Kinases of the MARK/Par-1 family of S/T protein kinases are regulators of diverse cellular processes in Caenorhabditis elegans, Drosophila, yeast, and mammalian cells. They are involved in nematode embryogenesis, epithelial cell polarization, cell signaling, and neuronal differentiation. MARK phosphorylates microtubule-associated proteins such as tau and is a key regulator of microtubule-based intracellular transport. Hyperphosphorylation of tau causes defects in neuronal transport and may induce abnormal aggregation of tau in Alzheimer disease and other tauopathies. Recent high-resolution structure analysis of MARK fragments covering the kinase domain and accessory regulatory domains has revealed important details regarding the autoregulation of MARK, but their interpretation has remained controversial. Here we focus on the structural aspects of MARK activity and autoregulation. Comparison of the available MARK structures with related kinases of the AMPK family and with new structures of MARK isoforms (MARK2 and 3) reveals unexpected structural similarities between these kinases that may help to resolve the existing controversies.

The MAP/microtubule affinity regulating kinase MARK/Par-1 phosphorylates microtubule associated proteins tau, MAP2, and MAP4, and is involved in the regulation of microtubule-based transport. Par-1, a homologue of MARK in Drosophila and C. elegans, is essential for the development of embryonic polarity. Four isoforms of MARK are found in humans. Recently, we reported the crystal structure of the catalytic and ubiquitinassociated domains of MARK2, an isoform enriched in brain (Panneerselvam et al., Structure (2006) 14, 173-183). It showed that the UBA domain has an unusual fold and binds to the N-terminal lobe of the catalytic domain. This is at variance with a previous lowresolution structure derived from small angle solution scattering (Jaleel et al., Biochem. J. (2006) 394, 545-555) which predicts binding of the UBA domain to the larger, C-terminal lobe. Here we report the crystal structure of the catalytic and UBA domain of another isoform, MARK1. Although the crystal packings of the two isoforms are unrelated, the overall conformations of the molecules are similar. Notably the UBA domain has the same unusual conformation as in MARK2 and it binds at the same site. Remarkable differences occur in the catalytic domain: at helix C, the catalytic loop, and the activation segment.

The Ser/Thr kinase MARK2 phosphorylates tau protein at sites that cause detachment from microtubules in Alzheimer neurofibrillary degeneration. Homologs of MARK2 include Par-1 in C. elegans and Drosophila, which generates embryonic polarity. We report the X-ray structure of the catalytic and ubiquitin-associated domains (UBA) of human MARK2. The activity was altered by mutations in the ATP binding site and/or activation loop. The catalytic domain shows the small and large lobes typical of kinases. The substrate cleft is in an inactive, open conformation in the inactivated and the wild-type structure. The UBA domain is attached via a taut linker to the large lobe of the kinase domain and leans against a hydrophobic patch on the small lobe. The UBA structure is unusual because the orientation of its third helix is inverted, relative to previous structures. Possible implications of the structure for the regulation of kinase activity are discussed.

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.

Microtubules are the intracellular tracks for two classes of motor proteins: kinesins and dyneins. During the past few years, the motor domain structures of several kinesins from different organisms have been determined by X-ray crystallography. Compared with kinesins, dyneins are much larger proteins and attempts to crystallize them have failed so far. Structural information about these proteins comes mostly from electron microscopy. In this chapter, we mainly focus on the crystal structures of kinesin motor domains.

Members of the MARK subfamily of the CaMK protein kinases phosphorylate microtubuleassociated proteins tau, MAP2, and MAP4 which are involved in the regulation of the cytoskeleton and the microtubule-based transport by motor proteins of the kinesin family [1]. Hyperphosphorylation of neuronal tau by MARK is a potential cause for the aggregation of tau and the formation of paired helical filaments which are a hallmark of Alzheimer's disease. Homologues of MARK in Drosophila and C. elegans (Par-1) and in fission yeast (KIN1) are essential for the development of cell polarity.

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 β‐tubulin monomer and the two flanking α‐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.

Kinesin is a microtubule-based motor protein responsible for anterograde transport of vesicles and organelles in nerve axons and other cell types. The energy necessary for this transport is derived from the hydrolysis of ATP which is thought to induce conformational changes in the protein. We have solved the X-ray crystal structures of rat brain kinesin in three conditions intended to mimic different nucleotide states: (1) with ADP bound to the nucleotide-binding site, (2) with bound ADP in the presence of AIF(4)(-), and (3) with ADP hydrolyzed to AMP by apyrase. In contrast to analogous cases observed in GTP-binding proteins or the muscle motor myosin, the structure of kinesin remained nearly unchanged. This highlights the stability of kinesin's ADP state in the absence of microtubules. Surprisingly, even after hydrolysis of ADP to AMP by apyrase a strong density peak remains at the position of the beta-phosphate which is compatible either with a phosphate or a sulfate from the solvent and appears to stabilize the nucleotide-binding pocket through several hydrogen bonds.

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The "motor" or "head" domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.

We have decorated microtubules with monomeric and dimeric kinesin constructs, studied their structure by cryoelectron microscopy and three-dimensional image reconstruction, and compared the results with the x-ray crystal structure of monomeric and dimeric kinesin. A monomeric kinesin construct (rK354, containing only a short neck helix insufficient for coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin head per tubulin subunit (alpha-beta-heterodimer). The orientation of the kinesin head (an anterograde motor) on the microtubule surface is similar to that of ncd (a retrograde motor). A longer kinesin construct (rK379) forms a dimer because of the longer neck helix forming a coiled-coil. Unexpectedly, this construct also decorates the microtubule with a stoichiometry of one head per tubulin subunit, and the orientation is similar to that of the monomeric construct. This means that the interaction with microtubules causes the two heads of a kinesin dimer to separate sufficiently so that they can bind to two different tubulin subunits. This result is in contrast to recent models and can be explained by assuming that the tubulin-kinesin interaction is antagonistic to the coiled-coil interaction within a kinesin dimer.

The binding stoichiometry of kinesin to microtubules was determined using several biochemical and biophysical approaches (chemical crosslinking, binding assays, scanning transmission electron microscopy (STEM), image reconstruction, and X-ray scattering). The results show that each tubulin dimer associates with one kinesin head, irrespective of whether kinesin occurs in a monomeric or dimeric form in solution. Moreover, these heads appear to align along the protofilament axis generating a 16 nm periodicity of successive kinesin dimers. This is consistent with a “tightrope” model of movement where the first head of the dimer provides a guiding signal for the following one.