Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc

Abstract

The activity-regulated cytoskeleton-associated protein (Arc) is a complex regulator of synaptic plasticity in glutamatergic neurons. Understanding its molecular function is key to elucidate the neurobiology of memory and learning, stress regulation, and multiple neurological and psychiatric diseases. The recent development of anti-Arc nanobodies has promoted the characterization of the molecular structure and function of Arc. This study aimed to validate two anti-Arc nanobodies, E5 and H11, as selective modulators of the human Arc N-lobe (Arc-NL), a domain that mediates several molecular functions of Arc through its peptide ligand binding site. The structural characteristics of recombinant Arc-NL-nanobody complexes were solved at atomic resolution using X-ray crystallography. Both anti-Arc nanobodies bind specifically to the multi-peptide binding site of Arc-NL. Isothermal titration calorimetry showed that the Arc-NL-nanobody interactions occur at nanomolar affinity, and that the nanobodies can displace a TARPγ2-derived peptide from the binding site. Thus, both anti-Arc-NL nanobodies could be used as competitive inhibitors of endogenous Arc ligands. Differences in the CDR3 loops between the two nanobodies indicate that the spectrum of short linear motifs recognized by the Arc-NL should be expanded. We provide a robust biochemical background to support the use of anti-Arc nanobodies in attempts to target Arc-dependent synaptic plasticity. Function-blocking anti-Arc nanobodies could eventually help unravel the complex neurobiology of synaptic plasticity and allow to develop diagnostic and treatment tools.

Fig 1. Domains of full-length Arc and recombinant Arc constructs.

Full-length Arc (flArc) is constituted by two main domains: The N-terminal domain (NTD) and the C-terminal domain (CTD). Arc-NTD contains the oligomerization motif, while the Arc-CTD can be further divided into the N-lobe (NL) and the C-lobe (CL). Arc-NL contains a multi-peptide binding pocket that mediates several of the molecular functions of Arc [–41]. This study used three recombinant Arc constructs: Dimeric full-length Arc (rArcFL-7A), Arc-CTD without its C-terminal tail (Arc-CTdt) and Arc-NL. The N-terminal histidine-tags, fusion proteins and linker regions of the recombinant proteins are not shown in this figure.

Fig 2. Characterization of Arc-NL, E5 and H11.

(A) DLS analysis of Arc-NL, E5 and H11. (B) CD spectroscopy of Arc-NL, E5 and H11. (C) SRCD spectroscopy of H11 and E5. (D) SRCD spectroscopy of Arc-NL, and the Arc-nanobody complexes Arc-NL-H11 and Arc-NL-E5. (E) Thermal denaturation curves based on SRCD of H11 and E5; the analysis is based on ellipticity at 190 nm. (F) Thermal denaturation curves based on SRCD of Arc-NL and the Arc-nanobody complexes Arc-NL-H11 and Arc-NL-E5. The analysis is based on ellipticity at 208 nm.

Fig 3. Protein crystals and refined structures for Arc-NL-H11, Arc-NL-E5 and H11.

(A) Crystals of Arc-NL-H11, Arc-NL-E5 and unbound H11. (B) The structures of Arc-NL-H11 (top left), Arc-NL-E5 (top right) and unbound H11 (bottom left). The apo E5 structure (bottom right) was previously solved (PDB ID: 7R20) [40] and is shown for comparison with the Arc-NL-E5 complex.

Fig 4. Structural analysis of Arc-nanobody complexes.

(A) Both E5 and H11 bind to the multi-peptide binding pocket (orange shading) of Arc-NL. Left: An overlay of Arc-NL in both complexes. The Arc-NL-H11 complex (middle) and Arc-NL-E5 (right) show different conformations for the Arc-NL N terminus. (B) Overlay of bound and unbound H11 and E5, showing the conformational changes, especially in CDR3, that each nanobody displays when binding Arc-NL. The aromatic residue of the segment binding to the Arc-NL pocket is shown. (C) Intermolecular interactions between each nanobody and Arc-NL (stereo view). The CDR3 of both nanobodies is the main contributor to the protein complex formation. Top: Arc-NL bound to H11. P/Y: The PxY motif in H11. Bottom: Arc-NL bound to E5. P/W: The PxW motif in E5. Red asterisk: Glu²¹⁵, which forms a salt bridge in both complexes. Interactions are shown with dashed lines: Hydrogen bonds/salt bridges in yellow and C-H…π interactions in green. An animated view of both complexes can be found in S1 and S2 Movies.

Fig 5. Thermodynamic characterization of the binding dynamics between Arc-NL, TARPγ2-derived peptides, and anti-Arc-NL nanobodies.

(A) Titration of E5/H11 into Arc-NL; both nanobodies display a similar thermodynamic binding profile. Only one representative replicate of each assay is shown. (B) Titration of P1/P2 into Arc-NL; binding is selective for P1, while P2 is valid as a negative control. (C) Displacement assay for E5. An apparent reduction in K_D and ΔH can be observed. (D) Displacement assay for H11. The apparent reduction of the K_D and ΔH values is seen. (E) Multiple sequence alignment of P1 and the CDR3s of H11 and E.

Fig 6. Comparative structural analysis of Arc-NL bound to different peptides derived from endogenous Arc ligands.

The Arc N-lobe adopts a similar conformation while interacting with different ligand peptides: TARPγ2 (PDB ID: 6TNO) [39], CaMK2A (PDB ID: 4X3I) [37], GKAPR4 (PDB ID: 6TNQ) [39] and GKAPR5 (PDB ID: 6TQ0) [39]. The same conformation is present in the complex between E5 and Arc-NL.

Fig 7. Binding of two nanobodies simultaneously.

(A) Analytical SEC of Arc-CTdt with nanobodies C11 and H11 indicates simultaneous binding. (B) SDS-PAGE of purified ternary complex rArcFL-7A+E5+C11. Arc is shown with the thick arrow and the two nanobodies with thin arrows. (C) SEC-MALS of rArcFL-7A with nanobodies E5 and C11. (D) SAXS data; displaced along the y axis for clarity. (E) Zoom-in of the low-angle region indicates different shapes for the scattering curves, with an increase in size upon addition of each nanobody. The crossover point is indicated by an arrow. The SAXS curves were scaled together for the analysis. (F) Ab initio dummy atom models for Arc (gray), Arc+E5 (blue), Arc+C11 (green), Arc+E5+C11 (magenta). Apparent positions of extra density upon nanobody addition are shown with arrows.

Fig 8. Identification of a putative Arc-NL binding site on PICK1.

(A) AlphaFold2 model of the PICK1 BAR domain shows that the distal loop carries a PxY motif (pink). The side chains of the Pro and Tyr residues in the motif are shown. (B) Sequence alignment between the H11 CDR3 loop and the PxY motif of PICK1. Bold residues are identical, and the PxY motif is highlighted in red.