (red), and PKCbIIRBX-np (salmon) of 150 ns simulation run. [Color figure can be viewed in the onlineissue, which is available at wileyonlinelibrary.com.]

Table VSummary of H-Bonding Interactions of RBX in the RepresentativeStructures from the 10 Clusters in PKCbIIRBX-p and PKCbIIRBX-np

Complexes

Cluster no. PKCbIIRBX-p* PKCbIIRBX-np*

1 Glu421, Val423, Thr404, Asp470 Glu4212 Glu421, Val423, Asp470 Glu421, Val4233 Glu421, Val423, Asp470 Glu421, Val4234 Glu421, Asp470 Glu421, Val4235 Glu421, Val423, Asp470 Glu4216 Glu421, Val423, Asp470 Glu421, Val4237 Glu421, Val423, Asp470 Glu421, Val4238 Glu421, Val423, Asp470 Glu421, Val4239 Glu421, Val423, Asp470 Glu421, Val42310 Glu421, Val423, Asp470 Glu421, Val423

*Interacting residues with the RBX in each cluster.

B.K. Grewal et al.

14 PROTEINS

result in an increased ability to restrict the conformational

changes competent to full activation in PKCbII enzyme.

CONCLUSIONS

Comparative crystal structure analysis of protein

kinases with PKCbII in different conformations high-

lighted that overall conformational changes in active and

inactive forms are due to changes in key structural fea-

tures. These features are glycine loop, activation loop,

aC-helix, distance between Ser352:CA and Gly486:CA,

Arg465–Thr500 interactions, substrate binding site, and

orientation of Thr500. The molecular dynamics analysis

showed that the conformational changes in PKCbIIATP-p

were competent toward active state of PKCbII, while

PKCbIIATP-np being relatively unstable toward the active

state. In PKCbIIATP-np, changes toward the inactive state

were also observed. The PKCbII complexes with RBX

covered less conformational space in comparison to

PKCbII complexes with ATP. The conformational space

of PKCbII complexes with RBX includes most of the

states toward inactive form along with a small number

of active states in PKCbIIRBX-p. This shows the conflict-

ing behavior between the ATP competitive inhibitors to

restrict the further conformational changes toward active

site, and fully phosphorylated enzyme to achieve confor-

mational changes competent to full activation. In

PKCbIIRBX-np, the complex showed the changes toward

the inactive state. But the conformational space covered

by PKCbII complexes with RBX was very less in compar-

ison to the space covered by PKCbII complexes with

ATP. Role of Thr500 in activation loop conformation is

also explained. Conclusively, from this study, phospho-

rylation appears to be the main factor required for the

activation of PKCbII, and RBX type inhibitors are essen-

tial for inhibiting the enzyme to attain the conforma-

tional state competent with its full activation. The better

interaction of ATP competitive inhibitors with Asp470

and Phe353 will enhance their capacity to restrict confor-

mational changes in enzyme toward full activation. All

the analyses were based on 150 ns molecular simulations.

Few observations, like change in position of residues

Gly503 in phosphorylated complex, Lys371:Nz-Glu390

ion pair formations both in phosphorylated and non-

phosphorylated complexes can be explained clearly by

increasing the time-period of molecular simulation.

REFERENCES

1. Battaini F, Pascale A. Protein kinase C signal transduction regula-

tion in physiological and pathological aging. Ann N Y Acad Sci

2005;1057:177–192.

2. Kawakami T, Kawakami Y, Kitaura J. Protein kinase Cb (PKCb):

nomal functions and dieases. J Biochem 2002;132:677–682.

3. Idris I, Donnelly R. Protein kinase C b inhibition: a novel therapeu-

tic strategy for diabetic microangiopathy. Diab Vasc Dis Res 2006;3:

172–178.

4. Koya D, King GL. Protein kinase C activation and the development

of diabetic complications. Diabetes 1998;47:859–866.

5. Danis RP, Sheetz MJ. Ruboxistaurin: PKC-b inhibition for com-

plications of diabetes. Expert Opin Pharmacother 2009;10:2913–

2925.

6. Aiello LP, Davis MD, Girach A, Kles KA, Milton RC, Sheetz MJ,

Vignati L, Zhi XE. Effect of ruboxistaurin on visual loss in patients

with diabetic retinopathy. Ophthalmology 2006;113:2221–2230.

7. Bridges AJ. Chemical inhibitors of protein kinases. Chem Rev 2001;

101:2541–2572.

Figure 11(A) Binding pose of RBX in ATP binding site residues of PKCbII; and (B) the residues in binding vicinity of PKCbII. [Color figure can be viewedin the online issue, which is available at wileyonlinelibrary.com.]

Conformational Variance in PKCbII

PROTEINS 15

8. Huse M, Kuriyan J. The conformational plasticity of protein kinases.

Cell 2002;109:275–282.

9. Smith IM, Hoshi N. ATP competitive protein kinase C inhibitors

demonstrate distinct state-dependent inhibition. PLoS One 2011;6:

1–9.

10. http://www.nature.com/scitable/topicpage/gleevec-the-breakthrough-

in-cancer-treatment-565.

11. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM,

Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S. Efficacy and

safety of a specific inhibitor of the BCR-ABL tyrosine kinase in

chronic myeloid leukemia. N Engl J Med 2001;344:1031–1037.

12. Kornev AP, Haste NM, Taylor SS, Ten Eyck LF. Surface comparison

of active and inactive protein kinases identifies a conserved activa-

tion mechanism. Proc Natl Acad Sci USA 2006;103:17783–17788.

13. Sonnenburg ED, Gao T, Newton AC. The phosphoinositide-

dependent kinase, PDK-1, phosphorylates conventional protein

kinase C isozymes by a mechanism that is independent of phospho-

inositide 3-kinase. J Biol Chem 2001;276:45289–45297.

14. Yonemoto W, McGlone ML, Grant B, Taylor SS. Autophosphoryla-

tion of the catalytic subunit of cAMP-dependent protein kinase in

Escherichia coli. Protein Eng 1997;10:915–925.

15. Grodsky N, Li Y, Bouzida D, Love R, Jensen J, Nodes B, Nonomiya

J, Grant S. Structure of the catalytic domain of human protein

kinase C b II complexed with a bisindolylmaleimide inhibitor. Bio-

chemistry 2006;45:13970–13981.

16. Leonard TA, R�o_zycki B, Saidi LF, Hummer G, Hurley JH. Crystal

structure and allosteric activation of protein kinase C bII. Cell

2011;144:55–66.

17. Schr€odinger L. PyMOL molecular graphics system, version 1.5. 0.4.

18. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz

DT, Repasky MP, Knoll EH, Shelley M, Perry JK. Glide: a new

approach for rapid, accurate docking and scoring. 1. Method and

assessment of docking accuracy. J Med Chem 2004;47:1739–1749.

19. Case D, Darden T, Cheatham III T, Simmerling C, Wang J, Duke R,

Luo R, Walker R, Zhang W, Merz K. AMBER 11. San Francisco:

University of California; 2010. p 142.

20. Bayly CI, Cieplak P, Cornell W, Kollman PA. A well-behaved electro-

static potential based method using charge restraints for deriving

atomic charges: the RESP model. J Phys Chem 1993;97:10269–10280.

21. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson

DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA. A second

generation force field for the simulation of proteins, nucleic acids,

and organic molecules. J Am Chem Soc 1995;117:5179–5197.

22. Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Develop-

ment and testing of a general amber force field. J Comput Chem

2004;25:1157–1174.

23. Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynam-

ics. J Mol Graphics 1996;14:33–38.

24. http://home.chpc.utah.edu/�cheatham/cluster-ptraj.pdf.

25. Hubbard SR. Crystal structure of the activated insulin receptor tyro-

sine kinase in complex with peptide substrate and ATP analog.

EMBO J 1997;16:5572–5581.

26. Hubbard SR, Wei L, Ellis L, Hendrickson W. Crystal structure of

the tyrosine kinase domain of the human insulin receptor. Nature

1994;372:746.

27. Knighton DR, Zheng J, Ten ELF, Ashford VA, Xuong NH, Taylor

SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic

adenosine monophosphate-dependent protein kinase. Science 1991;

253:407–414.

28. Akamine P, Wu J, Xuong NH, Eyck LFT, Taylor SS. Dynamic fea-

tures of cAMP-dependent protein kinase revealed by apoenzyme

crystal structure. J Mol Biol 2003;327:159–171.

29. Narayana N, Diller TC, Koide K, Bunnage ME, Nicolaou K,

Brunton LL, Xuong NH, Ten ELF, Taylor SS. Crystal structure of

the potent natural product inhibitor balanol in complex with the

catalytic subunit of cAMP-dependent protein kinase. Biochemistry

1999;38:2367–2376.

30. Trafny EA, Xuong NH, Adams JA, Eyck LFT, Taylor SS, Sowadski

JM. cAMP-dependent protein kinase: crystallographic insights into

substrate recognition and phosphotransfer. Protein Sci 1994;3:176–

187.

31. Zheng J, Knighton DR, Taylor SS, Xuong NH, Sowadski JM, Eyck

LFT. Crystal structures of the myristylated catalytic subunit of

cAMP-dependent protein kinase reveal open and closed conforma-

tions. Protein Sci 1993;2:1559–1573.

32. Takimura T, Kamata K, Fukasawa K, Ohsawa H, Komatani H,

Yoshizumi T, Takahashi I, Kotani H, Iwasawa Y. Structures of the

PKC-kinase domain in its ATP-bound and apo forms reveal defined

structures of residues 533-551 in the C-terminal tail and their roles

in ATP binding. Acta Crystallogr Sect D: Biol Crystallogr 2010;66:

577–583.

33. Messerschmidt A, Macieira S, Velarde M, B€adeker M, Benda C,

Jestel A, Brandstetter H, Neuefeind T, Blaesse M. Crystal structure

of the catalytic domain of human atypical protein kinase C-iota

reveals interaction mode of phosphorylation site in turn motif. J

Mol Biol 2005;352:918–931.

B.K. Grewal et al.

16 PROTEINS