Structures of TOG1 and TOG2 from the human microtubule dynamics regulator CLASP1

Abstract

Tubulin-binding TOG domains are found arrayed in a number of proteins that regulate microtubule dynamics. While much is known about the structure and function of TOG domains from the XMAP215 microtubule polymerase family, less in known about the TOG domain array found in animal CLASP family members. The animal CLASP TOG array promotes microtubule pause, potentiates rescue, and limits catastrophe. How structurally distinct the TOG domains of animal CLASP are from one another, from XMAP215 family TOG domains, and whether a specific order of structurally distinct TOG domains in the TOG array is conserved across animal CLASP family members is poorly understood. We present the x-ray crystal structures of Homo sapiens (H.s.) CLASP1 TOG1 and TOG2. The structures of H.s. CLASP1 TOG1 and TOG2 are distinct from each other and from the previously determined structure of Mus musculus (M.m.) CLASP2 TOG3. Comparative analyses of CLASP family TOG domain structures determined to date across species and paralogs supports a conserved CLASP TOG array paradigm in which structurally distinct TOG domains are arrayed in a specific order. H.s. CLASP1 TOG1 bears structural similarity to the free-tubulin binding TOG domains of the XMAP215 family but lacks many of the key tubulin-binding determinants found in XMAP215 family TOG domains. This aligns with studies that report that animal CLASP family TOG1 domains cannot bind free tubulin or microtubules. In contrast, animal CLASP family TOG2 and TOG3 domains have reported microtubule-binding activity but are structurally distinct from the free-tubulin binding TOG domains of the XMAP215 family. H.s. CLASP1 TOG2 has a convex architecture, predicted to engage a hyper-curved tubulin state that may underlie its ability to limit microtubule catastrophe and promote rescue. M.m. CLASP2 TOG3 has unique structural elements in the C-terminal half of its α-solenoid domain that our modeling studies implicate in binding to laterally-associated tubulin subunits in the microtubule lattice in a mode similar to, yet distinct from those predicted for the XMAP215 family TOG4 domain. The potential ability of the animal CLASP family TOG3 domain to engage lateral tubulin subunits may underlie the microtubule rescue activity ascribed to the domain. These findings highlight the structural diversity of TOG domains within the CLASP family TOG array and provide a molecular foundation for understanding CLASP-dependent effects on microtubule dynamics.

Fig 1. H. s. CLASP1 TOG1 forms a structurally conserved α-solenoid composed of six HRs.

(A) Domain architecture of CLASP family members. H. sapiens (H.s.) CLASP1 and CLASP2, X. laevis (X.l.) Xorbit, D. melanogaster (D.m.) MAST, C. elegans (C.e.) CLS-1, and S. cerevisiae (S.c.) Stu1p. S.c. Stu1p’s two C-terminal domains are uniquely colored based on lack of homology to TOG3 and the CLIP-170 interaction domain (CLIP-ID) of animal CLASP family members. (B) Architecture of H.s. CLASP1 TOG1 shown in cartoon format. The helices of each of the six HRs (A-F) are colored across the spectrum. The image at top is rotated 90° about the x-axis to present the view shown at bottom which focuses on the intra-HR loops. N- to C-terminal directionality is shown from deep purple (N-terminus) to deep red (C-terminus). (C) Structural alignment of H.s. CLASP1 TOG1 with H.s. CLASP2 TOG1 and D.m. MAST TOG1 (PDB accession codes 5NR4 and 4G3A respectively, [25,41]).

Fig 2. H.s. CLASP1 TOG1 has a conserved face delineated by intra-HR loops.

(A) Sequence alignment of CLASP family TOG1 domains from H. sapiens (H.s.) CLASP1, X. laevis (X.l.) Xorbit, D. melanogaster (D.m.). MAST, and C. elegans (C.e.) CLS-1. H.s. CLASP1 TOG1 2° structure and solvent accessible surface area (SASA) are shown above the alignment. Rectangles represent α-helices. Residues are highlighted based on 100% identity (blue), 100% similarity (light green), and 75% similarity (orange)(see Materials and methods for amino acid similarity criteria). (B) Cross-species conservation delineated in A, mapped on the H.s. CLASP1 TOG1 structure, and rotated in 90° steps about the x-axis. The orientation at upper left corresponds to the upper orientation in Fig 1B. (C) View of the HR A loop residue V17 shown in stick format with 2mF_o-DF_c electron density shown in blue, contoured at 1.0 σ. (D) Intra-HR residues shown in stick format with conservation color-coded as in A and B.

Fig 3. H.s. CLASP1 TOG2 forms a convex α-solenoid, structurally homologous to H.s. CLASP2 TOG2.

(A) Architecture of H.s. CLASP1 TOG2 shown in cartoon format. The helices of each of the six HRs (A-F) are colored across the spectrum. The image at top is rotated 90° about the x-axis to present the view shown at bottom which focuses on the intra-HR loops. N- to C-terminal directionality across the HR α-solenoid is shown from deep purple (N-terminus) to deep red (C-terminus), with the N-terminal α2N helix colored blue. (B) Structural alignment of H.s. CLASP1 TOG2 and H.s. CLASP2 TOG2 (PDB accession code 3WOY, [44]) using the Dali server [52].

Fig 4. H.s. CLASP1 TOG2 has a conserved face delineated by intra-HR loops.

(A) Sequence alignment of CLASP family TOG2 domains from H. sapiens (H.s.) CLASP1, X. laevis (X.l.) Xorbit, D. melanogaster (D.m.) MAST, and C. elegans (C.e.). CLS-1. H.s. CLASP1 TOG2 2° structure and solvent accessible surface area (SASA) are shown above the alignment. Rectangles represent α-helices. Residues are highlighted based on 100% identity (blue), 100% similarity (light green), and 75% similarity (orange)(see Materials and methods for amino acid similarity criteria). (B) Cross-species conservation delineated in A, mapped on the H.s. CLASP1 TOG2 structure and rotated in 90° steps about the x-axis. The orientation at upper left corresponds to the upper orientation in Fig 3A. (C) View of the HR A loop residue W338 shown in stick format with 2mF_o-DF_c electron density shown in blue, contoured at 1.0 σ. (D) Intra-HR residues shown in stick format with conservation color-coded as in A and B.

Fig 5. CLASP family TOG1, TOG2, and TOG3 domains each have unique architectures.

(A) Comparison of H.s. CLASP1 TOG1, H.s. CLASP1 TOG2, and M.m. CLASP2 TOG3 (PDB accession code 3WOZ, [44]) structures using rmsd analysis of corresponding Cα atoms across the three domains (Table 2, Dali server [52]). Pairwise analysis was performed, comparing HRs A-C, D-F, and all six HRs: A-F. (B) Pairwise structural comparison of CLASP family TOGs 1–3. The H.s. CLASP1 TOG2 and M.m. CLASP2 TOG3 HR A-C triad was structurally aligned to the H.s. CLASP1 TOG1 HR A-C triad using the Dali server and respective pairwise comparisons generated [52]. Images at left are oriented with each domain’s intra-HR loops positioned at the top of the domain as shown. The images at right were produced after a 90° rotation about the x-axis and focus on the respective surfaces composed of intra-HR loops. (C) Electrostatic surface potential mapped on the structures of H.s. CLASP1 TOG1, H.s. CLASP 1 TOG2, and M.m. CLASP2 TOG3. The surface of each domain presented is the surface composed of intra-HR loops, oriented as presented in the images at right in panel B.

Fig 6. Distinct CLASP family TOG domain architectures predict distinct tubulin binding properties.

(A) CLASP family TOG domains modeled on tubulin based on the structure of the XMAP215 microtubule polymerase family member S.c. Stu2 TOG2 in complex with αβ-tubulin (PDB accession code 4U3J [37], shown at top; S.c. Stu2 TOG2 in red, α- and β-tubulin shown in wheat and lavender respectively). To generate models of CLASP family TOG domains bound to tubulin in a similar mode, the first HR triad (A-C) from each of the CLASP family TOG domain structures analyzed was structurally aligned to the S.c. Stu2 TOG2 HR A-C triad using the Dali server [52]. TOG domains at left are shown in cartoon format along with a transparent molecular envelope. The images at right were generated after a 90° rotation about the y-axis and depict each TOG domain in surface representation. The M.m. CLASP2 TOG3 structure is from PDB accession code 3WOZ [44]. (B) Comparative analysis of the intra-HR loop residues of each of the CLASP family TOG domains analyzed, which in S.c. Stu2 TOG2 are used to bind tubulin. The orientation of each domain, relative to the orientation shown in A (right panel) was generated after a 180° rotation about the y-axis, followed by a 90°Counterclockwise rotation about the z-axis.

Fig 7. M.m. CLASP2 TOG3 is predicted to engage laterally associated tubulin on the microtubule lattice.

(A) Model of M.m. CLASP2 TOG3 superpositioned on a microtubule. Shown are two laterally-associated tubulin heterodimers from neighboring protofilaments. M.m. CLASP2 TOG3 is shown in dark purple, modeled bound to the tubulin heterodimer shown at right (akin to the single-tubulin heterodimer binding mode depicted in Fig 6A). The model generated of M.m. CLASP2 TOG3 bound to free tubulin (Fig 6A) was superpositioned onto the lattice coordinates of GMPCPP-bound tubulin (PDB accession code 3JAT [53]). The tops of the β-tubulin subunits are shown (plus end oriented towards the viewer), looking into the bore of the microtubule with the luminal region oriented above and the microtubule exterior oriented below. (B) Model as shown in (A), viewed from the microtubule exterior with the plus end oriented up. β-tubulin is shown in lavender, α-tubulin is shown in wheat. Potential M.m. CLASP2 TOG3 HR B and HR D contacts with the laterally-associated tubulin subunit are demarcated with red arrows.

Fig 8. Model of CLASP family TOG domain structures and potential tubulin binding modes.

TOG domains and tubulin are colored as presented in Fig 6A. From top to bottom: H.s. CLASP1 TOG1 (orange) is architecturally similar to the TOG domains of the XMAP215 family member S.c. Stu2. While no tubulin- or microtubule-binding activity has been detected for CLASP family TOG1 domains, a role in cellular localization and autoregulation has been reported. H.s. CLASP1 TOG2 (blue) has a convex TOG architecture across its tubulin-binding surface that may be used to engage tubulin in a hypercurved state and may underlie the anti-catastrophe and rescue activity reported for the structurally similar H.s. CLASP2 TOG2 domain. M.m. CLASP2 TOG3 (purple) also has a convex architecture across its tubulin-binding surface, but is not bent as dramatically as H.s. CLASP1 TOG2. The convex M.m. CLASP2 TOG3 architecture may be used to engage a curved tubulin state. Relative to the other CLASP family TOG domains presented, the M.m. CLASP2 TOG3 domain has a unique architectural shift in the plane tangential to the microtubule surface and orthogonal to its tubulin-binding surface. This shift may enable it to engage the laterally associated tubulin subunit on the neighboring protofilament and support the reported ability of H.s. CLASP2 TOG3 to promote microtubule rescue.