The Tyrosine Gate of the Bacterial Lectin FimH: A Conformational Analysis by NMR Spectroscopy and X‐ray Crystallography

Abstract

Urinary tract infections caused by uropathogenic E. coli are among the most prevalent infectious diseases. The mannose-specific lectin FimH mediates the adhesion of the bacteria to the urothelium, thus enabling host cell invasion and recurrent infections. An attractive alternative to antibiotic treatment is the development of FimH antagonists that mimic the physiological ligand. A large variety of candidate drugs have been developed and characterized by means of in vitro studies and animal models. Here we present the X-ray co-crystal structures of FimH with members of four antagonist classes. In three of these cases no structural data had previously been available. We used NMR spectroscopy to characterize FimH-antagonist interactions further by chemical shift perturbation. The analysis allowed a clear determination of the conformation of the tyrosine gate motif that is crucial for the interaction with aglycone moieties and was not obvious from X-ray structural data alone. Finally, ITC experiments provided insight into the thermodynamics of antagonist binding. In conjunction with the structural information from X-ray and NMR experiments the results provide a mechanism for the often-observed enthalpy-entropy compensation of FimH antagonists that plays a role in fine-tuning of the interaction.

KinITC—One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists

“…Modifications of the mannose moiety, known to form an extended hydrogen bond network in the deep binding pocket of FimH LD , affect the k off values (slope=0.268, R 2 =0.906) to a greater extent than those of the aglycone (slope=0.133, R 2 =0.801). In contrast, modifications of the aglycone, forming beneficial contacts with the tyrosine gate in the bound state, influence k on values (slope=−0.270, R 2 =0.943) to a larger degree than those of the mannose moiety (slope=−0.135, R 2 =0.710).…”

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI

“…As noted in prior co-crystal structures, [11a,16] the mannose ring is bound tightly in a deep hydrophilic pocket by an intricate network of hydrogen bonds between the C2, C3, C4, and C6 hydroxy groups of mannose and the main chain of F1 and side chains of N46, D54, Q133, N135, and D140. The O-linked aglycone projects toward the lid of the binding pocket and positions functional groups for favorable interactions.…”

Rational design strategies for FimH antagonists: new drugs on the horizon for urinary tract infection and Crohn’s disease

“…Ernst [86] and Lindhorst [83, 87] designed mannoside ligands that possess a squarate (cyclobutene-based) B-ring, attached at the para position of a phenyl mannoside via an amide bond. This aglycone was predicted to increase van der Waals contact within the hydrophobic groove.…”

“…There are also several reports of other biaryl aglycones that contain a spacer between the A and B phenyl rings. For example, Lindhorst created a series of photoactivatable diazo-linked A and B phenyl rings [91-92], and Ernst synthesized mannosides with amide linked A-B rings [86]. …”