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Liu, Xiao (2009): Comprehensive bioinformatic analysis of kinesin classification and prediction of structural changes from a closed to an open conformation of the motor domain. Dissertation, LMU München: Faculty of Biology
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Abstract

Kinesins form a large microtubule-associated motor protein super-family that can be found in every eukaryotic genome sequenced so far. Not only is the translocation of a large number of organelles, protein complexes and mRNAs carried out by them, but also the formation of the meiotic spindle and mitotic spindle integrity are strongly dependent on the kinesins. Fourteen different sub-families of kinesin have been reported. However, previous analyses were based on a relatively small number of selected kinesins (<600 sequences). Whether new classes of kinesin exist or the old classification system will hold as new sequence data become available is unknown. In this project, comprehensive computational analyses were performed on a large kinesin dataset (2,530 sequences). Sixteen conserved motifs were identified within the motor domain, including the ATP-binding motifs, microtubule-binding interface and many conserved secondary structural elements. Phylogenetic analysis confirmed the fourteen sub-family classification scheme. Thirteen of sub-families were well defined and statistically supported. The kinesin-12 sub-family had less support, with a clade confidence of 73%. In addition, a profile-based, automatic classification program was implemented according to the fourteen kinesin sub-groups. The accuracy of the program is over 85%, which makes the detection and classification of new kinesin sequences fast and easy. Kinesin-1, formerly known as conventional kinesin, is the best-studied member of the kinesin super-family. Motility studies have revealed an interesting phenomenon that the fungal kinesin-1s move 4-5 times faster than the animal kinesin-1s in general. Determining the sequence and structural factors that are responsible for the velocity difference is a topic of current research. Previous protein-chimera experiments have determined that the motor domain is essential for speed control. However, detailed analyses of the motor domain through mutagenesis have presented many challenges to biologists, because it is still unknown whether the speed is controlled by one particular amino acid residue or by a complex combination of several residues. With comparative analyses of the primary sequences of fungal and animal kinesin-1s, many group-specific residues were identified. Several of them are located inside II functionally important motifs such as the ATP-binding pocket and potential microtubule binding motifs, which appear to be responsible for the functional differences. The others are widely distributed in many important secondary structural elements. The mapping of these residues onto the fungal and animal three-dimensional crystal structures (1BG2 and 1GOJ) has led to the discovery of several structural changes from a closed to an open conformation of the motor domain. Most of the group-specific residues are involved in the spatial interactions with other group-specific residues or conserved residues. Many of these interactions can be detected only in the closed conformation. They contain functional elements, such as the switch-I, loop-11, β5 etc that lie within the core structure of the motor domain. When the structure changes into the open conformation, these elements are released and become active for binding to the microtubule. At the same time, many new interactions made by the group-specific residues are formed for the stabilization of the motor domain. These structurally crucial interaction-pairs of residues and the group specific residues found in the ATP-binding pocket provide insight into the potential control of kinesin velocity. The different structures of the fungal and animal ATP-binding pockets appear to be vital for ATP hydrolysis, but cannot control the velocity by itself. Some of the detected combinations of residues must interact within the ATP-binding pocket. They could be used as guidance for the biologists to design experiments to eventually discover the mechanism of velocity control. Many useful methods are implemented in the web-server, such as a classification tool, a conservation calculation tool, a motif search tool, and a discriminating residues (group-specific residues) search tool. The web-server is accessible at http://www.bio.uni-muenchen.de/~liu/kinesin_new/