Biopolymer s

Relating Nucleotide-Dependent Conformational Changes in Free TubulinDimers to Tubulin Assembly

Kathiresan Natarajan, Jagan Mohan, Sanjib SenapatiDepartment of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India

Received 12 June 2012; accepted 29 August 2012

Published online 10 September 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22153

This article was originally published online as an accepted

preprint. The ‘‘Published Online’’ date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

Microtubules (MTs) are cytoskeletal polymers that

play key roles in diverse array of cellular func-

tions, such as mitosis and meiosis, motility,

maintenance of cell shape, and intracellular

transport of organelles. The a,b-tubulin dimer is

the basic structural unit of MTs. Essential to the microtubule

stability and function, the dimer contains two nonidentical

guanine-nucleotide binding sites—a nonexchangeable (N)

site at a-tubulin that always binds a GTP molecule, and

an exchangeable (E) site at b-tubulin that can bind either a

GTP or GDP.1,2 The bound nucleotide at E site stays

in equilibrium with free nucleotides in cytosol, where the

GDP $ GTP exchange at E site can take place freely.3,4

In vitro, MT assembly from purified tubulin solutions

involves polymer nucleation and elongation to reach a steady

state, which is characterized by a constant ratio of polymeric

tubulin to free tubulin dimers. According to the existing

model, nucleation is the crucial initial phase where several

GTP-tubulin dimers assemble to form oligomers that subse-

quently combine to form 2D sheets and microtubules.5,6

Thus, a detailed knowledge of the structural differences

between free GTP- and GDP-tubulin dimers in solution

has direct relevance in understanding the MT assembly and

function.

Relating Nucleotide-Dependent Conformational Changes in Free TubulinDimers to Tubulin Assembly

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

article.Correspondence to: Sanjib Senapati; e-mail: [email protected]

ABSTRACT:

The complex dynamic behavior of microtubules (MTs) is

believed to be primarily due to the ab-tubulin dimer

architecture and its intrinsic GTPase activity. Hence, a

detailed knowledge of the conformational variations of

isolated a-GTP-b-GTP- and a-GTP-b-GDP-tubulin

dimers in solution and their implications to interdimer

interactions and stability is directly relevant to

understand the MT dynamics. An attempt has been made

here by combining molecular dynamics (MD)

simulations and protein–protein docking studies that

unravels key structural features of tubulin dimer in

different nucleotide states and correlates their association

to tubulin assembly. Results from simulations suggest that

tubulin dimers and oligomers attain curved

conformations in both GTP and GDP states. Results also

indicate that the tubulin C-terminal domain and the

nucleotide state are closely linked. Protein–protein

docking in combination with MD simulations suggest

that the GTP-tubulin dimers engage in relatively stronger

interdimer interactions even though the interdimer

interfaces are bent in both GTP and GDP tubulin

complexes, providing valuable insights on in vitro finding

that GTP-tubulin is a better assembly candidate than

GDP-tubulin during the MT nucleation and elongation

processes. # 2012 Wiley Periodicals, Inc. Biopolymers 99:

282–291, 2013.

Keywords: tubulin dimer; nucleotide state; tubulin

assembly; molecular dynamics simulation; protein–

protein docking

Contract grant sponsor: Council of Scientific and Industrial Research (CSIR)

VVC 2012 Wiley Periodicals, Inc.

282 Biopolymers Volume 99 / Number 5

Although crystal structures of a,b-tubulin dimer bound

to different antimitotic agents are available,1,2,7–13 till date

there is no report of experimentally determined structures of

isolated GTP- and GDP-tubulin dimers. Such structures will

be of immense interest since predominantly the isolated

tubulin dimers regulate the microtubule function, as dis-

cussed above. The crystal structures of tubulin show three

functionally distinct domains in each monomer (Figure 1).

An N-terminal domain (N-domain) that constitutes the nu-

cleotide-binding region, an intermediate domain (I-domain)

that contains the taxol binding site, and a C-terminal domain

(C-domain) that has been implicated in the binding of sev-

eral motor proteins. The N-terminal domain comprises of

alternating parallel b-strands (S1–S6) and a-helices (H1–

H6). The loops, T1–T6 that join each strand and helix along

with the core helix H7, form the nucleotide binding pocket.

The intermediate domain formed by three helices (H8 –

H10) and a mixed b sheet (S7 – S10), involves in lateral and

longitudinal contacts with neighboring dimers. The loop

between S7 and H9, commonly known as M loop, comprises

part of the taxol-binding site in b-tubulin. The loop between

helices H7 and H8 (loop T7) and H8 itself are seen to inter-

act with the nucleotide of next subunit along the protofila-

ment. The C-terminal domain is formed by two antiparallel

helices, H11 and H12 that fold across the other two domains.

The small helix, H11’ connecting these two helices is also im-

portant for the interaction with the next monomer along the

protofilament.

Computational methods have proven to be a valuable tool

for biological research. Recent computational studies have

shed light on various properties of tubulins and MTs, which

were otherwise difficult to explore experimentally.14–30 The

role of nucleotides on structural rearrangements of tubulin

dimers has recently been investigated by Gebremichael et

al.21 and Bennett et al.22 These simulation studies have indi-

cated intrinsic bending in tubulin dimers. Bennet et al. also

have noted that the dimer flexibility plays an important role

in tubulin assembly. Simulations of model protofilaments

have helped to understand the structural features of tubulin

oligomers present in solution,23 binding of drugs24,25 and

antimitotic peptides,26,27 and the elastic properties of tubu-

lins in assembly.28–30

In this study, we use all-atom MD simulations and pro-

tein–protein docking to explore the binding characteristics of

tubulin dimers to produce tubulin oligomers, in different

nucleotide states. Specifically, we aim to elucidate the struc-

tural variations that make GTP-tubulin a better assembly

candidate than GDP-tubulin during MT nucleation and

elongation processes. Results suggest that GTP fine-tunes the

functional loop regions at interdimer interface to achieve

better binding with the next GTP-tubulin. Results also imply

that the tubulin C-terminal domain and the nucleotide state

are closely linked. Moreover, GTP-tubulins are found to

engage in relatively stronger interdimer interactions even

though the interdimer interfaces are bent in both GTP and

GDP tubulin oligomers.

RESULTS AND DISCUSSIONSTo begin with, we have performed two all-atom MD simula-

tions of ab-tubulin dimer liganded to GTP at N site and

GDP or GTP at E site. The simulations were initiated from

the straight crystal structure of tubulin dimer bound to taxol

(PDB ID: 1JFF1). Taxol was removed from the crystal struc-

ture, and the resultant conformation was equilibrated in

explicit water via 20 ns MD run, to obtain a model structure

for a-GTP-b-GDP-tubulin dimer. This equilibrated structure

was further engineered to generate the starting structure for

the second simulation, where we modified the GDP at E site

to GTP (by this means we wanted the GDP-to-GTP-exchange

to take place without the influence of taxol, as presumably

happens in cytosol). The resultant structure was then equili-

FIGURE 1 Taxol-bound straight crystal structure of ab-tubulin

heterodimer (PDB ID: 1JFF). For clarity, only the functionally rele-

vant loops T2, T3, T4, T5, H1-S2 from N-domain (red); loops M,

H6-H7, T7, helix H8 from I-domain (purple); and helices H11, H12

from C-domain (blue) are highlighted.

Nucleotide-Dependent Changes in Free Dimers to Tubulin Assembly 283

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brated via 20 ns MD run to obtain a model structure for a-

GTP-b-GTP-tubulin dimer. Both the model structures were

further simulated to generate 250 ns MD data, upon which

all analyses were performed. The simulation details are pre-

sented under ‘‘Materials and Methods’’ section.

Conformational Changes in GTP- and GDP-Tubulin

Dimers

The b-subunit is found to undergo relatively larger confor-

mational changes than the a-subunit in both GTP-tubulin

and GDP-tubulin simulations (Supporting Information Fig-

ure S1). This is not surprising, considering the fact that both

taxol binding and GTP-hydrolysis take place within the b-

subunit. Moreover, a comparison of the root mean squared

displacements (RMSD) of the residues of b-subunits implies

that the GTP-tubulin residues deviate slightly more than the

GDP-tubulin residues from the starting crystal conformation

(Supporting Information Figure S2). A detailed comparison

of the model structures with the crystal conformation is pre-

sented in Figure 2. The figure is generated by a stereo super-

position of the crystal structure and the final 100 ns time-

averaged structures from simulations, according to Ca atoms

of the b-subunits. In a 3D representation of the structural

elements, the figure highlights the most notable variances

among the three states of tubulin dimer. The sites of maxi-

mum variances include the nucleotide binding loops T1, T2,

T3, T5, and some of the allosteric loops, M loop, H6-H7

loop, H1-S2 loop in b-monomer. The T1, T2 loops close

down over the nucleotide in GTP-tubulin under the influ-

ence of additional c-phosphate. The T3 and T5 loops, which

are known to be involved in longitudinal contacts, show

larger deviation in GTP-tubulin dimer. The M loop that

comprises part of the taxol binding site, exhibits a great deal

of variation. Although it is found to protrude outward later-

ally in GTP-tubulin dimer as similar to the taxol-bound

tubulin structure (though in larger extent in the former), it

protrudes inward in GDP-tubulin dimer. The H1-S2 loop

that resides opposite to M loop also demonstrates a greater

outward movement, when the protein b-subunit is bound to

GTP or taxol. The H6-H7 loop, which is known to play a key

role in longitudinal interactions along the protofilaments,

shows greater extendibility in GTP-tubulin dimer than in the

other two systems. These changes are shown in Figure 2. To

visualize the differential movements of these loops more

clearly, we have plotted their distances from the core helix

H7 as a function of time in GTP and GDP states. As Figure 3

indicates, these loops are more flexible and tend to extend

farther in GTP state. The greater tendency of some of these

loop regions for outward longitudinal movements in GTP-

tubulin might suggest that GTP binding favors interdimer

interactions and hence the protofilament elongation. It is

generally believed that largely extended protein residues are

more free to move and hence can readily approach to the

neighboring subunits for stronger interactions. It is also

reported that GTP binding favors protofilament elonga-

tion.31 The movements of the allosteric loops due to nucleo-

tide exchange are not simply stochastic, as shown by recent

mutational and protein–drug interaction studies where

strong correlations between tubulin N- and I-domains were

noted.24,32

When this work was in progress, the crystal structures of

GTP- and GDP-tubulins in complex with a stathmin-like do-

main were reported.31 In accordance with the crystal struc-

tures, our simulation results show a great deal of difference

between GTP- and GDP-tubulins in the T3 and T5 loop

region. Both the loops, in GTP-tubulin, show greater propen-

sity to involve in longitudinal interactions by extending out-

ward longitudinally (Figures 2 and 4a). The direct interaction

FIGURE 2 Time-averaged structures of the model GTP- and

GDP-tubulin dimers, superposed on the taxol-bound tubulin crystal

structure, based on the Ca atoms of b-subunits. Secondary struc-

tural elements which underwent the most significant conforma-

tional changes are highlighted. Color scheme—orange: GTP-tubu-

lin, green: GDP-tubulin, blue: crystal structure.

284 Natarajan, Mohan, and Senapati

Biopolymers

of GTP c-phosphate with the T3 loop helps the b:Asn101

side chain swinging out, which in turn modulates the posi-

tion of b:Thr180 in T5 loop. As a result, the H-bond between

b:Asn101 and b:Thr180 breaks and the T5 loop flips out in

GTP state, as similar to the crystal structure of GTP-tubulin.

This makes b:Asn101, which is the most conserved residue

across the species and known to make longitudinal contact,

more available for interactions. The T5 loop residue

b:Asp179 is also seen to become more exposed in GTP-tubu-

lin, similar to the crystal structure. These changes are shown

in Figure 4a and Supporting Information Movies S1 and S2.

Our simulation data could also capture both ‘‘in’’ and ‘‘out’’

conformations of T5 loop in GDP-tubulin as similar to the

crystal structure, even though the flip-out movement was

short lived (Figure 3b).

It is noteworthy that the core elements of the protein also

have undergone changes under the influence of the nucleo-

tide. This is shown in Figure 4b, where the superposition of

N- and C-terminal domains of the b-subunit demonstrates

variations in the intermediate domain. This domain is found

FIGURE 3 Differential movements of functionally relevant loops in GTP and GDP states. A dis-

tance metric is used, in which the Ca distance of the central residue of a loop is measured from the

centre of core helix H7, over time. The regions of interest include: (a) T3 loop, (b) T5 loop, (c) H6-

H7 loop, (d) M loop, and (e) H1-S2 loop. Color scheme—Red: GTP-tubulin; green: GDP-tubulin.

All the loop regions appear to be more flexible and to extend farther in GTP state. In (b), the T5

loop of GDP-tubulin attains ‘‘out’’ conformation similar to the GTP state during 0–30 ns and 100–

130 ns of simulation time. Otherwise, this loop attains ‘‘in’’ conformation in GDP state.


Biopolymers

to rotate slightly toward N-domain in GTP state but rotates

in opposite direction in the GDP state. A very similar rota-

tion in the intermediate domain was noted, while comparing

the classical curved (PDB ID: 1SAO) to straight tubulin

structures (PDB ID: 1JFF) by tubulin N- and C-terminal

superposition. The observed change in the intermediate

domain is also consistent with the proposal of Amos and

Lowe that ‘‘hydrolysis (GTP to GDP state) could vary the rel-

ative orientation of the N terminal domain and intermediate

domain.’’33

Nucleotide-Dependent Intradimer Bending

The controversy over straight versus curved conformation of

ab-tubulin dimer remains unresolved over the decades.

Although the allosteric model postulates that GTP-tubulin

dimer is straighter and prestructured in solution for assembly

onto the microtubule wall,34–37 the lattice model proposes

that the tubulin dimers adopt the curved conformation in

solution irrespective of the nucleotide state.38–43 In this

work, following the prescription of Nogales and Wang,36 we

have estimated the intradimer bending in tubulin by com-

puting the angle subtended by the axis of two monomers

within the dimer. The bending deformation is quantified by

computing the instantaneous angle assumed by two vectors

A and C, drawn on the molecular frames. The origin of the

coordinate system was chosen to be the centre of mass

(COM) of the overlapping region of a- and b-monomers,

primarily comprised of a:H6-H7, a:H11-H12, a:T5, a:T3,

a:T2, b:H10, b:H10-S9, b:S9, b:H8, and b:T7. The vector

from the origin to the COM of a-subunit (excluding the resi-

dues considered in overlapping region) was termed as A, and

the one from the origin to the COM of b-subunit (excluding

the residues in overlapping region) was termed as C. The vec-

tors are set to point to the COM of the subunits. The angle

subtended by the axes of two subunits was then computed as:

H ¼ 180o � Cos�1ðA:CÞ=jAjjCj:

The computed angular distributions of the model dimers,

sampled over the final 100 ns production phase, are pre-

sented in Figure 5. The result from the control simulation of

taxol-bound GDP-tubulin crystal structure is also included

for comparison. The profiles show a similar shape with the

distribution nearly Gaussian, peaking at an angle of 208 for

GDP-tubulin and 178 for GTP-tubulin. The taxol-bound

structure is seen to distribute around 148, in comparison to

its value of 118 in the lattice-constrained conformation in the

crystal structure. The angle values thus indicate that tubulin

relaxes from straight crystal conformation to more curved

conformations in isolated states and both the GTP and GDP

tubulin dimers are naturally bent. This finding is consistent

with the recent crystal structures, where both GTP- and

GDP-tubulins, bound to RB3-SLD protein, were found to

attain curved conformations.31 The advantage of the present

simulation data is that it covers a range of distribution of the

intradimer angle, out of which the curved conformation

stands out to be the most probable state. The time-averaged

FIGURE 4 (a) Close-up view of the nucleotide binding region.

Loops T3, T5 from both GTP- (orange) and GDP-tubulins (green)

are highlighted. Also shown is the nucleotide in GTP state. The

presence of an Mg21 ion along with its coordinating water was

observed around the GTP in GTP-tubulin. (b) Change in intermedi-

ate domain (orange: GTP-tubulin, green: GDP-tubulin) is high-

lighted by superposing the N- and C-domains (light green).


Biopolymers

structures of the model GTP- and GDP-tubulins are com-

pared with the curved crystal structures of tubulin dimer

from the work of Nawrotek et al.31 and are shown in Sup-

porting Information Figure S3. The RMSD values for the

GTP- and GDP-states are only 1.82 A and 1.64 A, suggesting

that tubulin dimer is indeed bent in both states. The direc-

tion of bending is outward, almost tangential to the MT wall.

Interestingly, the C-domain in GTP-tubulin dimer slightly

misaligns from the crystal structure. We will discuss it in a

subsequent section.

Nucleotide State and Tubulin Assembly

Recent electron microscopy studies due to Mozziconacci et

al.44 have suggested that the tubulin dimers can exist as short

oligomers in solution. Moreover, tubulin oligomers form the

nuclei for MT nucleation process.5,6 These reports encour-

aged us to construct the tubulin dimer–dimer complexes

from the model tubulin structures and investigate their bind-

ing characteristics in GTP- and GDP-states. The dimer–

dimer complexes were initially built from the time-averaged

structures, using the protein–protein docking program

HADDOCK.45 The lowest energy complex with correct lon-

gitudinal pitch from each category was subjected to 50 ns

MD simulation for structure refinement. Figure 6 depicts the

time-averaged conformations of the complexes, obtained

from the final 20 ns simulation data. We found that the con-

formations of our model protofilaments resemble the RB3-

SLD crystal structure very closely, with a few additional lon-

gitudinal contacts. The calculated interdimer contact angle in

model GDP-protofilament is � 128, which is very close to

the value of 12.68 in the crystal structure. The GTP-protofila-

ment is also found to be bent with the interdimer angle is

� 88. The range of angles could be attributed to the plasticity

at the interdimer interface. It is also possible that all contacts

could not be formed in the docking experiment or during

the subsequent simulation time in GTP state.

It is worth mentioning here that, in the HADDOCK out-

put, even though the cluster-1 (i.e., energetically ranked 1) in

GTP-tubulin docking contained the lowest energy complexes

with correct pitch, in case of GDP-tubulin only cluster-3 and

cluster-4 (i.e., energetically ranked 3 and 4) contained the

conformations with correct longitudinal pitch. These cor-

rect-pitch-conformations represent 63% and 59% of the total

docked complexes in GTP- and GDP-state, with average

intermolecular energy 2678.85 6 30.74 and 2303.98 6

89.12 kcal/mol, respectively. For GDP-tubulin docking, the

lower energy conformations in cluster-1 and cluster-2 had

average intermolecular energies 2481.82 6 69.88 and

2366.81 6 66.47, respectively. In these dimer–dimer com-

plexes, however, the top dimer rotated substantially about

the vertical axis, which presumably could be an artifact of

defining the contact area through the choice of active

residues in HADDOCK. Hence, we warn the readers that

the output of HADDOCK is very sensitive to the choices of

FIGURE 6 Time-averaged structures of the dimer–dimer com-

plexes of (a) GTP-tubulins and (b) GDP-tubulins along longitudinal

direction. Secondary structural elements, primarily involved in lon-

gitudinal interactions include—loops H6-H7, H11-H12, T2, T3, T5

from b-tubulin (red) and loops H10-S9, T7, helices H8, H10 from

a’-tubulin (yellow). Also shown are the bound nucleotides, GTP

(magenta) and GDP (orange).

FIGURE 5 The distribution of intradimer bending angles in

model GTP- (solid line) and GDP-dimers (dashed line). The distri-

bution of taxol-bound structure is also included (dotted line).


Biopolymers

ambiguous intermolecular restraints (AIRs), and recommend

a detailed inspection of the output, plus a set of explicit sol-

vent MD simulations on the docking structures.

Figure 7 demonstrates the interdimer interface contacts

present in GTP- and GDP-tubulin complexes. The contact

map is obtained by calculating the areas of residue–residue

contacts at the dimer–dimer interface.46 As the figure indi-

cates, the total number of contacts in the two complexes is

nearly similar. However, a relatively stronger interdimer

binding in GTP state stems from the larger residue–residue

contact areas, as shown in Table S1. Table S1 lists the major

contacts present at the dimer–dimer interfaces, along with

the residue–residue contact areas. The total contact area at

the GTP and GDP dimer–dimer interface was found to be

2290 A2 and 1738 A2, respectively. The computed interface

contact areas are very similar to that in the crystal structure

(2550 A2; Ref. 31) and previously modeled protofilaments

(2000 A2; Ref. 23). The larger strength of GTP-tubulin longi-

tudinal contacts agrees well with their larger bending stiffness

in protofilaments, as noted by Grafmuller and Voth.23 This

finding is also consistent with in vitro studies, which impli-

cate GTP-tubulin to be a better assembly candidate than

GDP-tubulin during MT nucleation and elongation proc-

esses. Moreover, a closer look at Figure 7 indicates the direct

involvement of T5 and H6-H7 region from b-subunit of

lower tubulin dimer with helix H10 from a-subunit of upper

tubulin dimer in longitudinal assembly (the number of con-

tacts is, however, more in GTP state). This finding is also in

consistent with the proposal of Nawrotek et al. that GTP

binding promotes protofilament elongation, through the

‘‘out’’ conformation of T5 loop.31

Comparing the results to the previous MD studies of

tubulin dimers by Gebremichael et al. and Bennett et al. and

of tubulin oligomers by Grafmuller and Voth, it became evi-

dent that the intradimer bending angles found in this study

are larger than Gebremichael et al. and Bennett et al., but

comparable to the study of Grafmuller and Voth. This can

mainly be due to the fact that, while the former studies were

carried out for 20 ns or less, the later and this study are per-

formed for more than 100 ns. As noted by Grafmuller and

Voth and also in this study that, larger changes in bending

angle and direction often takes place on timescales of 50 ns

and longer. In this study, the direction of bending is seen to

be very similar to the direction described by Bennett et al.

and Grafmuller and Voth. The interdimer bending is also

comparable to the work of Grafmuller and Voth (88–128 in

this work versus 58–108 in Grafmuller and Voth).

Nucleotide States and Carboxy Terminal Domain

The carboxy terminal domain is an interesting but not thor-

oughly understood region of ab-tubulin dimer. Various b-

tubulin isoforms are known to vary sequence in this region.47

Further, this region serves as a binding site for microtubule

motors, associated proteins, and cationic molecules.48,49 Lit-

erature has suggested the possibility that the C-terminal do-

main and the nucleotide state of b-tubulin could be linked,

with the nucleotide state affecting the interaction capability

of the C-terminal region.50–52 Here, we attempt to explore

such a link by investigating the correlation between N- and

C-terminal domains in GTP and GDP states. This was done

FIGURE 7 Interdimer interface contacts in longitudinal dimer–

dimer complexes—(a) GTP-tubulin complex (b) GDP-tubulin com-

plex. Blue squares indicate the contact between pair of residues.


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by examining the stability of the salt-bridge present at the

interface of N- and C-domain and also by measuring the

angle constituted by these two domains. The time evolution

of the salt-bridge, in Figure 8a, shows that the distance

between the bridging residues b:Lys105 in helix H3’ (N-do-

main) and b:Glu411 in helix H11’ (C-domain) is always sig-

nificantly smaller in GTP-dimer. This implies that the dislo-

cation of C-domain of GDP-dimer from straight orientation

in taxol-bound structure is more frequent. The distribution

of angles (Figure 8b), constituted by the vectors passing

through the interfacial helices H3’ and H11 also shows a dis-

tinct difference, where GTP-tubulin exhibits a bimodal distri-

bution. Although majority of tubulin conformations in GDP

state possess a C-domain bent by an angle of about 678,more than 50% of tubulin conformations in GTP state attain

a less-curved conformation with an angle of about 508. This

might suggest that GTP state prevents the outward curving

of C-domain in a greater extent, which thereby can engage in

more favorable interdimer association. The corresponding

angle found to be 65.38 in 1JFF and 78.68 and 79.38 in RB3-

SLD bound GTP- and GDP-tubulin crystal structures. The

similar and little larger angle values in GTP- and GDP-tubu-

lin crystals could be due to the bound RB3-SLD, which wraps

around the C-domain of tubulin.

SUMMARYAND CONCLUSIONSWe present a comprehensive study of tubulin dimers and

oligomers in different nucleotide states to examine the

intrinsic conformational changes in isolated dimers and

understand how these changes modulate the tubulin assem-

bly. Data show that functionally relevant loops, T3, T5, H6-

H7 exhibit greater tendency for outward longitudinal move-

ments in GTP-tubulin, suggesting that GTP binding can

facilitate interdimer interactions and protofilament elonga-

tion. In consistent with recent crystal structures, both GTP-

and GDP-bound tubulin dimers and oligomers are found to

have curved conformations. The range of intra- and inter-

dimer bending angles is also very similar to the recently

reported model protofilaments, 88–208 in this study com-

pared to 58–148 in the work of Grafmuller and Voth. Results

also suggest that GTP-tubulin dimers engage in relatively

stronger interdimer interactions along the filament. The

larger strength of GTP-tubulin longitudinal contacts agrees

well with their larger bending stiffness in protofilaments.

This finding is also consistent with in vitro studies, which

implicate GTP-tubulin to be better assembly candidate than

GDP-tubulin. Lastly, we show that the tubulin C-terminal

domain and the nucleotide state are closely linked with the

GTP state preventing the outward curving of C-domain.

However, this remains a testable hypothesis for future

research. The study also provides a platform for evaluating

the empirical force fields to capture the large-scale changes in

complex biological systems, such as the one investigated

here. The similar changes observed in this study using

AMBER parameters and the previous studies using

CHARMM parameters are encouraging.

The accuracy of the model tubulin structures was also

examined by performing a second control simulation. In the

model GTP-tubulin structure, the GTP ligand was switched

to GDP and the evolution of the bending angle was noted. As

Supporting Information Figure S4 shows, the original GDP-

tubulin structure was reproduced and the bending angles

switched back to the GDP bent state during the course of 100

ns simulation. Similarly, the protein–protein docking results

were checked by repeating the calculations with different set

of GTP- and GDP-tubulin structures, corresponding to the

conformations of free dimers at 50, 100, 150, and 200 ns of

FIGURE 8 (a) Time evolution of the salt-bridge between

b:Glu411 in C-domain and b:Lys105 in N-domain. Locations of the

residues are highlighted in the inset. (b) The distribution of angles

constituted by the interfacial helices H3’ and H11. Color scheme—

Red: GTP-tubulin; green: GDP-tubulin.


Biopolymers

simulation time. The dimer–dimer complexes were found to

be consistently bent in both states, even though the GTP-

tubulin complexes were relatively stronger.

MATERIALS AND METHODSThe coordinates for missing residues a:1, b:1, and a:35-60 in the

crystal structure were modeled using the InsightII graphics pack-

age.53 The hydrogens for heavy atoms were added by leap program

in Amber 9.0 package. Added hydrogens were energy minimized for

2000 steps using the conjugate gradient algorithm. The protonation

states of histidines—HID or HIE—were determined by the local

hydrogen bonding network using WHATIF program.54 A set of par-

tial atomic charges for GDP and GTP was obtained via quantum

electronic structure calculations. Using the Gaussian 03 program

with the 6-311G* basis set, we performed a Hartree-Fock geometry

optimization procedure. The atom-centered RESP charges were

determined via fits to the electrostatic potentials obtained from the

calculated wave functions. The missing interaction parameters in

the nucleotides were generated using antechamber tool in Amber.

After relaxing the added atoms in gas phase, the structure was

solvated in an octahedral periodic box of explicit water with water

molecules extending 12 A outside the protein on all sides. The trans-

ferable intermolecular potential three point (TIP3P) model was

chosen to describe the water molecules. To neutralize the system

and to maintain an ionic strength of 140 mM, 125 potassium and

89 chloride ions were incorporated. The system thus contained a

total of 34994 particles. Particle-Mesh Ewald summation55 with a 10

A cutoff was used to treat the long-range electrostatics. Noting that

the crystal structure used to initiate the MD simulations was deter-

mined at low resolution (e.g., in 1JFF, 15% of the rotamer and 10%

of the backbone torsion angles were flagged as outliers), an exten-

sive set of minimization and thermalization of the engineered struc-

ture was performed to allow the system to remediate the bad geom-

etry and to relax from its lattice-constrained conformation. For this,

a further 2000 steps of conjugate gradient minimization was per-

formed followed by successive heating to 310K with an temperature

increment of 25K and maintaining Ca restraints for a total duration

of 5 ns. The resulting structure was further minimized after remov-

ing the restraints and heated to 310K in 10 steps of 1 ns each. Then,

the system was equilibrated for 20 ns in NPT ensemble at 1 atm.

During this period, the potential energy of the system was seen to

be converging (Supporting Information Figure S5). The resulting

structure, thus, produces us a reliable starting model for the free

GDP-tubulin dimer. This structure was (i) further simulated to gen-

erate the 250 ns production data for GDP-tubulin system and (ii)

further engineered to prepare the starting conformation for a-GTP-

b-GTP-tubulin simulation, as the following.

In the equilibrated structure of GDP-tubulin, a c phosphate was

introduced to the bound GDP at E site to convert it to GTP. Subse-

quently, one Mg21 ion was added and the number of KCl was

adjusted to maintain the ionic strength. This new system was then

equilibrated for 20 ns following the exact procedure as described

above, which produces the starting model for free GTP-tubulin. The

simulation was extended until a trajectory of 250 ns length in NPT

ensemble was achieved. As a control, the taxol-bound tubulin crys-

tal structure was also simulated for 250 ns. The coordinate files of

the time-averaged GTP- and GDP-tubulin dimers are provided as

Supporting Information. All simulations were performed using the

NAMD package with Amber ff99SB force field56 on 64 processors of

an Infiniband Xeon E5472 linux cluster.

Models of the tubulin dimer–dimer complexes were obtained by

performing protein–protein docking calculations, using HAD-

DOCK.45 HADDOCK is a high ambiguity driven protein–protein

docking program that makes use of experimental data to search the

conformational space efficiently. The experimental information on

the interacting residues is introduced as AIRs, defined as an ambig-

uous intermolecular distance (diAB) with a maximum value of 3 A

between any atom m of an active residue i of protein A (miA) and

any atom n of both active and passive residues k of protein B (nkB)

and vice versa. The residues which are directly involved in binding

are termed active residues and the nearest neighbors are passive resi-

dues. The effective distance deffiAB for each restraint is calculated as:

deffiAB ¼

� XNatoms

miA¼1

XN Bres

k¼1

XNatoms

nkB¼1

1

d6miAnkB

��16

where Natoms indicates all atoms of a given residue and NBres the sum

of active and passive residues for the second protein. Thus, AIRs ena-

ble the search through all possible configurations around the interact-

ing site to predict the most favorable interacting pair of amino acids.

The docking protocol consists of three stages: (i) randomization of

orientations and rigid body energy minimization, (ii) semirigid simu-

lated annealing (SA) in torsion angle space, and (iii) final refinement

in Cartesian space with explicit solvent. After calculation, the struc-

tures are ranked according to their intermolecular energy which is

sum of electrostatic, van der Waals, and AIR energy terms.

In this study, the active residues for dimer–dimer docking were

chosen based on the electron crystallographic data.57 Passive resi-

dues were chosen as the nearest neighbors of the active residues

with [50% solvent accessibility, which also include the nucleotides

and the Mg21 ion. In each case, 1000 initial complex structures

were generated by randomizing the orientations of two partner

proteins whose translational and rotational movements were kept

completely free. The structures were subjected to rigid body energy

minimization, and the best 200 solutions were selected for SA

refinements. The resultant structures were classified into four clus-

ters. The lowest energy complex with correct longitudinal pitch

from GTP and GDP-tubulin docking was subjected to 50 ns MD

simulation for final refinement.

The computer resources of Computer Centre, IIT Madras are gratefully

acknowledged. KN acknowledges CSIR for the research fellowship.

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