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

Description

Ligand-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.

Abstract

Ministerio de Economía y Competitividad de España. CTQ2012-31247 y CTQ2016-77270-R

Abstract

Junta de Andalucía-FQM-345

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