Nucleotide-induced conformational changes in CtRoco

(A) Schematic representation of the domain arrangement of CtRoco (top), and its anticipated conformational cycle linked to GTP binding and hydrolysis (bottom) (Deyaert, Wauters, et al., 2017). (B) The previously published crystal structure of nucleotide-free CtRoco showing the protein as a compact homodimer, where the LRR domains fold back on the Roc-COR domains (PDB: 6hlu) (Deyaert et al., 2019). (C) NbRoco1 and NbRoco2 specifically bind to the CtRoco monomer in its “GTP state” (top) (Leemans et al., 2020). The cryo-EM map of GTPγS-bound full-length CtRoco in complex with NbRoco1 and NbRoco2 at 7.7 Å resolution shows an elongated monomeric arrangement (bottom-left). A composite map, obtained by superposing the individual cryo-EM maps focused on the C-terminal (3.9 Å) and N-terminal (3.6 Å) part of the protein, was generated (bottom-right). This map is colored according to the corresponding domains and Nbs. (D) Final model of CtRoco-NbRoco1-NbRoco2 based on the maps shown in (C).

Cryo-EM data collection, refinement and validation statistics

The structural changes occurring upon GTP-driven CtRoco activation.

(A) Superposition of a subunit of the nucleotide-free CtRoco dimer (labeled “NF”; beige) and the GTPψS-bound monomeric CtRoco-NbRoco1-NbRoco2 (labeled “GTP”, colored by domain). The Roc domains of both structures were used for the superposition. Nbs were removed in the representation of CtRoco-Nb Roco1-NbRoco2 for clarity. The conformational change occurring in the LRR domain and α0-helix is indicated by an arrow. (B) Close-up view on the displacement of the LRR, α0-helix and CORA upon CtRoco activation. The position of CORA in the “GTP” conformation is incompatible with the position of the α0-helix in the nucleotide-free conformation, providing a mechanism to relay conformational changes from Roc and CORA toward the α0-helix and LRR domain. The LRR, α0 and CORA of nucleotide-free and GTPψS-bound CtRoco are colored in different shades of yellow, orange and blue, respectively (C) Mesh representation of the cryo-EM map around the Switch 2 region and the preceding β-strand in the GTPψS-bound Roc domain. (D) Superposition of Roc domains and surrounding regions of nucleotide-free (beige) and GTPψS-bound CtRoco (colored according to domain). The conformation of the Switch 2 region in GTPψS-bound CtRoco would sterically clash with the “dimerization loop” of the adjacent protomer of the nucleotide-free dimer, providing an initial trigger for nucleotide-induced monomerization. (E) Upon activation, the CORB domain of GTPψS-bound CtRoco (blue) displays a 30° rotational movement with regard to corresponding domain of nucleotide-free CtRoco (beige). The H554-Y558-Y804 triad (corresponding to N1437-R1441-Y1699 in LRRK2), located at the Roc-CORB interface, acts as a hinge point for this rotational movement.

Mechanisms of the allosteric activation of CtRoco by NbRoco1 and NbRoco2.

(A) NbRoco1 binds at the interface of the Roc, CORA and LRR domains, close to the α0-helix and the hinge region of the LRR movement. (B) Superposition of NbRoco1 on the conformation adopted by the nucleotide-free CtRoco shows considerable sterical clashes between the CDR3 region of NbRoco1 and the LRR domain and α0-helix of nucleotide-free CtRoco (see inset). This provides a mechanism for the NbRoco1-mediated stabilization of CtRoco in its GTP-bound active conformation. (C) Superposition of NbRoco2 on the conformation adopted by the nucleotide-free CtRoco shows that the position of NbRoco2 in the curvature of the LRR domain is not compatible with the closed conformation of CtRoco, due to severe sterical clashes with the CORB domain.

Cross-linking mass spectrometry (CX-MS) experiments show the stabilization of GTPψS-bound CtRoco in its elongated active conformation by NbRoco1 and/or NbRoco2.

(A) Intramolecular DSSO-crosslinks within GTPψS-bound CtRoco in the absence of nanobodies. (B) Effect of NbRoco1 on the intramolecular DSSO-crosslinks within GTPψS-bound CtRoco. The intermolecular cross-links between CtRoco and NbRoco1 are also shown. (C) Effect of NbRoco2 on the intramolecular DSSO-crosslinks within GTPψS-bound CtRoco. The intermolecular cross-links between CtRoco and NbRoco2 are also shown. (D) Effect of the combination of NbRoco1 and NbRoco2 on the intramolecular DSSO-crosslinks within GTPψS-bound CtRoco. The intermolecular cross-links between CtRoco, NbRoco1 and NbRoco2 are also shown.

NbRoco1 and NbRoco2 have a synergistic effect on CtRoco activation that is potentiated by the bi-valent nanobody construct NbR2-R1.

(A) NbRoco2 enables the binding of NbRoco1 to the nucleotide-free CtRoco. The fluorescence anisotropy signal of the FITC-labelled NbRoco1 is monitored upon titration with increasing concentrations of nucleotide-free CtRoco in presence of an excess of NbRoco2. The equilibrium dissociation constant (Kd ± standard error), obtained by fitting of triplicated data with a quadratic binding equation, is given. (B) NbR2-R1 has a 3-fold increased affinity towards nucleotide-free CtRoco in comparison to NbRoco2. FITC-labelled NbR2-R1 or NbRoco2 were titrated with nucleotide-free CtRoco, and fluorescence anisotropy data was analyzed as in (A). (C) Single turn-over GTP (5µM) hydrolysis rate of CtRoco-L487A (5 µM) in absence of nanobodies (orange), or in presence of either NbRoco1 (blue) or both NbRoco1 and NbRoco2 (green). (D) Single turn-over GTP (5 µM) hydrolysis of CtRoco-L487A (5 µM) in presence of Nb R2-R1, all GTP was converted within the dead time (20s) of the experiment.

Similarities in the activation mechanisms of CtRoco and LRRK2.

Comparison of the conformation of the LRR domain and the α0-helix adopted within GTPγS-bound CtRoco-NbRoco1-NbRoco2 (this work, colored by domain), nucleotide-free CtRoco (beige) (PDB: 6hlu) (Deyaert et al., 2019), and GDP-bound LRRK2 (green) (PDB: 7lhw) (Myasnikov et al., 2021). The Roc domain of the three structures was used for the superposition.

Cryo-electron microscopy (Cryo-EM) workflow to obtain the maps corresponding to CtRoco-NbRoco1-NbRoco2.

Flow chart of the cryo-EM data processing, including 2D and 3D classifications, focused map strategies and density map reconstructions. Local resolution maps, FSC curves, and cryo-EM density maps are also shown. Unless otherwise stated, all analyses were performed using cryoSPARC v3.3. Details are provided in the Methods section.

Cryo-electron microscopy (Cryo-EM) workflow to obtain the map corresponding to CtRoco-NbRoco1.

Flow chart of the cryo-EM data processing, including 2D and 3D classifications and density map reconstructions. Local resolution map, FSC curve, and cryo-EM density map are also shown. Unless otherwise stated, all analyses were performed using cryoSPARC v3.3. Details are provided in the Methods section.

2D classification of CtRoco-NbRoco1-NbRoco2 (representative classes).

(A) 2D classes from particles extracted in a box size of 384 Å. Only some of these classes display the full particle, most are centered on one half of the protein showing blurred density for the rest of the particle. (B) Selected classes displaying the pull particle. (C) 2D classes from particles selected for a focused map centered on the LRR domain, extracted in a box size of 243 Å. Blurred density for the rest of the particle is also visible. (D) 2D classes from particles selected for a focused map centered on the Roc-COR domains, extracted in a box size of 243 Å. Blurred density for the rest of the particle is also visible.

Quality of the cryo-EM map of CtRoco-NbRoco1-NbRoco2.

(A) Map focused on the N-terminal (LRR) domain in complex with NbRoco2. The inset shows a zoom-in on the NbRoco2 binding region. (B) Mesh representation of the cryo-EM map around the nucleotide-binding region of the Roc domain (left) and around the bound GTPγS molecule (right). (C) Cryo-EM map around NbRoco1 in two orientations. The CDR loops are highlighted in brown (CDR1), green (CDR2) and magenta (CDR3).

Fourier shell correlation (FSC) plots of refined atomic models against the focused cryo-EM density maps of CtRoco-NbRoco1-NbRoco2.

(A) FSC plot of refined LRR-NbRoco2 model. The resolution at which the FSC curve drops below 0.5 is indicated. (B) FSC plot of refined Roc-COR-NbRoco1 model. The resolution at which the FSC curve drops below 0.5 is indicated.

The rotational movement of the CORB domain upon GTP-driven CtRoco activation induces monomerization.

Superposition (on the Roc domain) of a GTPψS-bound CtRoco monomer (“GTP”; colored by domain) on the nucleotide-free CtRoco dimer (“NF”, beige). Only the CORB domain of the superposed subunit of nucleotide-free CtRoco is shown for clarity. The rotational movement of the CORB domain upon GTPψS binding would cause sterical clashes with the Roc domain of the adjacent protomer of nucleotide-free CtRoco (brick red), providing a mechanism for monomerization.