Molecular

Dynamics Calculations of the Electrostatic Properties of Tubulin Their Consequences for Microtubules

and

J. A. Tuszynski,* E. J. Carpenter,* E. Crawford,* J. A. Brown,* W. Malinski,* and J. M. Dixont *Department of Physics, University of Alberta, Edmonton AB Canada, T6G 2Jl. fphysics Department, University of Warwick, Coventry, U K" CV4 TAL jtus@phys, ualberta. ca

Abstract We present the results of molecular dynamics computations based on the atomic resolution structures of tubulin published as lTUB and IJFF in the Protein Data Bank. Values of net charge, spatial charge distribution and Cartesian dipole moment components are obtained for the tubulin alpha-beta heterodimer. Physical consequences of these results and subsequent computations are discussedfor 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 the microtubule lattice and the tubulinkinesin binding strength. Keywords: tubulin, kinesin, molecular dynamics, microtubules, electrostatics 1. Introduction Microtubules (MTs) are protein filaments of the cytoskeleton with lengths that vary but commonly reach 5-10 ~m. Theyare 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 we.l established experimentally. A small difference between the (X- and 13-monomersof 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 this offset is only 0.92 nm because the (X- and 13-monomershave 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. [1, 2] Their

crystallographic results were made available through the Protein Data Bank (PDB) (entries: ITUB and IJFF) which allowed us to view the 3D atomic resolution structure oftubulin. (see Fig. I) 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 {X- and 13-monomers exist and are called isotypes. [3]. The biophysical properties of these variants have been examined and are discussedbelow in this paper. Fig. 1 is diagram of the tubulin molecule produced from the Nogales et al. [2] crystallographic data (file IJFF) shows the similarity between the {X-subunit (left halt) and the 13-subunit(right halt). The white spheres indicate the location of GTP , GDP and taxol when bound.

Figure

1. Tubulin

dimer,

prepared

using MolMol.

[4]

2. Electrostatic modelling of tubulin

Fig. 2 shows the results of our molecular dynamics simulations for the electric charge distribution on the surface of a tubulin dimer tha includes the two carboxytermini that are very flexible and which have not been resolved crystallographically.

2.1. C-termini
The (having C-termini up to of 10 tubulin net are strongly charges) electro-negative and interact

negative

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. 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. Fig. 4 is a reconstruction of the electric polarization of a microtubule that takes into account the dipole moments of the individual dipoles and their orientation with respect to the microtubule axis. It is interesting to note that while the net dipole moment of tubulin is very large, its 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.

Bearing in mind that tubulin is both highly charged and possessesa pennanent 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. First of all, we have perfonned calculations of the dimer-dimer interaction that arises due to the charge and dipole moment present on each independent protein in solution. The result of these calculations is shown in Fig. 5 in tenns of force lines of attraction between two neighboring dimers. 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.

Figure 5. A view of the attractive regions about a tubulin dimer as would be experienced by another dimer. The smallest principal moment of inertia of the dimers is perpendicular to the page, the middle one is aligned vertically, the largest principal moment horizontally. See text for more details. Figure prepared using Rasmol. [7]

3.1. Microtubule-Charge

interaction
of length

As an example, we considered a microtubule

5 ~m, outer radius 12.5 nm, and surface charge density a = 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.

Figure 4. The arrows indicate the orientation of the permanent dipole moments of individual tubulin dimers with respect to the surface of a microtubule. Figure prepared using MolMol. [4]

3. Electrostatic structure

effects

of

microtubule

5. Conclusions
In this paper we have considered the role of electrostatics in the interactions between tubulin, microtubules and other charged or polarized molecules. In particular, we have shown that in spite of Debye screening, a microtubule can exert a Coulomb force on a charged particle that is up to 5 nm away from its surface. The dipole-dipole forces that have been calculated are negligible for the most part. However, they can be felt by dipoles that are perpendicular to the microtubule surface and removed from the equatorial plane. When two microtubules are found in the same vicinity, they can exert significant forces of repulsion even in the presence of ionic screening. Since the negatively charged C-termini protrude perpendicularly to the microtubule surface this effect is additionally increased and explains the existence of the so-called 'zone of exclusion' [10] known to cell biologists for many years. We have shown that the microtubule structure, in particular the lateral binding between protofilaments, is consistent with the location of positive and negative segments of the electrostatic potential for optimal binding. It is worth mentioning that a recent paper [11] showed the electrostatic surface of the whole microtubule following computations involving the Poisson-Boltzmann equation. From these calculations a dramatic difference between the plus and minus ends of a microtubule has been revealed. It is very likely that this difference leads to the wellknown difference in polymerization kinetics involving these two ends. We hope that the new insights gained by performing these computations will be useful in our understanding of the cellular machinery as well as in the efforts to construct nanomachinery that uses biomolecular components or hybrid structures mimicking the effects observed in living cells.

[I] E. Nogales, S.G. Wolf, and K.H. Downing, "Structure of the a13Tubulin Dirner by Electron Crystallography", Nature, Nature Publishing Group, London, Jan. 08, 1998, pp. 199-203. [2] J. Lowe, H. Li, K.H. Downing, and E. Nogales, "Refined Structure of ~- Tubulin at 3.5 A Resolution", Journal of Molecular Biology, Academic Press-Elsevier, London, Feb. 26, 2002, pp. 1045-1057. [3] 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. [4] 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. 51-55. [5] B. Boeckmann, A. Bairoch, R. Apweiler, M.-C. Blatter, A. Estreicher, E. Gasteiger, M.J. Martin, K. Michoud, C. O'Oonovan, 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. [6] M.A. Marti-Renom, A. Stuart, A. Fiser, R. Sanchez, 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. 291-325. [7] R. Sayle and E.J. Milner-White, "RASMOL: Biomolecular Graphics for All", Trends in Biochemical Sciences (TIBS), Elsevier Science Ltd., London, Sep. 1995, pp. 374-376. [8] W. Wriggers and K. Schulten, "Nucleotide-Dependent Movements of the Kinesin Motor Domain Predicted by Simulated Annealing", Biophysics Journal, Biophysical Society, Bethesda. U.S.A., Aug. 1998, pp. 646--{)61. [9] M. Kikkawa, E.P. Sablin, Y. Okada, H. Yajima, R.J. Fletterick, and N. Hirokawa, "Switch-Based Mechanism of Kinesin Motors", Nature, Macmillan Publishers Ltd., England, May 24,2001, pp. 439-445. [10] Dustin, p ., Microtubules, Springer- V erlag, Berlin, 1984. [11] N.A. Baker, D. Sept, S. Joseph, M.J. HoIst, and J.A. McCammon, "Electrostatics of Nanosystems: Application to Microtubules and the Ribosome", Proceedings of the National Academy of Sciences, National Academy of Sciences, Washington U.S.A., Aug. 21,2001, pp. 10037-10041.

6. Acknowledgments
This research was supported by grants from NSERC and MITACS-MMPD. Discussions with Dr. Dan Sackett ofNIH are gratefullyacknowledged. I. M. D. would like to thank the staff of the Theoretical Physics Institute of the University of Alberta for all their kindness during his visit.

7. References