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Dissertation zugänglich unter
URN: urn:nbn:de:gbv:18-26165
URL: http://ediss.sub.uni-hamburg.de/volltexte/2005/2616/

Structural Studies on Kinesin-1 Motor Domain and Light Chain from Rattus norvegicus and Neurospora crassa

Strukturelle Studie an der Kinesin-1 Motordomäne und der leichten Kette aus Rattus norvegicus und Neurospora crassa

Mylonas, Spyridon

 Dokument 1.pdf (2.790 KB) 

SWD-Schlagwörter: Kinesin , Zellskelett , Kristallographie
Freie Schlagwörter (Deutsch): Motorproteine , Vesikeltransport
Basisklassifikation: 42.13
Institut: Biologie
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Dissertation
Hauptberichter: Mandelkow, Eckhard (Prof. Dr.)
Sprache: Englisch
Tag der mündlichen Prüfung: 02.09.2005
Erstellungsjahr: 2005
Publikationsdatum: 19.09.2005
Kurzfassung auf Englisch: Kinesin-1, the founding member of the kinesin superfamily, is a plus-end-directed microtubule motor that mediates the fast anterograde transport in neurons, in a processive manner.

Kinesin-1 is a heterotetramer of two heavy (KHCs) and two light chains (KLCs). Each heavy chain comprises three domains: the N-terminal motor domain, the central stalk domain and the C-terminal tail domain. The motor domain contains both the microtubule and the nucleotide binding structural elements. The kinesin light chain consists of an N-terminal heptad repeat region and a six-repeat TPR domain that is known to interact with a variety of proteins and may prove to play a critical role in the attachment of kinesin to its cargoes. C-terminal isoform-specific sequences of the light chain target kinesin to different cargoes.

This thesis explored structural aspects of both the motor domain of the heavy chain from Rattus. norvegicus and Neurospora. crassa and the TPR domain of the light chain from Rattus. norvegicus.

The entire light chain was cloned but its expression yielded insoluble protein. Further investigation was carried by generating a series of constructs differing in the length of the TPR domain. In most cases the expression of these constructs yielded insoluble proteins and solubilization efforts were carried out. In cases where soluble proteins were obtained purification was then carried out. The purified proteins were used in crystallization trials screening numerous conditions. Crystallization of the KLC TPR domain is an aim that was not achieved.

Previous studies of the kinesin-1 motor domain in Drosophila revealed the importance of an arginine of the switch-1 region on the function of the motor. This arginine is suggested to participate in the formation of a critical salt bridge that positions the water molecule within the active site. Additionally, it is hypothesized to participate in a network of hydrogen bonds that are supposed to hold together the three essential and flexible regions for ATP hydrolysis: switch-1, switch-2 and helix α4.

A series of four mutant constructs were generated by substituting this arginine to an alanine and lysine respectively in the motor domains from Neurospora. crassa and Rattus. Norvegicus (Nk355R207A, Nk355R207K, Rk354R204Aand Rk354R204K). Moreover, a mutant construct was generated where -an important for microtubule binding- surface loop (L11) was deleted. The proteins were expressed and purified and used in crystallization trials.

In the case of Nk355R207A and Rk354R204A protein crystals, data sets were obtained at a resolution of 3.05 Å and molecular replacement produced unique solutions, using the structures of the wild type proteins as search models. Interestingly, the crystals of both mutants belonged to the same space group P21, in contrast to the crystals of the wild type proteins that belonged to the space group P212121. Furthermore, in the case of the Nk355R207A crystals the orientation of two of the molecules in the unit cell was found to be the same between the crystals of the wild type and the mutant proteins.

Aim of this study was to contribute structural information about different domains of the motor protein kinesin-1, using the X-ray crystallography technique. The crystallization of the motor domain mutants could provide interesting insight into the conformational changes in the active site of the motor that could increase our understanding of how the motor uses the energy from ATP hydrolysis to generate motion.

Moreover, crystallizing the TPR domain of the light chain of kinesin-1 could reveal how kinesin interacts with its cargo. In light of its association with proteins such as the APP or the JIPs, it would be interesting to determine the structural basis of these interactions. This could in turn aid the design of a new generation of molecular drugs, intended to exploit the kinesin-cargo interaction and get targeted to the cellular compartments - such as the synapse - where they are needed.


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