Role of Coiled-Coil Registry Shifts in the Activation of Human Bicaudal D2 for Dynein Recruitment upon Cargo Binding

Abstract

Dynein adaptors such as Bicaudal D2 (BicD2) recognize cargo for dyneindependent transport, and cargo-bound adaptors are required to activate dynein for processive transport, but the mechanism of action is unknown. Here we report the X-ray structure of the cargo-binding domain of human BicD2 and investigate the structural dynamics of the coiled-coil. Our molecular dynamics simulations support the fact that BicD2 can switch from a homotypic coiled-coil registry, in which both helices of the homodimer are aligned, to an asymmetric registry, where a portion of one helix is vertically shifted, as both states are similarly stable and defined by distinct conformations of F743. The F743I variant increases dynein recruitment in the Drosophila homologue, whereas the human R747C variant causes spinal muscular atrophy. We report spontaneous registry shifts for both variants, which may be the cause for BicD2 hyperactivation and disease. We propose that a registry shift upon cargo binding may activate autoinhibited BicD2 for dynein recruitment.

Graphical Abstract

Coiled-coils are dynamic structures. Coiled-coil registry shifts, in which one helix is vertically displaced by one helical turn with respect to the other helix of the coiled-coil, have been reported for only a few proteins, including dynein,^1–7 but may potentially be an inherent property of many coiled-coil structures with important physiological functions. Cytoplasmic dynein is the predominant motor complex that mediates the transport of almost all cargo that is transported toward the minus end of microtubules.⁸ Dynein adaptor proteins such as Bicaudal D2 (BicD2)⁹ recognize cargo for transport and link it to the motor complex,^10–15 and cargo-bound adaptors are also required to activate dynein for processive transport; however, the underlying mechanism is elusive.^13,15 Full-length BicD2 forms an autoinhibited dimer,¹⁴ in which the dynein binding site¹⁰ is masked by the C-terminal cargo-binding domain (CTD).^10–19 Once cargo binds to the CTD, autoinhibition is released.^10–15 It is of note that BicD2 autoinhibition is likely compromised by a classical dominant mutation of the Drosophila bicaudal gene (F684I) that causes increased dynein recruitment to the BicD2 homolog.^16,20 This variant results in anterior patterning defects, including double-abdomen fly embryos.

BicD2-dependent transport pathways are important for signaling, for neurotransmission at synapses,²¹ and for a fundamental process in brain development.^22–25 Furthermore, mutations of human BicD2 cause a subset of cases of the neuromuscular disease spinal muscular atrophy (SMA), which is the most common genetic cause of death in infants.^26–28 A structure of the human BicD2-CTD is currently not available but would provide insights into the underlying causes for SMA. Notably, the structure of the homologous mouse (Mm) BicD1- CTD has a homotypic coiled-coil registry in which the helices of the dimer are aligned,¹⁷ whereas Drosophila (Dm) BicD-CTD features an asymmetric coiled-coil structure, where a portion of one of the helices is shifted vertically.¹⁶ It is unknown if these conformations are species-specific or whether BicD2 homologues undergo coiled-coil registry shifts in distinct functional states.

Here we use an approach that combines X-ray crystallography, molecular dynamics (MD) simulations, and circular dichroism (CD) spectroscopy to probe structural dynamics in the BicD2-CTD coiled-coil. Our data support the notion that BicD2 may undergo coiled-coil registry shifts, which may play a role in the release of BicD2 autoinhibition upon cargo binding.

Here we have determined the X-ray structure of the human BicD2-CTD (residues 715–804) at 2.0 Å resolution (R_Free =25.7%, Figure S1 and Table S1). The BicD2-CTD forms a homodimeric coiled-coil (Figure 1A,B, Table S2).²⁹ Coiled-coils are characterized by heptad repeats “abcdefg” in the protein sequence, where residues in the “a” and “d” positions are usually hydrophobic and engage in characteristic knobsinto-holes interactions at the core. Each knob residue fits into a hole composed of four residues on the opposite chain.^30,31 The human BicD2-CTD features a homotypic coiled-coil registry, where identical knob residues at “a” and “d” positions of the heptad repeat from both chains intercalate to form a layer of knobs-into-holes interactions (Figure 1A,B, Figure S2A). In comparison, the Dm BicD-CTD coiled-coil has a homotypic registry in the C-terminal half (Figure 1C, green), where the α- helices are aligned, and a heterotypic registry in the N-terminal half, where each knob residue i from one chain intercalates with a knob residue i+4 in the second chain (yellow).¹⁶

Figure 1.

Human BicD2 may undergo coiled-coil registry shifts. (A) Schematic representation of coiled-coil registries. (B) X-ray structure of human BicD2-CTD in cartoon representation, which features a homotypic registry. Knob residues in the “a” position of the heptad repeat are shown as red spheres. (C) X-ray structure of Dm BicD (PDB ID 4BL6),¹⁶ which has an asymmetric coiled-coil registry (heterotypic registry in yellow, homotypic registry in green). (D,E) Equilibrated in silico structures of human BicD2-CTD with homotypic (D) and asymmetric (E) registries at 300 K. “a” and “d” position knob residues are shown as spheres in the left and right panels, respectively. See also Figures S2 and S5.

Interestingly, in all BicD2 homologues, the coiled-coil is interrupted between “a”-position residues A740 and C754 of Hs BicD2 (Figure 1 and Figures S2 and S3), and thus residues in this region do not engage in knobs-into-holes interactions. This interruption likely serves to accommodate two bulky aromatic residues (F743, F750) at the “d” positions of the heptad repeat (Figure 2A,B). It was previously suggested that the asymmetric coiled-coil structure of Dm BicD is stabilized by specific interactions of the homologous residues F684,F691, and Y698.^16,17 Indeed, in Hs BicD2, the aromatic rings of the F743 residues are stacked face-to-face, whereas in Dm BicD, the aromatic rings of F684 are stacked edge-to-face. Furthermore, distinct interactions of F750 and Y757 stabilize the homotypic and asymmetric registries (Figure 2).

Figure 2.

Distinct orientations of key aromatic residues stabilize distinct coiled-coil registries. (A) X-ray structure of the BicD2-CTD is shown in red cartoon representation. F743, F750, and Y757 are shown in yellow stick representation. The view is rotated by 90°. (B) X-ray structure of the Dm BicD-CTD¹⁶ shown in the same representation. Homologous aromatic residues are labeled. (C,D) Equilibrated in silico structures of human BicD2-CTD with homotypic (C) and asymmetric (D) registries at 300 K. (C) Note that F743 rotates outward in ~0.3 ns of simulation. F750 and Y757 point outward into solution. (D) In the structure with an asymmetric registry, F743, F750, and Y757 are all part of the dimer interface.

The structures of Hs BicD2-CTD and Mm BicD1-CTD¹⁷ were obtained by cocrystallization with cargo, which may have stabilized the homotypic registry, whereas the Dm BicD-CTD with an asymmetric registry was crystallized in the absence of cargo.¹⁶ Therefore, we compared the relative stability of human BicD2 with distinct coiled-coil registries in the absence of cargo. An MD simulation starting from the human BicD2- CTD structure maintained a homotypic coiled-coil registry (Figure 1D). The analysis of the equilibrated structure revealed a key difference compared with the crystal structure. The F743 side chains from both monomers that interact within the hydrophobic core of the dimer in the crystal structure rotated out to be solvent-exposed within ~0.3 ns of the simulation and adopted a conformation similar to that of F750 (Figure 2C).

The conformation with an asymmetric registry was created in silico by mutating the X-ray structure of the Dm BicD-CTD¹⁶ to the protein sequence of human BicD2. Furthermore, the conformation with the heterotypic registry was created by pulling one helix of the dimer downward with respect to the other by one helical turn. The resulting structures were equilibrated using MD simulations. The registries remained stable in the simulations, and the equilibrated structures exhibited a comparable number of knobs-into-holes interactions as the crystal structures (Figure 1E and Figure S4). In conjunction with excellent structure validation results and an absence of steric clashes (Table S3), this confirms that the resulting BicD2 structures with the asymmetric and heterotypic registries are bona fide coiled-coils (Figure S5).

Because the dimer interface is mainly stabilized by hydrophobic interactions, the interface area can be an indicator of stability (Table S4). The asymmetric registry has a larger interface area (2477 Ų) compared with the homotypic (2302 Ų) registry, which partly results from the difference in the orientation of F743, F750, and Y757. Furthermore, the asymmetric registry has a larger number of salt bridges and hydrogen bonds (8/14 respectively) compared with the homotypic (7/7) registry. The calculation of the free-energy profiles for the formation of dimers with different registries indicates that the asymmetric registry has similar stability to the homotypic registry (Figure S6) and hence is a competing alternative conformation of the coiled-coil. Greater stability of conformations with the asymmetric registry, as indicated by experiments and the structural analysis above, by a few kilocalories per mole, which is similar to the typical error bars in such calculations, may still be possible. A comparison of the thermal stability of Hs BicD2-CTD (homotypic registry) and Dm BicD-CTD (asymmetric registry) by CD spectroscopy also suggests comparable stability (Figure S7A–C).

A notable difference between BicD2 conformations with different registries is that each registry is stabilized by distinct side-chain orientations of F743, F750, and Y757. In the equilibrated homotypic registry, the F743 and F750 side chains are rotated out to be solvent-exposed (Figure 2C). In contrast, in the asymmetric (Figure 2D) and heterotypic (Figure S4) registries, these side chains are rotated toward the dimer core with a stabilizing edge-to-face interaction between the two F743 side chains and the two F750 side chains. These differences suggest that F743 and F750 may be key residues involved in determining the coiled-coil registry, hence facilitating a registry shift, as previously proposed.¹⁶

Explicit evidence of the role of these residues in facilitating a registry shift was provided by simulations of the human BicD2/R747C mutant, which causes SMA.²⁷ R747 is located at the “a” position of the heptad repeat and is sandwiched between residues F743 and F750, where the coiled-coil is interrupted. In the X-ray structure of human BicD2-CTD, the positively charged arginine side chain of this residue is turned toward the surface of the coiled-coil rather than engaging in knobs-into-holes interactions at the core (Figure 3A,B). From the structural analysis, we predict that the R747C mutation destabilizes the coiled-coil. The smaller and more hydrophobic cysteine side chain likely engages in knobs-into-holes interactions at the core, resulting in an interface that would not be able to accommodate the aromatic residues and thus would destabilize the coiled-coil.

Figure 3.

R747C and F743I mutations cause registry shifts in MD simulations of human BicD2-CTD. (A) Close-up view of the X-ray structure of the human BicD2-CTD near R747 (cyan, labeled), in the same representation as in Figure 2A; “a” position residues are shown as spheres. (B) Top view. (C) Left: Cartoon representation of the equilibrated human BicD2-CTD structure with the homotypic registry, colored by residue type (blue: positively charged, red: negatively charged, green: polar, white: nonpolar). R747 was mutated to cysteine (silver spheres). Note that F743 is part of the dimer interface, whereas F750 and Y757 point outward into solution. Middle: After ~30 ns of simulation, a local registry shift to the heterotypic registry is induced, and F750 becomes part of the dimer interface. Right: After ~79 ns of simulation, F743 and F750 point outward into solution, and the registry returns to homotypic. (C) Left: F743I mutation (silver spheres) in the equilibrated structure of human BicD2-CTD with the asymmetric (heterotypic/homotypic) registry. Right: The fully heterotypic coiled-coil registry after ~92 ns of simulation is shown.

Thus we carried out MD simulations of the R747C mutant. During the simulation, a spontaneous local coiled-coil registry shift from homotypic to heterotypic was observed, with the recovery of the homotypic registry later in the simulation. For the duration of ~79 ns, the F743 side chain in either monomer remained oriented toward the hydrophobic core of the dimer, and a local registry shift around F743 from homotypic to heterotypic was induced. This was accompanied by an inward rotation of the F750 side chains. Subsequently, the F743 and F750 side chains rotated outward and became solvent-exposed, which led to the recovery of the homotypic registry in this region (Figure 3C, Figure S5). These results suggest that distinct orientations of F743 and F750 are specific to BicD2 with homotypic and heterotypic registries. Furthermore, the fact that a spontaneous local coiled-coil registry shift is observed in simulations supports the idea that human BicD2 is capable of coiled-coil registry shifts.

Simulations of the F743I mutant provided further support for this idea. The homologous F684I mutation in Drosophila leads to the increased association of Dm BicD with dynein, thereby “hyperactivating” BicD-dependent transport.¹⁶ To gaininsight into this mutation, an MD simulation for BicD2-CTD/F743I was carried out. Within ~92 ns of the simulation, the asymmetric registry changed to a fully heterotypic registry (Figure 3D and Figure S5 and Table S5). This result indicates that the registry of the coiled-coil may switch in the F684I mutant, which may be the cause of the observed stronger association of Dm BicD with dynein.

An experimental investigation of the BicD2-CTD/R747C human disease variant as well as the E774G disease variant suggests that they are, to a large degree, misfolded to random coils (Figure 4A,B, Figure S7D). This confirms that these mutations destabilize the coiled-coil (Figure 3A,B, Figure S8), possibly causing the disease. CD wavelength scans of the BicD2-CTD/R747C disease variant lack the characteristic α- helical minima at 208 and 222 nm, suggesting misfolding (Figure 4B). In comparison, CD wavelength spectra and melting curves of a conservative R747K variant are very similar to those of the wild type (T_M = 47.4 ± 2.5 °C and T_M = 46.0 ± 1.8 °C; Figure 4B, Figure S7E). Since the F743I mutation induces a spontaneous but stable registry shift in BicD2-CTD in simulations (Figure 3), we hypothesized that the resulting F743I/R747C double mutant could rescue the folding of and stabilize BicD2 in a registry-shifted conformer. Notably, CD wavelength spectra of the BicD2-CTD/F743I/R747C double mutant overlay well with the wild type, suggesting comparable α-helical content and confirming that the combination of these two variants rescues the folding of BicD2 (Figure 4C). As a control, CD wavelength spectra of the BicD2-CTD/F743I mutant were recorded, which overlay well with those of the wild type (Figure 4D). Melting curves (Figure 4E) indicate that this double variant is substantially more stable (T_M = 58.7 ± 0.6 °C) compared with the wild type (T_M = 46.0 ± 1.8 °C). The double variant also has a higher T_M compared with the F743I single variant (T_M = 51.2 ± 0.8 °C, Figure S7F). This stabilization effect of the double variant is not due to disulfide bond formation as the BicD2 sample is kept in a reduced state and thereby is possibly due to the stabilization of the mutant in a registry-shifted conformer. One of the main transport cargoes for human BicD2 is Rab6^GTP, which recruits it to Golgi-derived vesicles.²¹ In a pull-down assay with GST-tagged Rab6^GTP, a comparable amount of the BicD2-CTD wild type and the BicD2-CTD/F743I/R747C variant was pulled down, respectively, indicating that the F743I/R747C mutation does not affect the binding of cargoes (Figure 4F). To conclude, folding is fully rescued by the F743I/R747C double mutation.

Figure 4.

Human disease variants of BicD2 are misfolded. (A–D) CD wavelength spectra of wild-type BicD2-CTD at 4 °C (red) and its variants are shown. The mean residue molar ellipticity [Θ] versus the wavelength is shown. (A) BicD2-CTD/E774G at 4 °C (native) and 90 °C (random coil). (B) BicD2-CTD/R747C (0.3 mg/mL at 4 °C in purple, 1.2 mg/mL at 4 °C in black, 0.3 mg/mL at 95 °C in gray). The spectra are overlaid with the conservative R747K variant (green). (C) BicD2-CTD/F743I/R747C (overlaid with WT and R747C variant). (D) BicD2-CTD/F743I.(E) Thermal unfolding curves of BicD2-CTD/F743I/R747C (cyan) and WT (red), monitored by CD spectroscopy at 222 nm. 0 and 100% protein unfolded represent the minimum and maximum values of [Θ], respectively. Apparent melting temperatures T_M are shown. (F) A GST pull-down assay was performed with GST-tagged Rab6^GTP and BicD2-CTD as well as its F743I/R747C variant. SDS-PAGE analysis of the elution fraction is shown. The location of molar mass standards (kDa) is indicated.

We have used a combined experimental/computational approach to investigate coiled-coil registry shifts in the cargo-binding domain of BicD2. Our data suggest that bona fide coiled-coil structures with alternative registries can be formed, which likely are of comparable stability as the experimentally determined structures. It is notable that cargo-bound adaptors such as BicD2 are required to activate dynein for processive transport.^10–15 Understanding how BicD2 modulates dynein motility is important because dynein facilitates a vast number of cellular transport events that are essential for chromosome segregation, neurotransmission, signaling, mRNA transport, and brain and muscle development.^8,21–25 Together, our data support the hypothesis that human BicD2 exists in equilibrium between distinct registry-shifted conformers. It is notable that our simulations for the R747C and F743I variants result in spontaneous coiled-coil registry shifts that establish a likely molecular mechanism for such registry shifts. In BicD2 with a homotypic registry, key phenylalanine residues rotate outward, narrowing the coiled-coil and allowing the α-helices to be aligned. However, in the asymmetric registry, these bulky residues are oriented toward the core and are accommodated by a vertical displacement of one helix against the other. Thus the MD simulations establish the dependence of the registry on the conformations of the phenylalanine residues, providing experimental support for a previously proposed idea.¹⁶

We propose that BicD2 with the asymmetric registry is the autoinhibited ground state because it is likely more stable. This conformation was also observed for Dm BicD that was crystallized in the absence of cargo,¹⁶ whereas homologues with a homotypic registry were crystallized in the presence of cargo.¹⁷ Cargo binding could induce a registry shift that converts the asymmetric coiled-coil into a symmetric one. Such a registry shift could activate autoinhibited BicD2 for dynein recruitment upon cargo binding. In vivo experiments are needed to validate this hypothesis.

Our results suggest that the BicD2-CTD/R747C and E774G disease mutations^26,27 lead to misfolding, which remains to be confirmed in the context of full-length BicD2. Whereas the MD simulations do not capture the misfolded state of BicD2- CTD/R747C due to the long time scales involved in forming such a state, they do indicate the instability of the R747C mutant relative to the wild type through spontaneous and transient registry shifts, which can be the cause for misfolding. It is notable that this is the first time that a human disease mutation has been linked to a coiled-coil registry shift. Previously, cargo-binding defects were reported for these mutations.^17,26,27 Our data emphasize that these mutations rather tamper with the coiled-coil structure because both folding and cargo binding are rescued when the R747C mutation is combined with a second registry-shift-inducing mutation (F743I). Whereas the recognition of the cargo Rab6^GTP is not affected by the F743I/R747C double mutation, it remains to be established whether a registry shift changes the selectivity of BicD2 toward other cargoes and whether such a mechanism regulates the cargo selection of BicD2 in vivo.

In conclusion, a registry shift facilitated by F743 and F750 could relieve the autoinhibition of BicD2 upon cargo binding and activate it for dynein recruitment. It is of note that residue F743 is mutated to isoleucine in the Drosophila homologue (F684I) in the variant that causes the bicaudal phenotype, which includes double-abdomen flies.²⁰ The mutation leads to an increased association of Dm BicD with dynein, thereby “hyperactivating” BicD-dependent transport^16,20 and linking a coiled-coil registry shift to a loss of autoinhibition. Furthermore, we provide evidence that disease-causing mutations in the BicD2 coiled-coil may alter the equilibrium between registry-shifted conformers, which we propose as a mechanism of pathogenesis that may also apply to other coiled-coils. The ability to undergo registry shifts may be an inherent property of coiled-coils. Therefore, our results provide novel insights into an important dynamic property of coiled-coils, which are one of the most common tertiary structures.

EXPERIMENTAL AND COMPUTATIONAL METHODS

For structure determination, the purified BicD2-CTD/Nup358 complex was set up for crystallization in hanging drops at 20 °C. Crystals of the human BicD2-CTD (residues 715–804) were obtained in space group C2.

MD simulations with implicit solvent were carried out using the CPU implementation of the PMEMD program in the AMBER16 package.³² The use of an implicit solvent model was justified by comprehensive comparisons of the results to those from explicit solvent simulations.