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Structural Basis for Kinesin-1:Cargo Recognition

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Science  19 Apr 2013:
Vol. 340, Issue 6130, pp. 356-359
DOI: 10.1126/science.1234264

Cargo-Motor Interaction

Kinesin-1 directs a diverse array of functions within axons by interacting with many proteins. To understand the mechanisms underpinning kinesin-cargo recognition, Pernigo et al. (p. 356, published online 21 March) solved the crystal structure of the tetratricopeptide repeat of kinesin light chain 2 bound to a W-acidic cargo peptide from SKIP—a critical host determinant in Salmonella pathogenesis that controls lysosome positioning.

Abstract

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 domain of kinesin light chain 2 in complex with a cargo peptide harboring a “tryptophan-acidic” motif derived from SKIP (SifA-kinesin interacting protein), 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 (KLCs), which harbour a tetratricopeptide repeat (TPR) domain, a versatile protein interaction platform (5, 6). The KLCTPR domain can recognize short peptide stretches within relatively disordered regions of its targets. These peptides are characterized by a tryptophan residue flanked by acidic residues (such as EWD) and are found in a growing list of KLC-binding proteins (716). [Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. In the mutants, other amino acids were substituted at certain locations; for example, R251D indicates that arginine at position 251 was replaced by aspartic acid.] 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 KLCTPR domain bound to a cargo W-acidic motif. We focused our attention on the SifA-kinesin interacting protein (SKIP) cargo for its importance in Salmonella pathogenesis. SKIP contains a pair of W-acidic motifs centered at amino acid positions 207 and 208 (WD) and 236 and 237 (WE) that fall within the N-terminal kinesin-1 binding region (residues 1 to 310) (Fig. 1A) (3, 7, 13). To assess the relative importance of the SKIP W-acidic motifs for KLC binding, we cotransfected HeLa cells with wild-type and WD/WE mutant constructs expressing green fluorescent protein (GFP)–SKIP(1-310) and hemagglutinin (HA)–KLC2 (Fig. 1B).

Fig. 1 Binding of SKIP to KLC2.

(A) Scheme of SKIP and KLC2. The two W-acidic motifs and KLC2 TPR repeats are highlighted. (B) Coimmunoprecipitation shows that alanine replacement in the first W-acidic motif (WD) abrogates SKIP:KLC2 binding, whereas the same substitution in the second motif (WE) has virtually no effect. (C) Fluorescence polarization measurements confirm that the first SKIP W-acidic motif (SKIPWD) has a higher affinity for the TPR domain of KLC2 than that of the second one (SKIPWE). A peptide encompassing both motifs (SKIPWDWE) binds with higher affinity than the best single motif. Kd values reported here were calculated at 150 mM NaCl. Binding affinity values at varying ionic strength values are given in fig. S1.

Disruption of the WD motif 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 10-amino-acid-long peptide centered on the WD motif (SKIPWD) (Fig. 1A) bound to KLC2TPR with a dissociation constant (Kd) of 24 μM, whereas the affinity of the equivalent SKIPWE peptide was above 110 μM (Fig. 1C). The presence of both motifs improves the affinity for KLC2TPR because a 40-amino-acid-long peptide SKIPWDWE that encompasses the W-acid pair sequence bound with an apparent affinity higher than that of the single SKIPWD motif (Kd = 4.9 μM). Binding affinity measurements at varying NaCl concentrations showed that electrostatic interactions play an important role in the recognition process (fig. S1).

For structural studies, we focused on the SKIPWD motif, and to facilitate the crystallization process, we engineered a chimeric construct in which the SKIPWD peptide was fused N-terminal to KLC2TPR via a flexible (TGS)4 linker (18). Crystals of SKIPWD-KLC2TPR were grown by using vapor diffusion techniques and diffracted at 2.9 Å by using synchrotron radiation. The final model of the cargo complex is characterized by R and Rfree values of 20.3 and 24.5%, respectively (table S1). KLC2TPR consists of six TPR repeats (TPR1 to TPR6), each contributed by a classical helix-turn-helix structural motif arranged in a right-handed super-helical conformation, with a non-TPR helix positioned between TPR5 and TPR6 (14). The structure of SKIPWD-KLC2TPR revealed the cargo peptide bound in an extended conformation at the N-terminal portion of the KLC2TPR concave surface, with a direction parallel to the external helix of the repeat (Fig. 2, A and B, and fig. S2). Although our construct includes the external helix of TPR1, this region, as well as other flexible stretches, could not be unambiguously interpreted in the electron density. Thus, KLC2TPR starts from TPR2 in our model.

Fig. 2 Structure of the SKIPWD-KLC2TPR cargo complex.

(A and B) Illustrated representations of the SKIPWD motif (displayed as stick model in green) bound to KLC2TPR domain (orange) in two orthogonal orientations. Simulated annealing (Fo-Fc) omit map for the W-acidic cargo motif is contoured at the 3σ level. Individual TPR repeats composed by helix-turn-helix elements (“I” and “E” for internal and external, respectively) are highlighted in (A). A non-TPR helix (αN) is between TPR5 and TPR6. (B) also shows the cargo-free KLC2TPR structure (gray transparent), with an orange arrow indicating the movement of the TPR2-TPR3 region with respect to the common TPR4-TPR6 reference frame. (C) Electrostatic potential surface representation of KLC2TPR with its SKIPWD-bound cargo. Positive and negative potential is shown in blue and red, respectively. Cargo recognition is achieved by a combination of charge complementarity and sequence specificity.

A comparison between cargo-free KLC2TPR (3CEQ) and cargo-bound KLC2TPR revealed structural differences considered to arise from a rigid jaw movement of the N-terminal TPR2-3 region, which closes upon cargo recognition engendering the binding surface and pockets for the SKIPWD peptide. (Fig. 2B and fig. S3). An analysis performed by using the Protein Interfaces, Surfaces and Assemblies (PISA) algorithm (19) indicates that all residues of the SKIPWD peptide are involved in formation of the complex, which is stabilized by residues from TPR2-TPR3 and the internal helix of TPR4 (Fig. 2C). This concave groove surface displays a positive electrostatic charge ideally poised to complement the negatively charged W-acidic cargo motifs (Fig. 2C and fig. S4). Overall, the interface area of the SKIPWD-KLC2TPR complex is ~770 Å2, stabilized by a mixture of H-bonds, salt bridges, and hydrophobic interactions (Fig. 3A).

Fig. 3 The SKIPWD-KLC2TPR interface and the effect of KLC2TPR mutations in cargo binding and cellular recruitment.

(A) Details of the SKIPWD:KLC2TPR interface. KLCTPR side chains stabilizing the SKIPWD cargo peptide (green) are shown as gray sticks emanating from the orange model. Noncarbon elements are nitrogen, dark blue; oxygen, red; and sulfur, yellow. Hydrogen bonds are represented by dotted light blue lines. (B) Coimmunoprecipitation assay showing the effect of KLC2TPR mutations at the SKIPWD:KLC2TPR interface on the interaction. (C) Fluorescence polarization measurements showing that R251D, N287L, and R312E mutations in KLC2 dramatically reduce the affinity of the TPR domain for the SKIPWD peptide. Kd values reported here were calculated at 150 mM NaCl. (D) Replacement of key KLC2TPR residues results in loss of GFP-KLC2 association with Arl8/SKIP–positive lysosomal membranes. Scale bar, 10 μm. In merge panels, GFP-KLC2, Arl8, and SKIP are shown in green, red, and blue, respectively.

The W residue central to the motif is generally flanked by amino acids bearing a carboxylate side chain. SKIPW207 (position 0, p0) is buried within a leucine-rich pocket positioned roughly in the middle along the TPR length and contributed by side chains from TPR2 (L248, R251, and L263) and TPR3 (N287, L290, and L291). In particular, the side-chain of N287 is in a conformation so that it serves the dual purpose of stabilizing SKIPW207 indole group by lining one side of the pocket while engaging at the same time in hydrogen bonds with the main chain amide and carbonyl oxygen of the residue at p+1. The latter position is almost invariably occupied by a glutamic or aspartic acid residue (7). The carboxylate side-chain of SKIPD208 points in the opposite direction to that of SKIPW207, engaging in a network of salt bridges and H-bonds with the positively charged R312 and K325 side chains of α3E and α4I, respectively. An acidic side chain at position p-1 (SKIPE206) of the motif is also very conserved in other W-acidic motifs. Like SKIPD208, the carboxylate side-chain of SKIPE206 faces the TPR3-TPR4 side of the KLC2TPR recognition groove, where it is stabilized by an ionic interaction with K325. Position p-2 of SKIPWD features a leucine residue (SKIPL205). The hydrophobic side chain of SKIPL205 is deeply buried in a hydrophobic pocket formed by residues from TPR3 and TPR4. The side chain of N329 plays a similar dual role as that of N287. Although lining the SKIPL205 pocket, it also H-bonds with the main chain at position p-1. Together, N287 and N329 act like a clamp on opposite sides of SKIPWD, providing 4 of the 10 hydrogen bonds that stabilize the complex. Residues at positions p-(3,4) are less important for complex stability consistent with their general lack of sequence conservation amongst W-acidic motif cargo (7). The C-terminal stretch of SKIPWD encompassing p+(2,3,4,5) is observed in a more compact, turn-like conformation, with SKIPD209,S210 at p+(2,3) making very minor contacts with the groove. As for the N-terminal peripheral region of SKIPWD, the exact nature of the amino acids at p+(4,5) does not seem critical for complex stability. Alternative side chains can be positioned at this topological position, possibly involving a rearrangement of the main chain.

To investigate the effect of amino acid replacements within the KLC2TPR region on the interaction with SKIP, we used immunoprecipitation as described above. All amino acid substitutions tested resulted in abrogation or near-abrogation of complex formation between KLC2 and SKIP (Fig. 3, B and C). The importance of electrostatic interactions in cargo recognition is underscored by charge-reversal mutations (Fig. 3, B and C). When coexpressed in cells, SKIP and its small guanosine triphosphatase–binding partner Arl8 associate strongly with lysosomes and promote their trafficking to the cell periphery. GFP-KLC2 associates with the same lysosomes (Fig. 3D and fig. S5) (13). This is essentially abolished by KLC2 mutations (R251D, N287L, and R312E) (Fig. 3D and fig. S5). The same mutations strongly inhibited the binding of KLC2TPR to the WD motif peptide (Fig. 3C). We conclude that optimal SKIP:KLC2 stability critically depends on a very conserved cargo recognition-interaction groove. We extended our analysis to the well-characterized Calsyntenin-1 (CSTN-1) cargo, which also exhibits two W-acidic motifs (9, 11, 12). Differently from SKIP, both CSTN-1 motifs bound to KLC2TPR with similar affinity (fig. S6). However, the mutations in the KLC2-binding groove that disrupted SKIPWD binding also abrogated recognition of CSTN-1 (fig. S6). A sequence alignment shows total conservation of the KLCTPR residues interacting with the cargo across the kinesin light chain family (fig. S7). Thus, the concave groove within the TPR domain where the SKIPWD peptide binds is likely to be the primary site of interaction for W-acidic cargo motifs in general. However, we cannot exclude that secondary sites also exist.

In the context of intracellular transport in which kinesin-1 functions as a tetramer containing two KLCs held together by a coiled-coil region and both SKIP motifs contribute to transport (7, 13), it is tempting to speculate that both chains can contribute to the binding of the W-acidic cargo pair. For SKIP, the higher-affinity WD motif would direct the first binding event to one of the KLCTPR, with avidity effect promoting the association of the WE motif to the other KLC (20, 21). It will be important to determine this relationship to the kinesin-1 tetramer and examine the importance of cargo-induced TPR conformational change in motor activation.

Supplementary Materials

www.sciencemag.org/cgi/content/full/science.1234264/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S7

Table S1

References (2233)

References and Notes

  1. Acknowledgments: Scientists of I24 beamline (Diamond Light Source, Didcot, UK) are gratefully acknowledged for their support during data collection. ARL8b-HA and myc-SKIP were a kind gift from S. Munro, (Laboratory of Molecular Biology–Medical Research Council, Cambridge). We thank T. Blundell and R. Nookala (University of Cambridge) for useful discussions. Coordinates and structure factor files for have been deposited in the Protein Data Bank under accession number 3ZFW. The authors declare no competing financial interests. M.P.D and A.L. are supported by a Wellcome Trust Research Career Development Fellowship to M.P.D. and a London Law Trust Medal Fellowship to M.P.D. S.P. is supported by a British Heart Foundation grant awarded to R.A.S.
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