j.a. tuszynski, e.j. carpenter, t. luchko, t. huzil and r.f. luduena: molecular dynamics...

8/3/2019 J.A. Tuszynski, E.J. Carpenter, T. Luchko, T. Huzil and R.F. Luduena: Molecular Dynamics Calculations of the Electros

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Proceedings ESA Annual Meeting 2005 217

Molecular Dynamics Calculations of the Electrostatic Properties of the

Tubulin Family of Proteins and Their Consequences for Drug Binding to

Microtubules

J. A. Tuszynski1, E. J. Carpenter

2, T. Luchko

2,

T. Huzil2, and Richard F. Luduea

3

1Department of Physics, University of Alberta,

Edmonton, Alberta, Canada, T6G 2J1

email: [email protected]

2Department of Physics, University of Alberta,

Edmonton, Alberta, Canada, T6G 2J1

3Department of Biochemistry, MSC 7760,University of Texas Health Science Center at San Antonio

7703 Floyd Curl Drive

San Antonio, Texas, 78229-3900, USA

Abstract

We present the results of molecular dynamics computations based on the

atomic resolution structures of tubulin published as 1TUB and 1JFF in the

Protein Data Bank. Values of the net charge, spatial charge distribution and

the dipole moment components are obtained for the tubulin alpha-beta

heterodimer. Physical consequences of these results and subsequent

computations are discussed for microtubules in terms of the effects on test

charges, test dipoles, and neighboring microtubules. Our calculations indicatetypical distances over which electrostatic effects can be felt by biomolecules,

ions, and other microtubules. We also demonstrate the importance of

electrostatics in the formation of bonds between microtubules and drugs such

as taxanes and colchicine.

I. INTRODUCTION

Microtubules (MTs) are protein filaments of the cytoskeleton with lengths that

vary but commonly reach 510 m [1]. They are composed of 12 to 17

protofilaments when self-assembled in vitro and almost exclusively of 13

protofilaments in vivo. These protofilaments are strongly bound internally and

are connected via weaker lateral bonds to form a sheet that is wrapped up into

a tube in the nucleation process. The general structure of MTs has been wellestablished experimentally. A small difference between the - and -monomers of tubulin allows the existence of at least two lattice types. Moving

around the MT in a left-handed sense, protofilaments of the A lattice have a

vertical shift of 4.92 nm upwards relative to their neighbors. In the B lattice

2005 Electrostatics Society of America


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218 Electrostatic Properties of Tubulin

this offset is only 0.92 nm because the - and -monomers have switchedpositions in alternating filaments. This change results in the development of a

structural discontinuity in the B lattice known as a seam.

In 1998 Nogales et al. reported crystallization of tubulin in the presence of

zinc ions. [2, 3] Their crystallographic results were made available through

the Protein Data Bank (PDB) (entries: 1TUB and 1JFF) which allowed us to

view the 3D atomic resolution structure of tubulin. Each tubulin monomer is

composed of more than 400 amino acids and, in spite of their similarity, slight

folding differences can be seen. It is worth stressing that several different

versions of both the - and -monomers exist and are called isotypes whenpresent in the same organism. [4]. Some of the biophysical properties of these

variants have been examined and are discussed below in this paper.

II. ELECTROSTATIC MODELING OF TUBULIN AND MICROTUBULES

The preliminary results of our simulations are illustrated in Fig.1 showing the

tubulin dimer with the two C-termini that are very flexible and which have not

been resolved crystallographically. The electrostatics of the surface of the

tubulin dimer including a reconstructed pair of C-termini is shown below.

Here the color blue is used to represent negative charge while red corresponds

to positive charges, white indicating neutral regions.

Figure 1. A map of the electric charge distribution on the surface of a tubulin

dimer with C-termini tails present.The C-termini of tubulin are strongly electrostatically negative (having up to

10 net negative charges) and interact electrostatically with several nearbycharged objects: (a) the surface of the tubulin dimer below (which is generally

negatively charged, with as many as 20 negative charges per monomer, but

which has a positively charged groove that can bind a C-terminus), (b) with


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J. A. Tuszynski, E. J. Carpenter, T. Luchko, T. Huzil 219

neighboring C-termini and (c) possibly with an adjacent protein such as

kinesin or a MAP (microtubule associated protein). Consequently, we have

found that the C-termini are likely to exist in several conformational states: (a)a rather flexible conformation pointing away from the MT surface and

subjected to random thermally agitated orientations, (b) two or more states in

which C-termini bind to the MT surface. Our molecular dynamics calculations

indicate that the energy difference between the two conformations of C-

termini bound to the surface is not very large, on the order of a few kBT at

room temperature. The latter ones are likely to exhibit collective properties

with a regular arrangement of binding locations on the surface of the

microtubule that they would decorate forming a fairly regular lattice structure.

It is an open question whether these collective states can be reversibly

switched which might be of potential significance in the operation of axonal

trafficking.Table 1 summarizes the results of our calculations of the tubulins net chargeand the dipole moment using the available data from 1TUB in the PDB, i.e.

excluding the C-termini that are known to account for approximately 40% of

the total charge of the protein. Note that the x-direction in Table 1 coincides

with the protofilament axis. The -monomer is in the direction of increasing x

values relative to the -monomer. The y-axis is oriented radially towards theMT center and the z-axis is tangential to the MT surface.

Table 1. Some electrostatic properties of tubulin based on the Nogales-

Downing structure that excludes the C-termini

Tubulin Properties Dimer -monomercharge (electrons) -10 -5

dipole (Debye)overall magnitude 1714 556

x-component 337 115

y-component -1669 554

z-component 198 -6

Bearing in mind that tubulin is both highly charged and possesses a permanent

dipole moment we have attempted to estimate the strength of electrostatic

effects on: a) a test charge, b) a test dipole, c) another microtubule in the

vicinity, and d) the dipole-dipole interaction between two microtubules.

Below we summarize our calculations [5]. First of all, we have performed

calculations of the dimer-dimer interaction that arises due to the charge and

dipole moment present on each independent protein in solution. It isinteresting to note that the lines of attraction appear consistent with the

presence of a hexagonal lattice structure on the surface of a microtubule.As an


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example, we considered a microtubule of length 5 m, outer radius 12.5 nm, $' & ) ( 0

= 0.5e/nm2. With a test charge of +5 e located at

a distance 5 nm from the surface of a microtubule we obtain a force ofelectrostatic attraction of 6 pN in water, which is reduced to only 0.5 pN in

standard ionic solutions with Debye lengths between 0.6 and 1.5 nm

depending on the ionic strength. This would indicate that the maximum

distance over which a microtubule can exert an influence on a charged particle

is on the order of 5 nm from its surface. Performing dipole-dipole interaction

energy calculations for a fictitious tubulin dimer in solution whose 2000 D

dipole moment is oriented along the axis perpendicular to the filament axis

and which is separated by 5 nm, we find that the interaction energy is now just

above 2 meV, a value smaller than room temperature thermal fluctuations. The

force of repulsion between two neighboring microtubules separated by a

distance of 40 nm between their centers in an aqueous environment is found to

be a staggering 0.2106 pN. However, when Debye screening due to ions at a

concentration of 150 mM is included the result is decreased to 9 pN. Taking asan approximately value of the polarization density 2000 D per dimer and a

separation between microtubule centers to be 40 nm and using the microtubule

length 5 m as well as accounting for Debye screening, results in an attractive

force between the two microtubules of 330 pN, which is very significant.

Moving the distance between microtubules to 90 nm (which is the mean

separation between axonal microtubules) reduces the attraction force by a

factor of 4000 to a mere 0.08 pN. It is also worth emphasizing that a

combined action of electric monopole-monopole and dipole-dipole forces will

have a competitive nature with Coulomb repulsion on short distances due to

negative charges of the microtubule surfaces and an attractive dipole-dipole

interaction that evidently extends over a larger range.

III. DEVELOPMENT OF TUBULIN ISOTYPE MODELS

We have constructed computational 3D models from the 290 available

sequences of different tubulins. We have exploited the high degree of

sequence and structure conservation that is observed within tubulin isotypes

and between the and subunits by using software such as the experimentalModeller and tubulin crystallographic data as structural templates to produce

3D models containing chosen amino acid sequences. As an initial step we

searched the Swiss-Prot database [http://ca.expasy.org/sprot/] for tubulin

amino acid sequences. Approximately 200 sequences representing a wide

range of species were downloaded. Of particular interest were the 15 human

sequences obtained. While only sequences in the Swiss-Prot database have

been obtained in a systematic manner, this initial set of sequences has since

been expanded by the addition of select sequences from the National Centerfor Biotechnology Information

[http://www.ncbi.nlm.nih.gov/Database/index.html]. The structures of- and


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-tubulin are known to be quite similar, being nearly indistinguishable at 6 .Since the sequences of the tubulins within an or tubulin family are moresimilar to each other than to those of the other family it is reasonable tobelieve that any given isotype sequence should produce a structure verysimilar to another member of that family. Accordingly, by substitutingappropriate amino acid side chains and properly adjusting to accommodateinsertion and deletions in the sequence of the published crystallographictubulin structure, this can be used as a framework to produce model structureswith different sequences [8]. In all of our modeling we work with the entiretubulin dimer, pairing with the various isotypes. Further experimentalsupport for the validity of this approach comes from the published structure,which is of a porcine sequence fit to bovine structural data. The porcine -tubulin sequence is largely the II isotype as was the majority of the tubulinwhose structure was determined by electron crystallography. To build such

3D structures of the isotypes Modeller (version 6.2) was used. This programuses alignment of the sequences with known related structures, used astemplates, to calculated desired isotype models. In detail, the providedsequence of the desired molecule is being aligned with sequences of thetemplate molecules. Then the full homology of the sequence was calculatedusing data of aligned sequences and the templates. We subsequently used thecomputer models to determine various physical properties by furthercomputation in appropriate molecular dynamics systems. For each of thetubulin isotypes, we have computed their volume, surface area, net charge andthe total dipole moment. The regularity of the obtained results indicates apossible correlation between structure and function.

We have recalculated the values of the dipole moments and net charges usingthe sequences of the various homologous isotypes of tubulin biochemicallycharacterized in the literature. This was done by creating probable structuresfor human tubulin isotypes listed in the Swiss-Prot [6] database using thecomputer program Modeller [7] with the Nogales structural data. Table 1shows values using the 1TUB file while Table 2 summarizes the results for thekey variants of tubulin heterodimers found in human cells including theC-terminal region. The Tubulin Dimer column uses the original databaselabels and the first three letters may be translated: TB = tubulin, A=, B=,|| is the total value of the dipole moment in debyes, Q is the total chargewhile M_z, M_r and M_theta are the cylindrical component value of thedipole moment (axial, radial and tangential, respectively).

Table 2. Values of the total dipole moment, ||, (in debye), the total charge,

Q, (in elementary charge units), and the dipole vector cylindrical componentsMz, Mr and M (in debye) for the human tubulin -dimers

Tubulin Dimer |M| Q Mz Mr M


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1 6413 49 5668 2910 729

2 5900 49 5119 2790 906

4 6336 49 5563 3030 122

5 6352 48 5618 2940 372

Q 5357 42 5096 1484 724

X 6761 48 5814 3446 201

1 6060 48 5202 2813 1329

2 5582 48 4653 2691 1506

4 5899 48 5097 2931 478

5 5963 47 5152 2840 973

Q 5010 41 4630 1384 1323

X 6359 47 5347 3346 800

1 6238 49 5434 2962 776

2 5732 49 4885 2843 953

4 6157 49 5329 3083 74

5 6174 48 5385 2992 419

Q 5157 42 4862 1536 771

X 6590 48 5580 3498 248

1 6069 48 5378 2655 929

2 5565 48 4829 2536 1106

4 5960 48 5274 2775 78

5 5994 47 5329 2685 571

Q 5046 41 4806 1229 923

X 6392 47 5524 3191 400

1 6252 49 5545 2581 1298

2 5761 49 4995 2462 1475

4 6090 49 5440 2701 447

5 6156 48 5495 2611 940

Q 5266 42 4973 1155 1292

X 6533 48 5690 3117 769

It is worth noting that there is a significant diversity in the values of dipole

moments and an even greater variation in the net charge per monomer. Note

also the predominance of the axial components of the dipole vector followed

by the radial component. A large variation in the values of the tangential

component should also be emphasized. We are currently pursuing a larger

scale study intended to provide conclusive evidence for the correlation

between the structure and function of this very important protein. It is

interesting to note that while the net dipole moment of tubulin is very large, its

near symmetrical ordering around the MT axis gives rise to a net cancellation

effect such that a minimal amount of torque is expected to arise from the

application of an external electric field to a microtubule in solution.

IV. ANALYSIS OF TUBULIN ISOTYPES FOR DRUG BINDING


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With the resulting initial models of tubulin structure, various computationalestimates of physical properties may be made. As an example, sequence

alignment indicates that among residues within 5 of the taxol molecule inthe Lwe data [3], only the III sequence shows any variation (Table 3). Thesubstitution of an alanine for a serine adjacent to an arginine that appears toprovide significant steric restrictions in taxoid binding kinetics potentiallyaffects the binding kinetics in the III isotype. We are currently performingmolecular dynamics simulations to try to determine what if any effect on thetemporal behavior of the arginine side chain this difference may have andwhat consequences this may have for taxoid binding.

Table 3. Human -Isotype Taxane-Binding Site Sequences

Residue 22-24 223-232 272-277 357-362I EVI GDLNHLVSAT PLTSRG PPRGLKII EVI GDLNHLVSAT PLTSRG PPRGLKIII EVI GDLNHLVSAT PLTARG PPRGLKIVb EVI GDLNHLVSAT PLTSRG PPRGLKV EVI GDLNHLVSAT PLTSRG PPRGLKVI EMI GDLNHLVSLT PLTAQG PPRGLSVII EVI GDLNHLVSAT PLTSQG PPRGLK

We have investigated the nature of taxol binding to tubulin by modeling thebinding pocket at a high resolution level. In the color plates below, we showthe view of the pocket highlighting the charge distribution and the presence ofspecific residues, in particular the arginine at position 276 that appears to playa crucial role in taxol binding. We have discovered that the arg 276 side-chain

forms a latch that appears to stabilize taxol in its binding pocket in -tubulin. Since it sticks out and around the taxoid and is very nearly in contactwith his 227 it would seem to hold the taxoid in place and would prevent itfrom binding in the first place. However, the arginine side-chain is quiteflexible and can be expected to move out of the way to allow the taxoid tobind and unbind. The replacement of ser 275 by alanine in III (Table 4)eliminates the -oxygen of ser 275. We currently investigate whether theabsence of this hydroxyl group affects the dynamics of arg 276. In thisconnection, we have also looked at the average distances between arg 276 andhis 227 in some of the isotypes and found some clear differences (Table 4).

Table 4. Distances between Arg 276 and His 227 in -Tubulin Isotypes

Isotype Average Distance ()


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I 12.34

III 11.59

IVa 12.31

IVb 9.02

When we modeled both paclitaxel and docetaxel at this site we found that the

average value of the arg 276his 227 distance does not correlate with the

strength of binding. For this reason, we have looked at thermal fluctuation

effects. We focused specifically on the fluctuation of the 2 atoms that close

the binding site (the NH2 of the guanidinium group of arg276 and the N 2 ofthe imidazole group of his227. These fluctuations are computed over a 100 ps

time period using 1 fs time steps. It is interesting that the values for III seemto be constant. Our models imply that the different conformations of the taxol

binding site indicate that, in principle, one should be able to construct analogs

of taxol that are specific for particular types of tubulin.

Our goal here is to define regions on the tubulin molecule that are likely to be

good targets for drugs. At present, we have five potential targets in mind.

These may be modified as further information about these sites becomes

available or new ones are discovered. The first target is the taxol-binding site

because the taxol-tubulin interaction has been experimentally determined in

three dimensions and is therefore the best known of our targets. A large

number of taxanes have already been synthesized although none of them has

been designed based on knowledge of the three-dimensional structure of the

binding site which is now finally available to us. Our models have shown that

the taxol site differs among the various isotypes. The second target is the high

affinity colchicine-binding site, located at the / interface. The third target isthe Vinca site. At present, this site has not been modeled in three dimensions;

however, photo-affinity labeling has shown that residues 175-213 in areinvolved in binding to vinblastine. The fourth target is the Fhit-binding site.

At the moment the location of the site is not known, but some further

experimentation should help to localize it. The fifth target is based on the

observation that the reactivities of cysteines 295, 305, 315, and 316 on aresignificantly inhibited by binding of colchicine to tubulin. This suggests that

these cysteines may be in a pocket, perhaps one that is created when

colchicine binds. Although this binding site is entirely on , our preliminary

results suggest that the conformation of differs depending on the nature of

the isotype in the dimer.

At this point all the human -tubulin isotypes have been modeled as described

above, each one as part of the / heterodimer. For the subunit, we haveused the structure of as described by Nogales et al [2-3]. For all the humanisotype models we have used a single a sequence.


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Sequence alignments of Human Class tubulin I, II, III, IVa, IVb, V, and VIhave identified a highly invariant sequence contained within the taxol binding

site, with the exception of class VI tubulin. In Figs. 2 and 3 the class III isshown with all 4 substitutions that occur within 10 angstroms of the bound

taxol molecule in the 1JFF structure. All substitutions are superimposible

(mutants have been shifted down slightly to show difference). These

differences occur at the following reside locations (numbering is as in 1JFF

structure file, not sequence), occurring 5-10 Angstroms from taxol:

Residue 82 Proline Alanine

Residue 241 Cysteine Serine

Residue 375 Alanine Serine

Occuring 5 or less Angstroms from Taxol

Residue 277 Serine

Alanine

Figure 2 Substitution of Residues from 1JFF to Class III. Structure wasenergy minimized using GROMACS following mutagenesis of specific side

chains.


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Figure 3 position of changes in III tubulin as compared to 1JFF sequence.Red residue Ser 277- Ala occurs within 5A of Taxol (side chain is buried away

from taxol). Green residues are found within 10 Angstroms. Blue residues aresubstitutions found outside the taxol binding pocket [9].

Tubulin classVI is more interesting (for identifying immediate taxolanalogs) as there are 5 differences that occur within 5 Angstroms of the bound

Taxol molecule (Figure 4). These differences are

Residue 23 Valine

Methionine

Residue 26 Aspartic Acid

Glutamic Acid

Residue 233 Alanine Leucine

Residue 277 Serine Alanine

Residue 278 Arginine

Glutamine

Figure 4 residues within 1JFF which are changed in Class VI tubulin.

Figure 5 represents the changes within the Class VI tubulin that are mostpromising. Here we see that there is a significant change in the volume of the


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binding pocket that interacts with the phenyl ring that becomes changed in

docetaxel. Reduction of the size of this functional group may increase binding

of taxol to this Class of tubulin. This could involve replacement of theNHCOPh group (bound to C3

" $ %( ' 0 ) 1 2 % 5# $ ) $ @ B D % H

a Methionine side chain in place of Valine 23. The presence of a surface

exposed sulfur atom at this position opens up the possibility for weak

hydrogen bonding or metal coordination at this site. Note that these

substitutions in Class VI tubulin do not affect the 3 I P R S T U V X Y T ` Y T b P d f g S h p

Knowing the dimensions of the binding site, we will use, as appropriate, three

separate approaches to design novel drugs for these sites. Let us assume that

we are designing a derivative specific for III. The first approach is acomputer-based modification of known structures. As an illustrative example,

we will use colchicine, a drug that binds preferentially to the IV dimer.Using the Ludi software module (MSI), we will then make modifications to

the colchicine structure so as to increase binding to III and decrease bindingto the other isotypes. For example, if there is a side chain in every humanisotype, projecting towards colchicine, and that side chain is absent in a

specific isotype of tubulin, we may add a group to colchicine that will occupy

the same space as that side chain and thus prevent binding to human III

tubulin but still allow binding to the selected tubulin isotype. Likewise, if

there is a hydrophobic side chain in the selected tubulin that is not present in

human III tubulins, we may add a non-polar group to colchicine so as toform a hydrophobic interaction with that side chain. The result of such

manipulations should be the generation of colchicine derivatives with high

specificity for selected tubulin isotypes. In the second approach, we will use

the DOCK program to search databases of small molecules whose structures

are known, such as the Cambridge Structural Database or the MDL Available

Chemicals Directory. DOCK is an algorithm that identifies small moleculesthat can fit into the ligand binding sites on proteins of known structure; it has

been used successfully to generate a variety of novel protein ligands. The


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DOCK program will allow us to choose small molecules that will fit into theactual or potential binding sites described above.

Our third approach will be to design molecules de novo. We will begin with amodel of the unoccupied drug binding pockets on the III model. Using theAffinity software module (MSI), we will identify groups that would haveoptimal interactions with the amino acid residues in the binding pockets oftrypanosome tubulin but not with those of human tubulin. The result of usingthe combination of all three approaches will be the design of completely novelcompounds that may be highly specific for selected isotypes of tubulin.

ACKNOWLEDGEMENTS

This research was supported by grants from NSERC and MITACS-MMPD,US Department of Defense and Technology Innovations, LLC of Rochester,NY.

REFERENCES

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Tubulin Dimer by Electron Crystallography, Nature, Nature PublishingGroup, London, Jan. 08, 1998, pp. 199203.

[3] J. Lowe, H. Li, K.H. Downing, and E. Nogales, Refined Structure of-Tubulin at 3.5 Resolution, Journal of Molecular Biology, AcademicPressElsevier, London, Feb. 26, 2002, pp. 10451057.

[4] Q. Lu, G.D. Moore, C. Walss, and R.F. Luduena, Structural andFunctional Properties of Tubulin Isotypes, Advances in Structural Biology,Jai Press, Stanford U.S.A., 1998, pp. 203227.

[5] J.A. Tuszynski, J.A. Brown, E. Crawford, E.J. Carpenter, M.L.A.Nip, J.M. Dixon and M.V. Sataric, Molecular Dynamics Simulations ofTubulin Structure and Calculations of Electrostatic Properties of Microtubules,Mathematical and Computer Modelling (to appear 2005).

[6] B. Boeckmann, A. Bairoch, R. Apweiler, M.-C. Blatter, A.Estreicher, E. Gasteiger, M.J. Martin, K. Michoud, C. ODonovan, I. Phan, S.Pilbout, and M. Schneider, The SWISS-PROT Protein Knowledgebase andits Supplement TrEMBL in 2003, Nucleic Acids Research, 2003, OxfordUniversity Press, Oxford, Jan. 1, 2003, pp. 365-370.

[7] R. Koradi, M. Billeter, and K. Wuthrich, MOLMOL: A Program forDisplay and Analysis of Macromolecular Structures, Journal of MolecularGraphics, Elsevier, London, Feb. 1996, pp. 5155.

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Comparative Protein Structure Modeling of Genes and Genomes, AnnualReview of Biophysics and Biomolecular Structrue, Annual Reviews, PaloAlto, USA, 2000, pp. 291325.


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