Oral 23rd International Society of Magnetic Resonance Conference 2023

Understanding ligand binding in a completely enclosed pocket of E. coli DsbA (#170)

BISWARANJAN MOHANTY 1 , Robert B. Fenwick 2 , Junya Yamamoto 3 , Geqing Wang 4 , Wesam S. Alwan 5 , Gaurav Sharma 5 , Martin L. Williams 5 , Olga Ilyichova 5 , Bradley C. Doak 5 , Begoña Heras 5 , Soichiro Kitazawa 3 , Ryo Kitahara 3 , Pramodh Vallurupalli 6 , Peter E. Wright 2 , Martin J. Scanlon 5
  1. THE UNIVERSITY OF SYDNEY, Darlington, NSW, Australia
  2. Department of Integrative Structural and Computational Biology, Scripps Research, 10550 North Torrey Pines Road, La Jolla, California, United States
  3. College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Japan
  4. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Bioscience, La Trobe University, Melbourne, Victoria, Australia
  5. Medicinal Chemistry & ARC Centre for Fragment-Based Design, Monash Institute of Pharmaceutical Sciences, Monash University(Parkville Campus), 381 Royal Parade, Victoria, Australia
  6. Tata Institute of Fundamental Research, 36/P, Gopanpally Village, Serilingampally Mandal, Ranga Reddy District, Hyderabad, Telangana, India

Proteins undergo conformational fluctuations to enable the binding of small ligands in the buried cavities (1). However, the detailed atomic-level characterization of how the ligand access into these cavities in proteins is often challenging. Here, we utilize relaxation dispersion, high-pressure, and HSQC-NMR to understand ligand binding in a pocket that is entirely enclosed within the protein structure of EcDsbA, a novel anti-virulence target (2). Using a chemical shift perturbation study by HSQC-NMR, we found that ligands preferably bind to the oxidized form (slow exchange on NMR chemical shift time scale with KD > 150 μM) more than the reduced EcDsbA despite the two states being structurally similar. In the oxidized and reduced DsbA, non-linear responses of 1HN and 15N chemical shifts were observed with pressurization to a similar extent at the ligand binding site, suggesting the pressure-dependent conformational changes are similar for both states. However, the relaxation dispersion NMR data identified the redox-dependent conformational exchange dynamics (μs-ms) at the active site helix near the binding site. We rationalized that this accounts for the critical binding difference in the two states, and the slow motion of the active site helix account for the slow exchange that is observed for such low-affinity compounds. Using chemical shift perturbation analysis by HSQC-NMR, we establish that the ligand-binding event into the enclosed pocket of EcDsbA is a two-step process. A fast exchange process forms an intermediate followed by a slow exchange process leading to the complex formation. Furthermore, we have demonstrated that ligands binding in the enclosed pocket inhibit the activity of EcDsbA in vitro. Since the pressure-dependence non-linear response of 1HN and 15N chemical shifts were significantly attenuated for the complex, we hypothesize that the attenuation of the active site helix conformational motion plays a role in inhibiting the EcDsbA-substrate interaction in the enzyme assay. These findings may contribute to our understanding of the importance of this internal cavity in the mechanism of DsbA and thereby help to guide the future development of more potent small molecule inhibitors of EcDsbA.

 

 

 

  1. V. A. Feher et al., Nat. Struct. Mol. Biol. 3, 516 – 521, 1996. 2. L. A. Adams et al., Angew. Chem. Int. Ed. 54, 2179 – 2184, 2015.