The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor

Abstract

Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ~34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.

Keywords: Centriole; Centrosome; Cytoskeleton; Dynein; Isothermal Titration Calorimetry (ITC); Protein Complex; Protein Structure; Structural Biology.

FIGURE 1.

Ana2 contains two conserved LC8 binding sites. A, the nascent centriole cartwheel with mapped components. The precise location of Ana2 on the cartwheel remains unknown, but it is known to bind the Sas-6 N-terminal region (12) and the Sas-4 C-terminal region (13). B, a conserved set of proteins drive centriole duplication. Conserved centriole duplication pathway components from D. melanogaster (D.m.), Homo sapiens (H.s.), and C. elegans (C.e.) are presented with orthologous proteins listed in the same row. Drosophila Asl (Asterless) recruits SAK/Plk4 (Polo-like kinase-4) to the site of nascent centriole formation via a direct interaction (50, 51), where it phosphorylates both a known and unknown set of substrates in the centriole duplication pathway (7, 52). Sas-6 (spindle assembly abnormal-6) oligomerizes to form the first structure observed using electron microscopy; this nine-spoked cartwheel is depicted on the left. In cells, Sas-6 oligomerization is Ana2 (anastral spindle-2)-dependent (9, 12, 17). Sas-4 (spindle assembly abnormal-4) is thought to recruit triplet microtubule blades and stabilize centriole elongation and maturation (mature centriole shown at the left) (5, 13, 14). C, comparison of Homo sapiens, D. rerio (D.r.), D. melanogaster, and C. elegans Ana2 orthologs reveals diversity in protein structure. Although the lengths of Ana2 orthologs differ, the presence of a Sas-4 binding domain (red), an Sas-6 binding domain (STAN domain; gray), and a predicted central coiled-coil region remain constant (domains shown as determined previously (12, 14)). Inset, alignments of individual Sas-4 binding domains and STAN domains between D. melanogaster and H. sapiens, D. rerio, or C. elegans reveal high percentages of invariant (first value) and similar (second value) residues. D, Ana2 and LC8 form a complex with Mud to orient the mitotic spindle during asymmetric divisions in the developing Drosophila neuroblast (24). Asymmetry is achieved, in part, via differential maturation of the centrosomes. The daughter centrosome forms the LC8-Ana2-Mud complex that coordinates spindle alignment with cortical polarity cues to maintain a stem population (ganglion mother cell (GMC)) (53, 24). E, full-length Drosophila Ana2 has an N-terminal Sas-4 binding region (13, 14) and a C-terminal STAN motif (12) conserved across functional Ana2 orthologs. The central predicted helical domain is flanked by two LC8 binding sites (site 1, residues 159–168; site 2, residues 237–246). Residue identity across Drosophila species is noted below in green. F, conservation within the Ana2 central helical domain and LC8 binding sites. Residues with 100% identity are highlighted in green, whereas those with 80% similarity are highlighted in yellow. Note that both the TQT (positions 165–166) and TQC (positions 243–244) sites are conserved within the genus.

FIGURE 2.

LC8 binds two Ana2 sites with different affinities. ITC isotherms of Ana2 peptide-LC8 interactions. A, 19 × 2 μl of 60 μm Ana2 peptide 1 was injected into 200 μl of 50 μm LC8. B, 18 × 2 μl of 2 mm Ana2 peptide 2 was injected into 200 μl of 100 μm LC8. Both Ana2 peptides display exothermic binding to LC8. The thermal profiles were integrated (top panels in A and B) and fit to a one-site binding model during iterative fitting until the model best fit the data. Each experiment was run in triplicate, with the K_D reported as the average (bottom right of bottom panels) with S.D. indicated.

FIGURE 3.

Structures of LC8-Ana2 complexes reveal LC8 homodimers bound to two parallel Ana2 peptides. A, LC8-Ana2 complex structures were determined using peptide-free LC8 search models. Initial F_o − F_c electron density for the Ana2 peptides is shown in green and contoured at 2.0σ (pep1) and 1.65σ (pep2). Final 2F_o − F_c electron density is shown below in gray with the final Ana2 pep1 and pep2 model included; electron density is contoured at 2.0 σ (pep1) and 1.0 σ (pep2). Final models of the respective LC8-Ana2 peptide complexes are presented in the top left (LC8-Ana2 pep1) and top right (LC8-Ana2 pep2) with peptides in the same orientation for reference. B and C, the final structures of LC8 bound to Ana2 pep1 (orange; B) and Ana2 pep2 (cyan; C) are shown looking down the complex's 2-fold axis (left) and after a 90° rotation about the y axis (right). The center schematic in B and C summarizes the secondary structure elements that comprise a single β-sheet in the LC8-peptide complexes. Each β-sheet is extended by the third β-strand contributed by the LC8 homodimeric mate (purple) as well as the bound Ana2 peptide (pep1 shown in orange (B); pep2 shown in cyan (D)). The final β-sheet comprises a total of six strands and is flanked by two α-helices (shown in mint, behind the sheet).

FIGURE 4.

Ana2 LC8-binding sites 1 and 2 employ both shared and unique LC8-binding determinants. A, interaction matrix displaying contacts between the LC8 homodimer (y axis) and Ana2 pep1 (orange; top x axis) or Ana2 pep2 (cyan, bottom x axis). Interactions are presented where atoms are less than or equal to 3.5 Å apart (hydrogen bonds and electrostatic interactions; shown in red for pep1 and pink for pep2) and 4.5 Å apart (van der Waals contacts; shown in dark gray for pep1 and light gray for pep2). Boxes completely filled in reflect similar LC8 interaction modes with each peptide, whereas those boxes that are half-filled indicate unique, peptide-specific interactions. B, conserved Gln¹⁶⁵ of Ana2 pep1 forms hydrogen bonds to LC8′ residues Glu³⁵′ and Lys³⁶′. C, Cys²⁴⁴ of Ana2 pep2 forms an electrostatic interaction with LC8 residue Arg⁶⁰. D, the Ana2 pep2 (cyan) C-terminal region forms extensive backbone hydrogen bonds with LC8 and is positioned differently than Ana2 pep1 (orange), which has been overlaid on the LC8-Ana2 pep2 structure for comparative purposes. In contrast to the Ana2 pep2 Cys²⁴⁴ backbone carbonyl and the Ile²⁴⁶ backbone amide that interact with LC8 Phe⁶² and Arg⁶⁰, respectively, the comparable Ana2 pep1 determinants (indicated with magenta arrows) are splayed and rotated away from LC8.

FIGURE 5.

The two LC8 binding sites of Ana2 differentially bind LC8. A, a comparison of the LC8 target-binding site among the apo, Ana2 pep1-bound, and Ana2 pep2-bound LC8 structures. Several LC8 residues within the binding pocket show conformational change upon binding peptides and are colored red: Asn¹⁰, Tyr⁶⁵, Thr⁶⁷, Phe⁷³, Tyr⁷⁵, Tyr⁷⁷, and Lys³⁶′. B, comparative panel showing the positioning of other peptides bound to Drosophila LC8: Nek9 (Protein Data Bank entry 3ZKE) (47), DIC (Protein Data Bank entry 2PG1) (36), and Pak1 (Protein Data Bank entry 3DVP) (48). C, alignment of Ana2 peptides with Nek9, DIC, and Pak1 as well as the canonical binding motifs G⁻²I⁻¹Q⁰V¹D² and K⁻³X⁻²T⁻¹Q⁰T¹. Conservation is shown in yellow, contoured to ≥70% similarity. D, Ana2 peptides 1 and 2 superimposed after aligning their respective, bound LC8 homodimers (not shown), viewed in two orientations. E, comparisons of the Ana2 peptides with Nek9 (periwinkle), DIC (lime), and Pak1 (salmon) peptides show that although relative positions of the side chains are conserved, the Ana2 pep2 C terminus uniquely bends toward the LC8 homodimer. Top, stick diagram; bottom, Cα trace. Measurements of the pep2 backbone show a 3.5 and 5.6-Å positional shift at the +1 and +2 Cα positions, respectively, for Ana2 pep2 versus Ana2 pep1. F, zoom view of the peptides at position +1 reveals the mechanism of the Ana2 pep2 Cys²⁴⁴ shift; the same position usually occupied by a +1-position threonine side chain hydroxyl is instead occupied by the Ana2 Cys²⁴⁴ backbone carbonyl group. This effectively positions the peptide deeper into the LC8 binding pocket.

FIGURE 6.

SEC-MALS of Ana2M co-purified with LC8 shows a stable complex corresponding to LC8₈-Ana2M₄. Purified Ana2M (residues 156–251, 11 kDa) remained soluble only when co-purified with excess LC8 and behaved as a single species throughout the purification, which included affinity tag chromatography followed by two sizing columns. A, detection of the LC8-Ana2M complex on a sizing column coupled with multiangle static light scattering shows a single peak (pink trace; Rayleigh ratio) at 117 ± 5.9 kDa (red; molecular weight measurement). The same experiments with a Q⁰T¹ to A⁰A¹ mutation show a single peak (light blue trace; Rayleigh ratio) at 84.8 ± 2.5 kDa (dark blue trace; molecular weight measurement). LC8 alone elutes as a dimer with a mass of 21.6 kDa (dark green trace; molecular weight measurement). B, SNAP-LC8 elutes as a single species (light green trace; Rayleigh ratio) at 59.3 kDa (dark green traces), corresponding to a dimer. Co-purification of SNAP-LC8 with Ana2M yielded a complex that eluted from the size exclusion column in a broad peak, with a shoulder characteristic of complex dissociation (light purple trace, Rayleigh ration). Experimentally determined molecular weight across the broad peak indicated complexes of varying size, ranging from 290 to 150 kDa (dark purple traces; different parts of the peak were integrated to determine the contributing sizes). All experiments are consistent with the formation of a stable LC8₈-Ana2M₄ complex.

FIGURE 7.

A proposed model of LC8-mediated Ana2 oligomerization. Our data indicate the formation of an LC8₈-Ana2₄ complex, which may have implications for the role of Ana2 in centriole duplication by clustering multiple Sas-4-binding (red ellipses) and Sas-6-binding (gray ellipses) domains. Each LC8 homodimer locally mediates parallel dimerization of Ana2. The model, as presented, portrays the central, predicted α-helix as a tetramerization domain. Whether this domain forms a tetrameric four-helix bundle remains to be determined, but it is presented as a parallel four-helix bundle (above) and an antiparallel four-helix bundle (below).