Structure determination of an unstable macromolecular complex enabled by nanobody-peptide bridging

Abstract

Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non-globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross-linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.

Keywords: complement; convertase; innate immunity; macromolecular complexes; nanobody; structural biology.

FIGURE 1

Strategies for stabilization of macromolecular complexes for structure determination. (a) Affinity maturation for a target subunit by display on, for example, a yeast cell. Target variants obtained by random mutagenesis are presented on the cells. The labeled binding partner is present in the fluid phase, and cells expressing target variants with improved affinity can be isolated by flow cytometry. (b) In gradient fixation, the labile complex is loaded on a preformed glycerol/cross‐linker gradient. After centrifugation, the cross‐linked complex is recovered and the relevant fraction used for preparation of electron microscopy (EM) grids. (c) A fusion protein containing at least two components of a labile complex increases the local concentration of interaction partners and thereby increases affinity. Shown here is the fusion of the MHC binding peptide to the N‐terminal end of the T‐cell receptor β‐chain through a flexible glycine rich linker. The peptide is optimally located for binding in the peptide binding groove of the MHC receptor prior to recognition of the MHC–peptide complex by the T‐cell receptor. Drawn from PDB entry 3PL6. (d) The BC2‐based strategy developed to tether factor properdin (FP) to the C3b:factor B (FB) complex. The BC2 nanobody recognizes the BC2 tag (red) inserted at the disordered N‐terminal end of FB (yellow, residues 26–34) and is fused to hFPNb1 that recognizes TSR4 within FP. (e) Schematic illustration of the bifunctional nanobody BC2‐hFPNb1 and BC2T‐FB

FIGURE 2

The alternative pathway of complement and biochemical characterization of the factor properdin–factor B (FP–FB) linking bifunctional nanobody. (a) The C3 convertase C4b:C2a is assembled upon activation of the upstream classical and lectin pathways. C3b generated by this convertase can associate with FB and FP to form the alternative pathway (AP) proconvertase complex C3b:FB:FP. Factor D cleavage converts this to the active AP C3 convertase C3b:Bb:FP, and since this convertase turns over multiple molecules of C3 before decaying, a strong amplification of the initial C3b deposition occurs. (b,c) C3 cleavage by the convertases C3b:Bb and CVF:Bb, panels (b) and (c), respectively, was monitored to compare the activity FB and BC2T‐FB. C3 cleavage to C3a and C3b was observed for both FB variants. (d) Size exclusion chromatography (SEC) analysis of the formation of the ternary complex BC2T‐FB:FPc:BC2‐hFPNb1 (gray chromatogram) and the quaternary complex C3b:BC2T‐FB:FPc:BC2‐hFPNb1 (red chromatogram). To the right, a nonreducing SDS‐PAGE analysis of the three peaks from the SEC analysis of the C3b:BC2T‐FB:FPc:BC2‐hFPNb1 complex (red chromatogram). An SDS‐PAGE analysis of the early peak of the ternary complex BC2T‐FB:FPc:BC2‐hFPNb1 (gray chromatogram) is presented in Figure S1b, (e) Left panel, biotinylated C3b was immobilized on a streptavidin coated biosensor and washed before binding to BC2T‐FB for 300 s. The biosensors were then washed for 10 s before exposed to a mixture of FPΔ3, BC2, and hFPNb1 in a 1:1:1 M ratio. Association and dissociation was monitored for 200 s. Right panel, association and dissociation phase after subtraction of the C3b:BC2T‐FB curve. (f) BLI experiment as in panel (e) but with the bispecific BC2‐hFPNb1 nanobody instead of the two separate nanobodies

FIGURE 3

Nanobody bridging allows structure determination of the properdin (FP) bound proconvertase. (a) Crystal structure of the complex between the 175 kDa complement C3b (green), the 100 kDa BC2T‐factor B (FB) (salmon), the 46 kDa two chain FPΔ3 monomer (dark gray) and its associated 15 kDa hFPNb1 nanobody (orange). The overall dimensions of the complex are approximately 25 × 12 × 10 nm. C3b is covalently linked to the complement activating surface through a covalent bond. In the orientation shown here, the activator is at the bottom of the panel linked to the C3b thioester domain. (b) Expanded view of the region outlined in panel (a). In focus are two α‐helices in the FB von Willebrand factor A (vWA) domain that adopt different conformations in the C3b:FB:FP complex compared to the C3b:FB complex. A 2mF_o‐DF_c electron density map contoured at 1.0 σ is displayed together with the model. (c) Docking of the BC2‐BC2T complex (nanobody in blue cartoon, peptide shown as cyan spheres) in residual electron density (light green) in a position compatible with fusion of the BC2T peptide to the N‐terminal end of FB. The C‐terminal end of the BC2 nanobody is closer to a symmetry related hFPNb1 (#2) compared to the hFPNb1 (#1) bound to the same complex as the nearby FB. (d) An overlay of FP (dark gray) and the C‐terminal domain of C3b (green) from the C3b:FB:FP complex and the C3b:Bb:FP complex (orange) demonstrates that the C3b‐FP interaction is preserved between the two functional states. For simplicity, labels FB and FP are used to represent BC2T‐FB and FPΔ3 in Figures 3 and 4.

FIGURE 4

The factor B (FB) von Willebrand factor A (vWA) domain and the key role of properdin (FP) thumb and index finger loops. (a) Comparison of the vWA domain in the FP bound proconvertase (salmon) and convertase (yellow). Proconvertase cleavage by FD and release of the Ba fragment induces a rotation of the vWA domain. The structures are superimposed on the C‐terminal domain of C3b. (b) A comparison of the proconvertase in the presence and absence of FP demonstrates the rotation of the FB vWA domain induced by FP binding. The structures are superimposed on the C‐terminal domain of C3b. (c) Magnified view documenting the conformational change difference internally in the FB vWA domain between the two states of the proconvertase. The side chain of the Phe286 residue is shown in sticks, while the Mg²⁺ ion in the FB metal‐ion dependent adhesion site (MIDAS) is displayed as a yellow sphere. The structures are superimposed on their FB vWA domains. (d) From the C3b:FB:FP structure, the FP thumb and index finger loops and the C‐terminal region of C3b is displayed from a FB point of view. The Mg²⁺ ion from the FB MIDAS is indicated. (e) Close‐up of the intermolecular interface showing a direct interaction between the FB vWA domain and the FP index finger loop. (f) Likewise, the FP thumb loop forms polar interactions with both C3b and FB.