(471) Implications of nanobody-mediated inhibition of the Pseudomonas virulence factor Cif

Background: Cif is a Pseudomonas aeruginosa epoxide hydrolase that mediates airway colonization [1] whose presence correlates with more severe obstructive lung disease [2]. By diverting recycling CFTR towards degradation pathways [3], Cif has the potential to attenuate the benefit of therapies that restore CFTR-mediated airway homeostasis. Cif also favors a hyperinflammatory environment by destroying a pro-resolving signaling intermediate [2]. A collaboration has led to the development of high-affinity a-Cif nanobodies and a high-throughput displacement assay that pits these nanobodies against inhibitors [4]. Using X-ray crystallography, we sought to characterize these Cif:nanobody interactions.

Methods: a-Cif nanobodies were crystallized, and also co-crystallized with Cif, using vapor diffusion in hanging drops. Oscillation X-ray diffraction data were collected at NSLSII and reduced using XDS. Initial phase estimates were obtained by molecular replacement and structure refinement was performed in PHENIX with manual rebuilding. Refined structures were superimposed by least-squares fitting to characterize interactions.

Results: Our structures of Cif co-crystallized with the nanobodies reveal two distinct modes of inhibition. Competitive nanobodies favor a mechanism where a hydrophobic sidechain is placed in the active site and overlaps with the steric volume of inhibitors. The non-competitive nanobody binds at the periphery of Cif, and interactions between this nanobody and Cif induce a mainchain shift of a12 towards the active site. Both competitive and non-competitive nanobodies can simultaneously bind to Cif. In this triple complex, the structure of Cif is reminiscent of Cif co-crystallized with competitive nanobodies.

Conclusions: The structure of Cif co-crystallized with competitive nanobodies validate the steric basis of the nanobody displacement assay. The interactions of Cif with the competitive nanobodies suggest that a nanobody-derived peptide inhibitor may be feasible. The structure of Cif co-crystallized with Nb219 and Nb108 unequivocally demonstrates competitive and non-competitive inhibition. Furthermore, steric coupling between residues near the active site and a12 provide evidence for a proposed nanobody-induced allosteric shift that restricts necessary conformational changes during substrate and inhibitor binding.

Acknowledgements: This work was made possible by the following funding sources: R01-AI091699, P20-GM113132, P30-DK117469, STANTO19R0, T32-AI007519 and the staff at NSLSII.

References: 1. Hvorecny KL, Dolben E, Moreau-Marquis S, Hampton TH, Shabaneh TB, Flitter BA, et al. An epoxide hydrolase secreted by Pseudomonas aeruginosa decreases mucociliary transport and hinders bacterial clearance from the lung. Am J Physiol Lung Cell Mol Physiol. 2018 01;314(1):L150–6.

2. Flitter BA, Hvorecny KL, Ono E, Eddens T, Yang J, Kwak DH, et al. Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators. Proc Natl Acad Sci USA. 2017 03;114(1):136–41.

3. Bomberger JM, Ye S, MacEachran DP, Koeppen K, Barnaby RL, O’Toole GA, et al. A Pseudomonas aeruginosa Toxin that Hijacks the Host Ubiquitin Proteolytic System. PLoS Pathog [Internet]. 2011 Mar 24 [cited 2020 Jul 8];7(3).

4. Vasylieva N, Kitamura S, Dong J, Barnych B, Hvorecny KL, Madden DR, et al. Nanobody-based binding assay for the discovery of potent inhibitors of CFTR inhibitory factor (Cif). Analytica Chimica Acta. 2019 Jan;S0003267019300273.