Selected Publications:

Interaction of kinesin motors, microtubules, and MAPs

Journal of Muscle Research and Cell Motility, 27(2), pages 125-137

A. Marxi, J. Muller, E.-M. Mandelkow

Abstract

Kinesins are a family of microtubule-dependent motor proteins that carry cargoes such as vesicles, organelles, or protein complexes along microtubules. Here we summarize structural studies of the “conventional” motor protein kinesin-1 and its interactions with microtubules, as determined by X-ray crystallography and cryo-electron microscopy. In particular, we consider the docking between the kinesin motor domain and tubulin subunits and summarize the evidence that kinesin binds mainly to β tubulin with the switch-2 helix close to the intradimer interface between α and β tubulin.

Kinesin's second step (pdf)

Proc. Natl. Acad. Sci., USA 101:3444-3449, (2004)

Klumpp, L.M., A. Hoenger, and S.P. Gilbert

We have identified dimeric kinesin mutants that become stalled on the microtubule after one ATP turnover, unable to bind and hydrolyze ATP at their second site. We have used these mutants to determine the regulatory signal that allows ATP to bind to the forward head, such that processive movement can continue. The results show that phosphate release occurs from the rearward head before detachment, and detachment triggers active-site accessibility for ATP binding at the forward head. This mechanism, in which the rearward head controls the behavior of the forward head, may be conserved among processive motors.

Microtubule-Kinesin interface mutants reveal a site critical for communication (pdf)

Biochemistry 43:2792-2803, (2004)

Klumpp, L.M., K.M. Brendza, J.E. Gatial, 3rd, A. Hoenger, W.M. Saxton, and S.P. Gilbert

Abstract

Strict coordination of the two motor domains of kinesin is required for driving the processive movement of organelles along microtubules. Glutamate 164 of the kinesin heavy chain was shown to be critical for kinesin function through in vivo genetics in Drosophila melanogaster. The mutant motor E164K exhibited reduced steady-state ATPase activity and higher affinity for both ATP and microtubules. Moreover, an alanine substitution at this position (E164A) caused similar defects. It became stalled on the microtubule and was unable to bind and hydrolyze ATP at the second motor domain. Glu(164), which has been conserved through evolution, is located at the motor-microtubule interface close to key residues on helix alpha12 of beta-tubulin. We explored further the contributions of Glu(164) to motor function using several site-directed mutant proteins: E164K, E164N, E164D, E164Q, and D165A. The results indicate that the microtubule-E164K complex can only bind and hydrolyze one ATP. ATP with increased salt was able to dissociate a population of E164K motors from the microtubule but could not dissociate E164A. We tested the basis of the stabilized microtubule interaction with E164K, E164N, and E164A. The results provide new insights about the motor-microtubule interface and the pathway of communication for processive motility.

Complex formation with kinesin motor domains affects the structure of microtubules (pdf)

J Mol Biol. 2004 Jan 2;335(1):139-53.

Krebs A, Goldie KN, Hoenger A.

Abstract

Microtubules are highly dynamic components of the cytoskeleton. They are important for cell movement and they are involved in a variety of transport processes together with motor proteins, such as kinesin. The exact mechanism of these transport processes is not known and so far the focus has been on structural changes within the motor domains, but not within the underlying microtubule structure.Here we investigated the interaction between kinesin and tubulin and our experimental data show that microtubules themselves are changing structure during that process. We studied unstained, vitrified samples of microtubules composed of 15 protofilaments using cryo electron microscopy and helical image analysis. 3D maps of plain microtubules and microtubules decorated with kinesin have been reconstructed to approximately 17A resolution. The alphabeta-tubulin dimer could be identified and, according to our data, alpha- and beta-tubulin adopt different conformations in plain microtubules. Significant differences were detected between maps of plain microtubules and microtubule-kinesin complexes. Most pronounced is the continuous axial inter-dimer contact in the microtubule-kinesin complex, suggesting stabilized protofilaments along the microtubule axis. It seems, that mainly structural changes within alpha-tubulin are responsible for this observation. Lateral effects are less pronounced. Following our data, we believe, that microtubules play an active role in intracellular transport processes through modulations of their core structure.

The C-terminus of Tubulin Modulates Nucleotide-Dependent Kinesin Binding (pdf)

EMBO J. 23: 989-999

Georgios Skiniotis, Jared C Cochran, Jens Müller, Eckhard Mandelkow, Susan P Gilbert, and Andreas Hoenger

Abstract

The flexible tubulin C-terminal tails (CTTs) have recently been implicated in the walking mechanism of dynein and kinesin. To address their role in the case of conventional kinesin, we examined the structure of kinesin–microtubule (MT) complexes before and after CTT cleavage by subtilisin. Our results show that the CTTs directly modulate the motor–tubulin interface and the binding properties of motors. CTT cleavage increases motor binding stability, and kinesin appears to adopt a binding conformation close to the nucleotide-free configuration under most nucleotide conditions. Moreover, C-terminal cleavage results in trapping a transient motor–ADP–MT intermediate. Using SH3-tagged dimeric and monomeric constructs, we could also show that the position of the kinesin neck is not affected by the C-terminal segments of tubulin. Overall, our study reveals that the tubulin C-termini define the stability of the MT–kinesin complex in a nucleotide-dependent manner, and highlights the involvement of tubulin in the regulation of weak and strong kinesin binding states.

Structure of a fast kinesin: implications for ATPase mechanism and interactions with microtubules (pdf)

EMBO J. 2001 Nov 15;20(22):6213-25

Song YH, Marx A, Müller J, Woehlke G, Schliwa M, Krebs A, Hoenger A, Mandelkow E.

Abstract

We determined the crystal structure of the motor domain of the fast fungal kinesin from Neurospora crassa (NcKin). The structure has several unique features. (i) Loop 11 in the switch 2 region is ordered and enables one to describe the complete nucleotide-binding pocket, including three inter-switch salt bridges between switch 1 and 2. (ii) Loop 9 in the switch 1 region bends outwards, making the nucleotide-binding pocket very wide. The displacement in switch 1 resembles that of the G-protein ras complexed with its guanosine nucleotide exchange factor. (iii) Loop 5 in the entrance to the nucleotide-binding pocket is remarkably long and interacts with the ribose of ATP. (iv) The linker and neck region is not well defined, indicating that it is mobile. (v) Image reconstructions of ice-embedded microtubules decorated with NcKin show that it interacts with several tubulin subunits, including a central beta-tubulin monomer and the two flanking alpha-tubulin monomers within the microtubule protofilament. Comparison of NcKin with other kinesins, myosin and G-proteins suggests that the rate-limiting step of ADP release is accelerated in the fungal kinesin and accounts for the unusually high velocity and ATPase activity.

Structural Analysis of the Microtubule-Kinesin Complex by Cryo-Electron Microscopy

Kinesin Protocols

December 2000, Methods in Molecular Biology, Vol 164 pages 235-254

Fabienne Beuron, Andreas Hoenger

Abstract

The structures of microtubule-kinesin complexes have been intensely studied within the last few years by using negative stain or cryo-electron microscopy and digital three-dimensional (3D) image reconstruction (2-4). On a working system, these methods constitute a straightforward approach to generate 3D data at around 20 å resolution within a few weeks. Such maps all ow the interpretation the 3D configuration of protein domains such as the binding geometry of kinesin motor heads to tubulin protofilaments (5) or the configuration of dimeric kinesin motor domains when bound to microtubules under different nucleotide conditions (6-8). More recently, the availability of near-atomic-resolution data of the components of microtubule-kinesin complexes, namely the αβ-tubulin dimer (9) and several monomeric and dimeric kinesin motor constructs, made it possible to interpret the structure of an intact microtubule (11) and the motor-tubulin interactions at near-atomic detail (8,12).

A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis

Cell. 1997 Jul 25;90(2):217-24.

Sosa H, Dias DP, Hoenger A, Whittaker M, Wilson-Kubalek E, Sablin E, Fletterick RJ, Vale RD, Milligan RA.

Abstract

Kinesin motors convert chemical energy from ATP hydrolysis into unidirectional movement. To understand how kinesin motors bind to and move along microtubules, we fit the atomic structure of the motor domain of Ncd (a kinesin motor involved in meiosis and mitosis) into three-dimensional density maps of Ncd-microtubule complexes calculated by cryo-electron microscopy and image analysis. The model reveals that Ncd shares an extensive interaction surface with the microtubule, and that a portion of the binding site involves loops that contain conserved residues. In the Ncd dimer, the microtubule-bound motor domain makes intimate contact with its partner head, which is dissociated from the microtubule. This head-head interaction may be important in positioning the dissociated head to take a step to the next binding site on the microtubule protofilament.

Three Different Approaches for Calculating the Three-Dimensional Structure of Microtubules Decorated with Kinesin Motor Domains

Sosa H., Hoenger A., Milligan R.A.

Source: Journal of Structural Biology, Volume 118, Number 2, March 1997 , pp. 149-158(10)

Abstract

We have used three different electron microscopy approaches to calculate three-dimensional maps of tubulin assemblies decorated with the motor domain of kinesin. The approaches used were: (1) Tilt series reconstruction of negatively stained tubulin sheets. (2) Back-projection reconstruction of microtubules in ice. (3) Helical reconstruction of microtubules in ice. The calculated maps show the overall configuration of the protofilaments and the interactions between the motor and the protofilaments at a resolution of 2-4 nm. The three methods revealed a similar binding configuration of the kinesin motor domain to the protofilament. We also found that seams can be present in potentially helical microtubules, limiting the use of helical reconstruction algorithms. Advantages and disadvantages of each of the three approaches are discussed.

Motor Domains of Kinesin and ncd Interact with Microtubule Protofilaments with the Same Binding Geometry

Source: Journal of Molecular Biology, Volume 265, Number 5, January 1997 , pp. 553-564(12)

Hoenger A., Milligan R.A.

Abstract

Kinesin and ncd (non-claret disjunctional) are microtubule associated motor proteins which share several structural features: both motors are dimers; each monomer is composed of a stalk region, a cargo binding domain and a motor domain; the motor domains have sim41% sequence identity. Despite these similarities the two motors have strikingly different movement properties: kinesin is a plus-end directed molecular motor, while ncd is minus-end directed. Here we compare the structure and the microtubule-binding properties of these oppositely directed molecular motors. We determined the three-dimensional structure of tubulin sheets decorated with the motor domains of either kinesin or ncd to a resolution of <20 Å by negative stain electron microscopy and tilt series reconstruction. Comparisons with a control structure of tubulin alone revealed that in both cases the motor domain binds to the outer crest of a single protofilament making contacts with both agr and bgr tubulin. Despite their opposite directionality, the geometry of attachment of the motor domain to the protofilament in the presence of AMP-PNP is very similar for both motors. These data rule out models for directionality which have the motors binding in an opposite orientation to the microtubules. Binding of the ncd as well as the kinesin motor domain appears to induce conformational changes in tubulin. This observation suggests an active role of tubulin in motor movement and/or in the determination of directionality.

Three-dimensional structure of a tubulin-motor-protein complex (pdf)

Letters to Nature, Nature 376, 271 - 274 , Vol 376 July 1995

Andreas Hoenger, Elena P. Sabliná, Ronald D. Vale, Robert J. Fletterick & Ronald A. Milligan

Abstract

The kinesin superfamily is a class of microtubule-based mechano-enzymes involved in intracellular transport and chromosome movements. Molecules that move towards either the plus end or the minus end of micro tubules are represented within the family. The motor domains of these molecules exhibit considerable sequence homology and contain both the ATP- and microtubule-binding sites (reviewed in refs 1, 2). Here we focus on non-claret disjunctional (ncd), a minus-end-directed motor involved in chromosome segregation in meiosis and early mitosis in Drosophila 3á¤-6. We have calculated a three-dimensional map of tubulin sheets decorated with monomeric recombinant ncd motor domains7 by negative-stain electron microscopy and image analysis. Comparisons with a control structure of tubulin alone reveal that each motor domain binds to the crest of a single protofilament, making extensive contacts with both the alpha and beta tubulin monomers. Binding of the motor domain results in significant conformational changes in both of the tubulin monomers.