Abstract
Glycoprotein 2 (GP2) and uromodulin (UMOD) filaments protect against gastrointestinal and urinary tract infections by acting as decoys for bacterial fimbrial lectin FimH. By combining AlphaFold2 predictions with X-ray crystallography and cryo-EM, we show that these proteins contain a bipartite decoy module whose new fold presents the high-mannose glycan recognized by FimH. The structure rationalizes UMOD mutations associated with kidney diseases and visualizes a key epitope implicated in cast nephropathy.
Highlights
Glycoprotein 2 (GP2) and UMOD are structurally related homopolymeric glycoproteins[1] (Extended Data Fig. 1a) that prevent bacterial pathogen adhesion[2,3] and are implicated in multiple pathologies of the intestine and the urinary tract, respectively[4,5]
We first expressed in mammalian cells the whole GP2 branch as well as the corresponding region of UMOD and assessed their ability to selectively capture the lectin domain of FimH (FimHL) from an Escherichia coli periplasmic extract
Molecular replacement with models generated by AlphaFold[2] allowed us to solve the structure, which was subsequently used to phase two additional crystal forms diffracting to ~1.4 Å resolution (Extended Data Figs. 3 and 4 and Supplementary Table 1)
Summary
GP2 D10C domain residues and mutations affecting the corresponding identical residues of UMOD are as follows: GP2 D61, P62, C163→UMOD D172H, P173L/R, C174R (c); GP2 R74, C285, D86, C4113, C10177→UMOD R185C/G/H/L/S, C195F/Y, D196N/Y, C223R/Y, C287F (d); GP2 P62, C163, W92, C8157, V163→UMOD P173L/R, C174R, W202C/S, C267F, V273F/L (e); GP2 C163, R94, C8157→UMOD C174R, R204G/P, C267F (f); GP2 Y164, C10177→UMOD Y274C/H, C287F (g). To gain further insights into this process, which was previously visualized only at low resolution by cryo-electron tomography[8], we reconstituted in vitro the complex between UMOD and FimHL from uropathogenic E. coli (UPEC) UTI89 and studied it by single-particle cryo-EM (Extended Data Fig. 9 and Supplementary Table 3). This yielded a map with a nominal resolution of 7.4 Å, whose comparison with that of free UMOD showed density for a single copy of FimHL bound to the D10C region that presents the N275 glycan (Fig. 2d and Supplementary Table 3). Consistent with our binding studies (Extended Data Fig. 2b), the majority of the UMOD/FimHL interface is clearly made by the decoy module; the density of the complex hints at the possibility that the C-terminal region of EGF III may contribute to the interaction with the lectin. Received: 19 August 2021; Accepted: 21 January 2022; Published online: 10 March 2022
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