Frank Kozielski

A model of the microtubule–kinesin complex based on electron cryomicroscopy and X-ray crystallography

BACKGROUNDnMotor proteins of the kinesin superfamily play an organising role in eukaryotic cells and participate in many crucial phases of the cell cycle by moving along microtubules and thereby changing the position of attached organelles. In their standard form, kinesin motors are elongated heterotetrameric protein complexes composed of two identical heavy chains and two light chains; the central regions of the heavy chains intertwine, forming a coiled coil, with the globular heads of the microtubule-interacting motor domains at one end. In order to understand how kinesin motors interact with and move along microtubules, we have combined electron cryomicroscopy and X-ray crystallographic data to build a model of the complex.nnnRESULTSnUsing electron cryomicroscopy and image reconstruction, we have obtained three-dimensional maps of complexes of kinesin motor domain dimers and microtubules. Motor domain dimers interact one to one with tubulin dimers, with one head attached--lying along the microtubule protofilament--and the other unattached--pointing sideways and upwards towards the microtubule plus end. Using currently available crystallographic data, we have built an atomic resolution model of the motor domain dimer, which can be successfully docked into the three-dimensional framework of the maps from electron cryomicroscopy.nnnCONCLUSIONSnDocking the atomic resolution model into the map of the microtubule-kinesin complex with the coiled coil of kinesin pointing away from the microtubule surface shows that the attached and unattached heads have similar relative positions on the microtubule and in the crystal. Three regions of the attached head appear likely to interact with the microtubule.

The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations

BACKGROUNDnThe kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules.nnnRESULTSnThe crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains.nnnCONCLUSIONSnThe ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins.

Structural links to kinesin directionality and movement.

The kinesin motor proteins generate directional movement along microtubules and are involved in many vital processes, including cell division, in eukaryotes. The kinesin superfamily is characterized by a conserved motor domain of ∼320 residues. Dimeric constructs of N and C class kinesins, with the motor domains at opposite ends of the heavy chain, move towards microtubule plus and minus ends, respectively. Their crystal structures differ mainly in the region linking the motor domain core to the α-helical coiled coil dimerization domain. Chimeric kinesins show that regions outside of the motor domain core determine the direction of movement and mutations in the linker region have a strong effect on motility. Recent work on chimeras and mutants is discussed in a structural context giving insights to possible molecular mechanisms of kinesin directionality and motility.

First steps towards effective methods in exploiting high-throughput technologies for the determination of human protein structures of high biomedical value

The EC Structural Proteomics In Europe contract is aimed specifically at the atomic resolution structure determination of human protein targets closely linked to health, with a focus on cancer (kinesins, kinases, proteins from the ubiquitin pathway), neurological development and neurodegenerative diseases and immune recognition. Despite the challenging nature of the analysis of such targets, approximately 170 structures have been determined to date. Here, the impact of high-throughput technologies, such as parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens or the use of mass spectrometry to assist sample preparation, on the structural biology of mammalian protein targets is illustrated through selected examples.

Screening for inhibitors of microtubule-associated motor proteins.

The mitotic spindle is an important target for cancer chemotherapy. The main protein target for drugs in clinical use is tubulin, the building block of microtubules. In recent years, other proteins of the mitotic spindle have been identified as potential targets for the development of more specific drugs with the hope that these will have fewer side effects than known antimitotics (taxanes, vinca alkaloids). The human genome contains more than 40 members of the kinesin superfamily, with at least 12 of these involved in mitosis and cytokinesis. HsEg5 (also called KSP, kinesin spindle protein), a member of the kinesin-5 family, involved in the formation of the bipolar spindle, is a very promising target for cancer chemotherapy with specific inhibitors in Phase I and II clinical trails. Several successful approaches exist today to screen Eg5 for inhibitors, including phenotype-based assays and simple in vitro assays that explore the intrinsic enzymatic ATPase activity of Eg5. Here, we describe a robust and straightforward in vitro method to rapidly screen Eg5 for inhibitors. The assay can easily be adapted to other mitotic kinesins that may be identified in the future as potential drug targets, or simply to obtain specific kinesin inhibitors for use in chemical genetics to study the function of this important class of proteins.

The structure of human neuronal Rab6B in the active and inactive form

The Rab small G-protein family plays important roles in eukaryotes as regulators of vesicle traffic. In Rab proteins, the hydrolysis of GTP to GDP is coupled with association with and dissociation from membranes. Conformational changes related to their different nucleotide states determine their effector specificity. The crystal structure of human neuronal Rab6B was solved in its inactive (with bound MgGDP) and active (MgGTPgammaS-bound) forms to 2.3 and 1.8 A, respectively. Both crystallized in space group P2(1)2(1)2(1), with similar unit-cell parameters, allowing the comparison of both structures without packing artifacts. Conformational changes between the inactive GDP and active GTP-like state are observed mainly in the switch I and switch II regions, confirming their role as a molecular switch. Compared with other Rab proteins, additional changes are observed in the Rab6 subfamily-specific RabSF3 region that might contribute to the specificity of Rab6 for its different effector proteins.

Crystallization and preliminary crystallographic analysis of the motor domain of human kinetochore-associated protein CENP-E using an automated crystallization procedure

Human centromere-associated protein E, a member of the kinesin superfamily, is a microtubule-dependent motor protein involved in cell division that has been localized transiently to the kinetochore. The protein is thought to be responsible for the correct attachment and positioning of chromosomes to the mitotic spindle during the metaphase. The 312 kDa protein comprises four different domains. In this study, the focus was on the N-terminal motor domain, which includes the ATP-binding site and a region for microtubule binding. Crystals of the CENP-E motor domain have been obtained by high-throughput crystallization screening using an automated TECAN crystallization robot. The crystals (737 x 132 x 79 microm) belong to the space group P2(1), with unit-cell parameters a = 49.35, b = 83.70, c = 94.16 angstroms, beta = 103.05 degrees. They diffract to 2.1 angstroms resolution using synchrotron radiation.