Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms

et al. (2012) Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms. PLoS ONE 7(3): e33943. doi:10.1371/journal.pone.0033943 Crystal Structures of the Tetratricopeptide Repeat Domains of Kinesin Light Chains: Insight into Cargo Recognition Mechanisms Haizhong Zhu 0 Han Youl Lee 0 Yufeng Tong 0 Bum-Soo Hong 0 Kyung-Phil Kim 0 Yang Shen 0 Kyung 0 Jik Lim 0 Farrell Mackenzie 0 Wolfram Tempel 0 Hee-Won Park 0 Wenqing Xu, University of Washington, United States of America 0 1 Structural Genomics Consortium , Toronto, Ontario , Canada , 2 Department of Pharmacology, University of Toronto , Toronto, Ontario , Canada , 3 Philip Pocock Catholic Secondary School , Mississauga, Ontario , Canada Kinesin-1 transports various cargos along the axon by interacting with the cargos through its light chain subunit. Kinesin light chains (KLC) utilize its tetratricopeptide repeat (TPR) domain to interact with over 10 different cargos. Despite a high sequence identity between their TPR domains (87%), KLC1 and KLC2 isoforms exhibit differential binding properties towards some cargos. We determined the structures of human KLC1 and KLC2 tetratricopeptide repeat (TPR) domains using X-ray crystallography and investigated the different mechanisms by which KLCs interact with their cargos. Using isothermal titration calorimetry, we attributed the specific interaction between KLC1 and JNK-interacting protein 1 (JIP1) cargo to residue N343 in the fourth TRP repeat. Structurally, the N343 residue is adjacent to other asparagines and lysines, creating a positively charged polar patch within the groove of the TPR domain. Whereas, KLC2 with the corresponding residue S328 did not interact with JIP1. Based on these finding, we propose that N343 of KLC1 can form ''a carboxylate clamp'' with its neighboring asparagine to interact with JIP1, similar to that of HSP70/HSP90 organizing protein-1's (HOP1) interaction with heat shock proteins. For the binding of cargos shared by KLC1 and KLC2, we propose a different site located within the groove but not involving N343. We further propose a third binding site on KLC1 which involves a stretch of polar residues along the inter-TPR loops that may form a network of hydrogen bonds to JIP3 and JIP4. Together, these results provide structural insights into possible mechanisms of interaction between KLC TPR domains and various cargo proteins. - Funding: This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 371633-09 (H.-W.P). The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck and Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research, and the Wellcome Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors received funding from commerical sources (GlaxoSmithKline, Merck and Co., the Novartis Research Foundation). This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. . These authors contributed equally to this work. Kinesins are a family of molecular motor proteins that move along polarized microtubules to transport a macromolecular cargo using energy obtained from adenosine triphosphate (ATP) hydrolysis. Defects of microtubule-based transport are deleterious to neuronal activity and eventually fatal [1,2,3,4]. Kinesin-1 is responsible for over 40 different cargos and functions as a heterotetramer composed of two subunits: the kinesin heavy chain (KHC) and the kinesin light chain (KLC) [5]. The KHC consists of three domains: the N-terminal motor domain that contains the ATP and microtubule binding sites, the central coiled-coil domain responsible for dimerization, and the Cterminal tail domain that regulates the ATPase and microtubule binding activity [6]. The KLC also contains three domains: the Nterminal coiled-coil domain (heptad repeat) that binds to the KHC, a tetratricopeptide repeat (TPR), and the C-terminal domain. The latter two domains of the KLC are primarily involved in the binding of cargos, functioning as a physical linker between the KHC and its cargos [7]. Four isoforms of KLC exist in humans: KLC1, KLC2, KLC3, and KLC4. The KLC1 isoform is highly expressed in neurons and binds to several proteins that are associated with neurodegeneration or axonal outgrowth as it interacts with JNK-interacting proteins (JIPs), Huntingtin-associated protein-1 (HAP1), alcadein1 (ALC1) torsinA, collapsing response mediator protein-1 (CRMP2), KIDINS220, and Daxx [8,9,10,11,12,13,14,15]. The KLC2 isoform shares several cargo proteins with KLC1 [9,12,14], but cannot interact with torsinA [10]. This is particularly interesting as the TPR domain, the putative binding site for these cargos, shares high primary sequence homology with 87% identity between the two isoforms. TPR domains are known as a protein-protein interaction module, which consists of multiple tandem-repeats of 34 amino acids [16]. The structures of many TPR domains have been solved, including the TPR domains of protein phosphatase 5 (PP5), peroxin 5 (PEX5P), small glutamine-rich tetratricopeptide (SGT), p67phox, and Hsp 70/90 operating protein-1 (HOP1) [17,18,19,20,21]. The structures of these TPR domains reveal a helix-turn-helix arrangement for each TPR repeat and a superhelical conformation of multiple TPR repeats [16]. Furthermore, the ligand-bound structures of p67phox-TPR with Rac1 GTPase and HOP1-TPR with a C-terminal end of Hsp 70/90 peptide establish two different mechanisms for the TPR-ligand interaction [18,20]. The TPR domain of p67phox recognizes its ligand through the loops connecting the TPR repeats located on the outer edges of the superhelix On the other hand, HOP1 utilizes a carboxylate clamp formed by two asparagine residues surrounded by lysines in the inner groove of the TPR domain to interact with Hsp. In mammalian KLC1, it is known that the groove of the TPR superhelix binds to the C-terminal residues of JIP1 whereas the edge of the TPR domain binds to internal residues of JIP3, suggesting that mammalian KLC1 exploits both the established TPR domain binding modes [8,21,22,23]. With numerous cargos, competition for KLC1 binding arises between the cargos [14]. JIP1 suppresses the transport of ALC1 cargos while ALC1 blocks the JIP1 mediated transport of APPcontaining vesicles [14]. Not surprisingly, both the cargos require their amino acids with similar properties to inte (...truncated)