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. Carpenter2, T. Luchko2, T. Huzil2, and Richard F. Luduea3
1

Department of Physics, University of Alberta, Edmonton, Alberta, Canada, T6G 2J1 email: jtus@phys.ualberta.ca

Department of Physics, University of Alberta, Edmonton, Alberta, Canada, T6G 2J1

3

Department 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 indicate typical 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 well established 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

218 Electrostatic Properties of Tubulin

this offset is only 0.92 nm because the - and -monomers have switched positions 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 when present 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 nearby charged 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

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 Ctermini 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 charge and 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 the MT center and the z-axis is tangential to the MT surface. Table 1. Some electrostatic properties of tubulin based on the NogalesDowning structure that excludes the C-termini Tubulin Properties charge (electrons) dipole (Debye) overall magnitude x-component y-component z-component Dimer -10 1714 337 -1669 198 -monomer -5 556 115 554 -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 is interesting 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

220 Electrostatic Properties of Tubulin

example, we considered a microtubule of length 5 m, outer radius 12.5 nm, = 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 of electrostatic 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 as an 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 experimental Modeller 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 Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/Database/index.html]. The structures of - and
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J. A. Tuszynski, E. J. Carpenter, T. Luchko, T. Huzil 221

-tubulin are known to be quite similar, being nearly indistinguishable at 6 . Since the sequences of the tubulins within an or tubulin family are more similar to each other than to those of the other family it is reasonable to believe that any given isotype sequence should produce a structure very similar to another member of that family. Accordingly, by substituting appropriate amino acid side chains and properly adjusting to accommodate insertion and deletions in the sequence of the published crystallographic tubulin structure, this can be used as a framework to produce model structures with different sequences [8]. In all of our modeling we work with the entire tubulin dimer, pairing with the various isotypes. Further experimental support 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 tubulin whose structure was determined by electron crystallography. To build such 3D structures of the isotypes Modeller (version 6.2) was used. This program uses alignment of the sequences with known related structures, used as templates, to calculated desired isotype models. In detail, the provided sequence of the desired molecule is being aligned with sequences of the template molecules. Then the full homology of the sequence was calculated using data of aligned sequences and the templates. We subsequently used the computer models to determine various physical properties by further computation in appropriate molecular dynamics systems. For each of the tubulin isotypes, we have computed their volume, surface area, net charge and the total dipole moment. The regularity of the obtained results indicates a possible correlation between structure and function. We have recalculated the values of the dipole moments and net charges using the sequences of the various homologous isotypes of tubulin biochemically characterized in the literature. This was done by creating probable structures for human tubulin isotypes listed in the Swiss-Prot [6] database using the computer program Modeller [7] with the Nogales structural data. Table 1 shows values using the 1TUB file while Table 2 summarizes the results for the key variants of tubulin heterodimers found in human cells including the C-terminal region. The Tubulin Dimer column uses the original database labels 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 charge while M_z, M_r and M_theta are the cylindrical component value of the dipole 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 components Mz, Mr and M (in debye) for the human tubulin -dimers

Tubulin Dimer

|M|

Mz

Mr

222 Electrostatic Properties of Tubulin

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

           

1 2 4 5 Q X 1 2 4 5 Q X 1 2 4 5 Q X 1 2 4 5 Q X 1 2 4 5 Q X

6413 5900 6336 6352 5357 6761 6060 5582 5899 5963 5010 6359 6238 5732 6157 6174 5157 6590 6069 5565 5960 5994 5046 6392 6252 5761 6090 6156 5266 6533

49 49 49 48 42 48 48 48 48 47 41 47 49 49 49 48 42 48 48 48 48 47 41 47 49 49 49 48 42 48

5668 5119 5563 5618 5096 5814 5202 4653 5097 5152 4630 5347 5434 4885 5329 5385 4862 5580 5378 4829 5274 5329 4806 5524 5545 4995 5440 5495 4973 5690

2910 2790 3030 2940 1484 3446 2813 2691 2931 2840 1384 3346 2962 2843 3083 2992 1536 3498 2655 2536 2775 2685 1229 3191 2581 2462 2701 2611 1155 3117

729 906 122 372 724 201 1329 1506 478 973 1323 800 776 953 74 419 771 248 929 1106 78 571 923 400 1298 1475 447 940 1292 769

J. A. Tuszynski, E. J. Carpenter, T. Luchko, T. Huzil 223

With the resulting initial models of tubulin structure, various computational estimates of physical properties may be made. As an example, sequence alignment indicates that among residues within 5 of the taxol molecule in the Lwe data [3], only the III sequence shows any variation (Table 3). The substitution of an alanine for a serine adjacent to an arginine that appears to provide significant steric restrictions in taxoid binding kinetics potentially affects the binding kinetics in the III isotype. We are currently performing molecular dynamics simulations to try to determine what if any effect on the temporal behavior of the arginine side chain this difference may have and what consequences this may have for taxoid binding. Table 3. Human -Isotype Taxane-Binding Site Sequences Residue I II III IVb V VI VII 22-24 EVI EVI EVI EVI EVI EMI EVI 223-232 GDLNHLVSAT GDLNHLVSAT GDLNHLVSAT GDLNHLVSAT GDLNHLVSAT GDLNHLVSLT GDLNHLVSAT 272-277 PLTSRG PLTSRG PLTARG PLTSRG PLTSRG PLTAQG PLTSQG 357-362 PPRGLK PPRGLK PPRGLK PPRGLK PPRGLK PPRGLS PPRGLK

We have investigated the nature of taxol binding to tubulin by modeling the binding pocket at a high resolution level. In the color plates below, we show the view of the pocket highlighting the charge distribution and the presence of specific residues, in particular the arginine at position 276 that appears to play a 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 contact with his 227 it would seem to hold the taxoid in place and would prevent it from binding in the first place. However, the arginine side-chain is quite flexible and can be expected to move out of the way to allow the taxoid to bind and unbind. The replacement of ser 275 by alanine in III (Table 4) eliminates the -oxygen of ser 275. We currently investigate whether the absence of this hydroxyl group affects the dynamics of arg 276. In this connection, we have also looked at the average distances between arg 276 and his 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 ()

224 Electrostatic Properties of Tubulin

I III IVa IVb

12.34 11.59 12.31 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 N2 of the 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 seem to 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 is the Vinca site. At present, this site has not been modeled in three dimensions; however, photo-affinity labeling has shown that residues 175-213 in are involved 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 are significantly 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 have used the structure of as described by Nogales et al [2-3]. For all the human isotype models we have used a single a sequence.

J. A. Tuszynski, E. J. Carpenter, T. Luchko, T. Huzil 225

Sequence alignments of Human Class tubulin I, II, III, IVa, IVb, V, and VI have 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 is shown 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 was energy minimized using GROMACS following mutagenesis of specific side chains.

226 Electrostatic Properties of Tubulin

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 are substitutions found outside the taxol binding pocket [9]. Tubulin classVI is more interesting (for identifying immediate taxol analogs) 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 most promising. Here we see that there is a significant change in the volume of the

J. A. Tuszynski, E. J. Carpenter, T. Luchko, T. Huzil 227

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 the NHCOPh group (bound to C3 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

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 a computer-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 binding to the other isotypes. For example, if there is a side chain in every human isotype, 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 to form 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 molecules that 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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228 Electrostatic Properties of Tubulin

DOCK program will allow us to choose small molecules that will fit into the actual or potential binding sites described above. Our third approach will be to design molecules de novo. We will begin with a model of the unoccupied drug binding pockets on the III model. Using the Affinity software module (MSI), we will identify groups that would have optimal interactions with the amino acid residues in the binding pockets of trypanosome tubulin but not with those of human tubulin. The result of using the combination of all three approaches will be the design of completely novel compounds 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 [1] Dustin, P., Microtubules, Springer-Verlag, Berlin, 1984. [2] E. Nogales, S.G. Wolf, and K.H. Downing, Structure of the Tubulin Dimer by Electron Crystallography, Nature, Nature Publishing Group, 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, Academic PressElsevier, London, Feb. 26, 2002, pp. 10451057. [4] Q. Lu, G.D. Moore, C. Walss, and R.F. Luduena, Structural and Functional 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 of Tubulin 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 and its Supplement TrEMBL in 2003, Nucleic Acids Research, 2003, Oxford University Press, Oxford, Jan. 1, 2003, pp. 365-370. [7] R. Koradi, M. Billeter, and K. Wuthrich, MOLMOL: A Program for Display and Analysis of Macromolecular Structures, Journal of Molecular Graphics, Elsevier, London, Feb. 1996, pp. 5155. [8] M.A. Marti-Renom, A. Stuart, A. Fiser, R. Snchez, F. Melo, A. Sali, Comparative Protein Structure Modeling of Genes and Genomes, Annual Review of Biophysics and Biomolecular Structrue, Annual Reviews, Palo Alto, USA, 2000, pp. 291325.

J. A. Tuszynski, E. J. Carpenter, T. Luchko, T. Huzil 229

[9] R. Sayle and E.J. Milner-White, RASMOL: Biomolecular Graphics for All, Trends in Biochemical Sciences (TIBS), Elsevier Science Ltd., London, Sep. 1995, pp. 374376.