Structural basis of myosin V Rab GTPase-dependent cargo recognition

Abstract

Specific recognition of the cargo that molecular motors transport or tether to cytoskeleton tracks allows them to perform precise cellular functions at particular times and positions in cells. However, very little is known about how evolution has favored conservation of functions for some isoforms, while also allowing for the generation of new recognition sites and specialized cellular functions. Here we present several crystal structures of the myosin Va or the myosin Vb globular tail domain (GTD) that gives insights into how the motor is linked to the recycling membrane compartments via Rab11 or to the melanosome membrane via recognition of the melanophilin adaptor that binds to Rab27a. The structures illustrate how the Rab11-binding site has been conserved during evolution and how divergence at another site of the GTD allows more specific interactions such as the specific recognition of melanophilin by the myosin Va isoform. With atomic structural insights, these structures also show how either the partner or the GTD structural plasticity upon association is critical for selective recruitment of the motor.

Keywords: DIL motif; GTPases; Rab effector; intracellular traffic.

Fig. 1.

Crystal structure of the Myo5 GTD. (A) Cartoon representation of the Myo5A–GTD structure, SD-1 is highlighted in blue and SD-2 in red, the intersubdomain linker in green. The main characterized binding sites for Rab11, RILPL2, MLPH, Sec15, and MD (motor domain) are indicated with arrows. (B) Superimposition of Myo5A and Myo5B GTDs. (C) Linker region between H1 and H2 helices; Myo5B (Upper) and Myo5A, which contains the H1′ helix (Lower).

Fig. 2.

Crystal structure of Rab11–GTP bound to Myo5B–GTD. (A) Overall structure of the complex. (B) The Upper diagram illustrates the real-time surface plasmon resonance (SPR) analysis of the interaction between GST–Myo5B–GTD (250 nM) and tethered 6× His–Rabs (K_dRab11GTP = 254 ± 39 nM, K_dRab11GDP = 8,500 ± 1,900 nM, K_dRab25GTP = 537 ± 66 nM, Rab14GTP binding is not detected). Fluorescence anisotropy measurements of Myo5B–GTD binding to Rab11:mantGppNHp, K_d = 620 ± 100 nM; or Rab11:mantGDP, K_d = 19,000 ± 2,000 nM are shown below. (C) Myo5B residues interacting with the active form Rab11a–GTP. The Myo5B Rab11 binding epitope is formed by two hydrophobic patches surrounded by a few polar residues. (D) Rab11–GTP residues involved in Myo5B binding. (E) Schematic view of the Rab11–GTP–Myo5B interactions. Mutations in the underlined residues result in Rab binding deficiency (9, 17, 22). Residues conserved between yeast Myo2p and vertebrate Myo5, as well as residues conserved within Rabs, are labeled in black. Hydrophobic contacts are shown as dashed lines, hydrogen bonds as double lines. The switch-1 I44 and the switch-2 Y73 of Rab11 bind to the Myo5B hydrophobic patch formed by residues of the H9 and H10 helices, whereas the other Rab residues bind to the patch formed mostly by residues of the H8 helix. The residues that do not form interactions in the Rab11–GDP to Myo5B interface are highlighted in yellow (see also Fig. S6).

Fig. 3.

Structural rearrangement of the Rab11 switches is required to bind to Myo5B. (A) Rab11 isoforms unbound structures in the GTP bound state [Protein Data Bank (PDB) codes: 1OIX, 1YZK, 2F9M, and 1OIW] exhibit well-ordered switches with a very similar conformation (pale colors). Both Rab11–GTP switches undergo significant rearrangement upon Myo5B binding (red). (B) Description of the specific interactions stabilizing the unbound active Rab11 structures. The T67 residue (red) forms an H bond with the G45 that results in a slight bulge of the switch-1 I44 residue (blue). The space between the switch-2 helix and the surface of the beta-sheet is occupied by the Y80 side chain (part of the hydrophobic triad) that forms an H bond with the carbonyl of L16. The conserved hydrophobic residues I44 and Y73 from switch 1 and switch 2 are thus spaced far apart. (C) In the Myo5B-bound Rab11–GTP structure, T67 (red) interacts with the main chain oxygen of L16. The Thr side chain thus fills the space between the beta-sheet and the switch-2 helix, pushing away the Y80 side chain and drastically changing the conformation of the hydrophobic triad (F48,W65,Y80). Interestingly, an hydrogen bond forms between the switch-1 G45 and switch-2 A68 residues maintaining the two switches close together, and this promotes the formation of a hydrophobic patch on the surface of Rab11 by aligning the conserved hydrophobic residues I44 and Y73 so that they interact with a complementary hydrophobic patch on the surface of Myo5B. The observed Rab11 conformation is similar to what is observed for most Rab GTPases in their active state to promote effector binding. It drastically differs from the previously observed conformation for active Rab11 unbound or bound to effectors (described in Fig. S5B).

Fig. 4.

Binding of MLPH to Myo5A GTD. (A) The MLPH GTBD (yellow) binding site is located within SD-1 (blue) of Myo5A GTD. (B) The Myo2p structure is not compatible with a peptide ligand binding at the same site. (C) Two MLPH hydrophobic residues, F191 and L189, are anchored into the hydrophobic cleft of the Myo5A–GTD identified between the H3 and H5 helices. In addition, the MLPH main chain is stabilized by multiple polar interactions with myosin. The side chains of E1595 from the H5 helix and R1528 from the H3 helix capture the peptide backbone from both sides by forming hydrogen bonds (see also E). The MLPH residues 187–189 and the Myo5A residues 1590–1592 form a hydrogen-bonding network corresponding to a parallel beta-structure. The side chains of positively charged residues K186 and R187 make hydrogen bonds to the L1588 carbonyl group and the N1590 and N1522 side chains, respectively. (D) The N-terminal tail of Myo5B from a symmetry-related molecule makes interactions in the hydrophobic site found between helices H3 and H5 in a manner similar to that observed in the Myo5A:MLPH complex but the directionality of the peptide bound in the cleft is opposite. (E) Structural rearrangements of the Myo5A–GTD upon MLPH binding. Local conformational changes of Myo5 residues Y1596 and R1528 are necessary to accommodate the MLPH peptide in its binding pocket. The apo–Myo5A is shown in white and the Myo5A in the complex is colored. (F) Same region as found in the Myo5B structure. Differences in the sequence of core residues in particular L1556 contribute to destabilize the Y1590 conformation necessary for MLPH binding. (G) The D1519 residue, mutated to glycine in Griscelli syndrome, stabilizes the MLPH-binding loop conformation. (H) Model of the off-state of Myo5B, indicating how each N-terminal tail of a GTD could interact with the SD-1 region (“MLPH” binding site) of the other GTD in the dimer and could stabilize the folded conformation as well as mask interaction with partners. GTD subdomains are labeled with numbers.