Kinesin-mediated cargo transport is required for many cellular functions and plays a key role in pathological processes. Structural information on how kinesins recognize their cargoes is required for a molecular understanding of this fundamental and ubiquitous process. Here we present the crystal structure of the tetratricopeptide repeat of kinesin light chain 2 in complex with a cargo peptide harboring a 'tryptophan-acidic' motif derived from SKIP, a critical host determinant in Salmonella pathogenesis and a regulator of lysosomal positioning. Structural data together with biophysical, biochemical and cellular assays allow us to propose a framework for intracellular transport based on the binding by kinesin-1 of W-acidic cargo motifs through a combination of electrostatic interactions and sequence-specific elements, providing direct molecular evidence of the mechanisms for kinesin-1:cargo recognition.The plus-end directed motor, kinesin-1 plays a critical role in the intracellular transport of diverse protein, ribonuclear protein complexes and membrane compartments on microtubules (1). Its functions are also usurped by bacteria and viruses to aid in their replication (2, 3). Kinesin-1 can perform this diverse range of functions by virtue of its ability to interact with many different cargo proteins (4). Diversity of cargo recognition is accomplished largely through the kinesin light chains (KLC) which harbour a tetratricopeptide repeat (TPR) domain, a versatile protein interaction platform (5, 6). The KLC TPR domain can recognise short peptide stretches within relatively disordered regions of its targets. These peptides are characterized by a tryptophan residue flanked by acidic residues (e.g. EWD) and are found in a growing list of KLC binding proteins (7-16). Although W-acidic motifs often occur in pairs, single motifs are also functional and can support microtubule-based transport even when explanted from their host protein (7,12,17). We set out to solve the structure of a KLC TPR domain bound to a cargo W-acidic motif. We focused our attention on the SKIP cargo for its importance in Salmonella pathogenesis.SKIP contains a pair of W-acidic motifs centred at amino acid positions 207-208 (WD) and 236-237 (WE) that fall within the N-terminal kinesin-1 binding region (residues 1-310)(3, 7, 13) (Fig. 1A). To assess the relative importance of the SKIP W-acidic motifs for KLC binding we co-transfected HeLa cells with wild-type and WD/WE mutant constructs expressing GFP-SKIP(1-310) and HA-KLC2 (Fig. 1B). Disruption of the WD motif significantly reduced GFP-SKIP interaction with HA-KLC2 whereas abrogation of the WE motif had no obvious effects. A double mutant with both motifs disrupted displayed HA-KLC2 binding similar to the single WD mutant. Thus the WE motif has a very low affinity for KLC2. Indeed, a ten amino acid long peptide centered on the WD motif (SKIP WD , Figure 1A) bound to KLC2 TPR with a K D of 24 μM whereas the affinity of the equivalent SKIP WE peptide was above 110 μM (Fig. 1C). The presenc...

The light chains (KLCs) of the microtubule motor kinesin-1 bind cargoes and regulate its activity. Through their tetratricopeptide repeat domain (KLC TPR ), they can recognize short linear peptide motifs found in many cargo proteins characterized by a central tryptophan flanked by aspartic/glutamic acid residues (W-acidic). Using a fluorescence resonance energy transfer biosensor in combination with X-ray crystallographic, biochemical, and biophysical approaches, we describe how an intramolecular interaction between the KLC2 TPR domain and a conserved peptide motif within an unstructured region of the molecule, partly occludes the W-acidic binding site on the TPR domain. Cargo binding displaces this interaction, effecting a global conformational change in KLCs resulting in a more extended conformation. Thus, like the motor-bearing kinesin heavy chains, KLCs exist in a dynamic conformational state that is regulated by self-interaction and cargo binding. We propose a model by which, via this molecular switch, W-acidic cargo binding regulates the activity of the holoenzyme.kinesin | KLC | TPR domain | microtubule motor | cytoskeleton

SummaryCytoplasmic dynein, the major motor driving retrograde axonal transport, must be actively localized to axon terminals. This localization is critical as dynein powers essential retrograde trafficking events required for neuronal survival, such as neurotrophic signaling. Here, we demonstrate that the outward transport of dynein from soma to axon terminal is driven by direct interactions with the anterograde motor kinesin-1. In developing neurons, we find that dynein dynamically cycles between neurites, following kinesin-1 and accumulating in the nascent axon coincident with axon specification. In established axons, dynein is constantly transported down the axon at slow axonal transport speeds; inhibition of the kinesin-1-dynein interaction effectively blocks this process. In vitro and live-imaging assays to investigate the underlying mechanism lead us to propose a new model for the slow axonal transport of cytosolic cargos, based on short-lived direct interactions of cargo with a highly processive anterograde motor.Video Abstract

In the sarcomeric M-band, the giant ruler proteins titin and obscurin, its small homologue obscurin-like-1 (obsl1), and the myosin cross-linking protein myomesin form a ternary complex that is crucial for the function of the M-band as a mechanical link. Mutations in the last titin immunoglobulin (Ig) domain M10, which interacts with the N-terminal Ig-domains of obscurin and obsl1, lead to hereditary muscle diseases. The M10 domain is unusual not only in that it is a frequent target of disease-linked mutations, but also in that it is the only currently known muscle Ig-domain that interacts with two ligands-obscurin and obsl1-in different sarcomeric subregions. Using x-ray crystallography, we show the structural basis for titin M10 interaction with obsl1 in a novel antiparallel Ig-Ig architecture and unravel the molecular basis of titin-M10 linked myopathies. The severity of these pathologies correlates with the disruption of the titin-obsl1/obscurin complex. Conserved signature residues at the interface account for differences in affinity that direct the cellular sorting in cardiomyocytes. By engineering the interface signature residues of obsl1 to obscurin, and vice versa, their affinity for titin can be modulated similar to the native proteins. In single-molecule force-spectroscopy experiments, both complexes yield at forces of around 30 pN, much lower than those observed for the mechanically stable Z-disk complex of titin and telethonin, suggesting why even moderate weakening of the obsl1/obscurintitin links has severe consequences for normal muscle functions.immunoglobulin domain | protein complex | x-ray crystallography | mechanosensor | myopathy S arcomeres are the smallest contractile units of striated muscles. They are highly ordered assemblies of precisely tailored actin and myosin filaments that are crosslinked at Z-disks and Mbands, respectively. The assembly of hundreds of protein subunits into ordered sarcomeres forms the structural basis for striated muscle contraction. The global layout of sarcomere assembly requires a giant ruler protein, titin, a 3000 kDa modular protein with a length of over 1.2 μm, displaying binding sites for proteins along the entire distance from Z-disk to M-band (1-3). Titin is composed of hundreds of immunoglobulin-and fibronectin-3-like (Ig and Fn3) domains that are arranged in specific patterns in the subcompartments of the sarcomere (3). The Ig-domains in sarcomeric proteins like titin, myomesin, or obscurin present a functionally versatile surface around a highly stable structural scaffold (4).At the M-band, the centers of myosin filaments are crosslinked into a mechanically stable network that involves titin, the cytoskeletal protein myomesin (5), and the giant obscurin (6), the latter acting as a linker to the sarcoplasmic reticulum (SR) (7). Obscurin, with a mass of around 800 kDa, was initially discovered as a ligand of Z-disk titin (3). Like titin, it is a modular protein composed of Ig and Fn3 domains. Intriguingly, obscurin localizes predominantly to the M-band ...

The molecular interplay between cargo recognition and regulation of the activity of the kinesin-1 microtubule motor is not well understood. Using the lysosome adaptor SKIP (also known as PLEKHM2) as model cargo, we show that the kinesin heavy chains (KHCs), in addition to the kinesin light chains (KLCs), can recognize tryptophan-acidic-binding determinants on the cargo when presented in the context of an extended KHC-interacting domain. Mutational separation of KHC and KLC binding shows that both interactions are important for SKIP–kinesin-1 interaction in vitro and that KHC binding is important for lysosome transport in vivo. However, in the absence of KLCs, SKIP can only bind to KHC when autoinhibition is relieved, suggesting that the KLCs gate access to the KHCs. We propose a model whereby tryptophan-acidic cargo is first recognized by KLCs, resulting in destabilization of KHC autoinhibition. This primary event then makes accessible a second SKIP-binding site on the KHC C-terminal tail that is adjacent to the autoinhibitory IAK region. Thus, cargo recognition and concurrent activation of kinesin-1 proceed in hierarchical stepwise fashion driven by a dynamic network of inter- and intra-molecular interactions.