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Saturation transfer difference (STD) NMR spectroscopy stands out as one of the most versatile techniques, allowing screening of fragments libraries and providing structural information on binding modes. Recently, it has been shown that a multi\u2010frequency STD NMR approach, differential epitope mapping (DEEP)\u2010STD NMR, can provide additional information on the orientation of small ligands within the binding pocket. Here, the approach is extended to a so\u2010called DEEP\u2010STD NMR fingerprinting technique to explore the binding subsites of cholera toxin subunit\u2005B (CTB). To that aim, the synthesis of a set of new ligands is presented, which have been subject to a thorough study of their interactions with CTB by weak affinity chromatography (WAC) and NMR spectroscopy. Remarkably, the combination of DEEP\u2010STD NMR fingerprinting and Hamiltonian replica exchange molecular dynamics has proved to be an excellent approach to explore the geometry, flexibility, and ligand occupancy of multi\u2010subsite binding pockets. In the particular case of CTB, it allowed the existence of a hitherto unknown binding subsite adjacent to the GM1 binding pocket to be revealed, paving the way to the design of novel leads for inhibition of this relevant toxin.<\/jats:p>","DOI":"10.1002\/chem.202001723","type":"journal-article","created":{"date-parts":[[2020,5,25]],"date-time":"2020-05-25T06:35:30Z","timestamp":1590388530000},"page":"10024-10034","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Exploring Multi\u2010Subsite Binding Pockets in Proteins: DEEP\u2010STD NMR Fingerprinting and Molecular Dynamics Unveil a Cryptic Subsite at the GM1 Binding Pocket of Cholera Toxin\u2005B"],"prefix":"10.1002","volume":"26","author":[{"given":"Serena","family":"Monaco","sequence":"first","affiliation":[{"name":"School of Pharmacy University of East Anglia  Norwich Research Park NR4 7TJ Norwich UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Samuel","family":"Walpole","sequence":"additional","affiliation":[{"name":"School of Pharmacy University of East Anglia  Norwich Research Park NR4 7TJ Norwich UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hassan","family":"Doukani","sequence":"additional","affiliation":[{"name":"School of Pharmacy University of East Anglia  Norwich Research Park NR4 7TJ Norwich UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ridvan","family":"Nepravishta","sequence":"additional","affiliation":[{"name":"School of Pharmacy University of East Anglia  Norwich Research Park NR4 7TJ Norwich UK"},{"name":"Department of Biochemistry &amp; Molecular Biology Sealy Center for Structural Biology &amp; Molecular Biophysics University of Texas Medical Branch  301 University Blvd Galveston TX 77555-1068 USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Macarena","family":"Mart\u00ednez\u2010Bail\u00e9n","sequence":"additional","affiliation":[{"name":"Department of Organic Chemistry Faculty of Chemistry University of Seville  41012 Seville Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ana T.","family":"Carmona","sequence":"additional","affiliation":[{"name":"Department of Organic Chemistry Faculty of Chemistry University of Seville  41012 Seville Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Javier","family":"Ramos\u2010Soriano","sequence":"additional","affiliation":[{"name":"Department of Organic Chemistry Faculty of Chemistry University of Seville  41012 Seville Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Bergstr\u00f6m","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biomedical Sciences Linnaeus University  391 82 Kalmar Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Inmaculada","family":"Robina","sequence":"additional","affiliation":[{"name":"Department of Organic Chemistry Faculty of Chemistry University of Seville  41012 Seville Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7250-5639","authenticated-orcid":false,"given":"Jesus","family":"Angulo","sequence":"additional","affiliation":[{"name":"School of Pharmacy University of East Anglia  Norwich Research Park NR4 7TJ Norwich UK"},{"name":"Department of Organic Chemistry Faculty of Chemistry University of Seville  41012 Seville Spain"},{"name":"Instituto de Investigaciones Qu\u00edmicas (CSIC-US)  Avda. 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