Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B

2022-05-272022-05-272020Monaco, S., Walpole, S., Doukani, H., Nepravishta, R., Martínez Bailén, M., Carmona Asenjo, A.T.,...,Angulo Álvarez, J. (2020). Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin B. Chemistry-A European Journal, 26 (44), 10024-10034.0947-65391521-3765https://hdl.handle.net/11441/133794Ligand-based NMRtechniquesto studyprotein–ligandinteractionsare potenttoolsin drugdesign.Satura-tiontransfer difference (STD)NMRspectroscopystandsoutas one of the mostversatiletechniques,allowing screeningof fragmentslibraries andproviding structural informationon bindingmodes.Recently,ithas beenshown that amulti-frequencySTD NMRapproach, differential epitopemapping(DEEP)-STDNMR,can provideadditionalinformationon theorientationof smallligandswithinthe bindingpocket.Here,the approachis extendedtoaso-calledDEEP-STDNMRfin-gerprintingtechniqueto explorethe bindingsubsitesofcholeratoxinsubunitB(CTB).To thataim,the synthesis of aset of newligandsispresented, whichhavebeensubjecttoathoroughstudy of theirinteractionswithCTB by weakaf-finitychromatography(WAC)andNMRspectroscopy.Re-markably,the combination of DEEP-STDNMRfingerprintingandHamiltonianreplicaexchangemolecular dynamicshasprovedtobean excellent approachto explore the geome-try,flexibility,and ligandoccupancy of multi-subsitebindingpockets.Inthe particularcaseof CTB,it allowed the exis-tenceof ahitherto unknownbindingsubsiteadjacent to theGM1bindingpocketto be revealed, pavingthe wayto thedesignof novelleadsfor inhibition of this relevanttoxin.application/pdf11 p.engAttribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/Exploring Multi-Subsite Binding Pockets in Proteins: DEEP-STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin Binfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccess10.1002/chem.202001723