Biochemical and Biophysical Research Communications
Crystal structure of the Candida albicans Kar3 kinesin motor domain fused to maltose-binding protein
Highlights
► The Candida albicans Kar3 motor domain structure was solved as a maltose-binding protein fusion. ► The electrostatic surface and part of the ATPase pocket of the motor domain differs markedly from other kinesins. ► The MBP–Kar3 interface highlights a new site for intramolecular or intermolecular interactions.
Introduction
Kinesin motor proteins transport intracellular cargos along microtubules (MTs) and influence the organization and dynamics of the microtubule (MT) cytoskeleton. They are typically composed of 4 domains; a motor domain, a neck or neck-linker, an α-helical coiled-coil forming region, and a cargo binding domain [1]. Of these domains, the motor domain is the most highly conserved (∼40% shared identity) and is the key element that binds and hydrolyses ATP as part of the mechanochemical cycle responsible for kinesin movement [2], [3]. During this cycle, conserved elements of the motor domain couple ATP binding, hydrolysis, and product release to conformational changes in the motor domain core that regulate MT affinity and guide re-orientation of the neck or neck-linker to produce direction-biased displacement of the kinesin and its cargo.
The kinesin superfamily is comprised of 14 different subfamilies, which are differentiated based on sequence, structure, function, and their directionality along MTs [1], [4]. The main role of some kinesin families is to mediate proper cell division through their involvement in mitotic spindle assembly and stabilization, as well as chromosome movement [5]. The Kar3 kinesin from the Saccharomyces cerevisiae is a well-studied mitotic kinesin, and is designated a member of the Kinesin-14 family due to its C-terminal motor domain and minus-end-directed movement on MTs [6], [7], [8]. Like several other members of the Kinesin-14 family, Kar3 has a second MT-binding site located in the cargo binding domain, allowing it to cross-link and slide MTs of opposite polarity past each other via a power-stroke motility mechanism [7], [9], [10]. This functionality allows it to mediate mitotic spindle organization by generating inward forces that pull the spindles toward the midzone and bundle the MTs [11], [12]. S. cerevisiae Kar3 is unique from most other kinesin isoforms because it interacts with two distinct kinesin-associated proteins (KAPs), named Cik1 and Vik1, which both lack the capacity to bind and hydrolyze ATP and yet can bind MTs and differentially influence the spatial and temporal localization of Kar3 [9], [11], [13], [14]. Moreover, the ScKar3Cik1 and ScKar3Vik1 complexes play discrete and important roles during mitotic division, meiosis, and karyogamy [9], [11], [15], [16], [17].
Recent studies have shown that deleting the KAR3 gene in the related diploid ascomycete fungus C. albicans interfered with normal morphogenesis of mating projections and abrogated nuclear fusion during karyogamy [18]. KAR3 knockout strains also exhibited longer generation times and lower cell viability of mitotically dividing yeast, suggesting that the Kar3 kinesin protein plays an important role in mitosis and mating of C. albicans. Given that this commensal fungus can cause debilitating mucosal infections, as well as life-threatening systemic infections [19], [20] in times of stress or in immune-compromised individuals, these insights highlight the potential for use of the C. albicans Kar3 kinesin as a novel target for antifungal drugs.
To begin building a molecular description of C. albicans Kar3, we have solved the X-ray crystal structure of its motor domain as a C-terminal fusion with maltose-binding protein (MBP). This is the first C. albicans motor protein structure to be determined and although its overall structure shows considerable homology with previously obtained structures of Kar3 from S. cerevisiae [10], [21], [22], and the filamentous fungus Ashbya gossypii [23], a number of unique features of the motor domain are apparent. Also, the close spatial arrangement of the MBP and CaKar3 motor domain (CaKar3MD) modules indicates the existence of an interface on CaKar3 that may be involved in intramolecular or intermolecular interactions that are new to the Kinesin-14 family.
Section snippets
Cloning, protein expression, and purification
cDNA for full-length CaKar3 was amplified from genomic DNA (ATCC number: 10231D-5), inserted into the TOPO cloning vector (Invitrogen), and sequenced to verify its identity. The CaKar3MD construct (Leu344–Lys687) was amplified by PCR and cloned into pET14d (Novagen) using NcoI and NotI restriction sites for untagged protein expression, or into pMal-MATa1 (Addgene) using HindIII and PstI for expression as a maltose-binding protein fusion [24]. Both vectors were expressed in the BL21-CodonPlus
Overall structure of the CaKar3 motor domain
To date, numerous kinesin motor domain structures, as well as structures of multi-subunit kinesin motor complexes, have been deposited in the PDB for a number of different organisms. Some of these include Kinesin-14 motors, such as Drosophila Ncd and Kar3 from S. cerevisiae [10], [21], [22], [32]. CaKar3 is 687 amino acids long and its motor domain resides between Leu344 and Lys687 (Fig. 1A) [33]. Sequence conservation between CaKar3 and other Kinesin-14 family members breaks down significantly
Acknowledgments
We thank Fred Faucher and Da Duan for assistance with diffraction data collection and processing. This work was supported by funding to J.S.A. from Canadian Institutes of Health Research and the Ontario Early Researcher Award program. J.S.A holds a Canada Research Chair (Tier 2) in Structural Biology.
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