{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T04:32:13Z","timestamp":1792729933312,"version":"4.5.0"},"reference-count":51,"publisher":"American Chemical Society (ACS)","issue":"17","license":[{"start":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T00:00:00Z","timestamp":1787616000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T00:00:00Z","timestamp":1787616000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T00:00:00Z","timestamp":1787616000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/501100001843","name":"Science and Engineering Research Board","doi-asserted-by":"publisher","award":["CRG\/2022\/008633"],"award-info":[{"award-number":["CRG\/2022\/008633"]}],"id":[{"id":"10.13039\/501100001843","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001843","name":"Science and Engineering Research Board","doi-asserted-by":"publisher","award":["MTR\/2022\/000664"],"award-info":[{"award-number":["MTR\/2022\/000664"]}],"id":[{"id":"10.13039\/501100001843","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004541","name":"Ministry of Education, India","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/501100004541","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Cryptic pockets are transient ligand-binding sites that remain hidden in apo protein structures and become accessible only through conformational change, making them difficult to identify and exploit in structure-based ligand discovery. Although recent AI\/ML approaches can identify residues associated with cryptic pocket formation, they do not directly yield the corresponding holo-like conformations. Enhanced-sampling and mixed-solvent strategies can promote pocket opening, but often under non-native conditions and without demonstrating stable ligand-bound holo-like states. Here, we present a computational framework in which residues with high cryptic-pocket propensity are used as mutation handles to perturb the free-energy landscape and expose ligand-compatible open states even in the absence of ligand binding. We establish this strategy on TEM-1 \u03b2-lactamase, a canonical cryptic-pocket system, and show that mutation-induced perturbations can shift the conformational ensemble toward an open pocket state prior to ligand binding. The resulting open state supports ligand binding in both docked structures and unbiased simulations, and the ligand remains stably bound after reversion to the wild-type sequence, consistent with recovery of a holo-like wild-type conformation. Importantly, the residues targeted for mutation are not the principal determinants of the ligand interactions observed in the bound state, indicating that their role is to reshape the conformational landscape and promote access to an open, ligand-compatible pocket rather than to form the binding interface itself. We further generalize this framework to LfrR, FtsZ, and Bombyx mori pheromone-binding protein, where mutation of predicted cryptic residues likewise generates open conformations capable of supporting ligand binding. Together, these results show that cryptic residue predictions can be used not only to identify hidden binding sites but also to recover holo-like conformations from apo structures, providing a practical framework for studying and targeting cryptic pockets.<\/jats:p>","DOI":"10.1021\/acs.jcim.6c01260","type":"journal-article","created":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T22:01:39Z","timestamp":1788213699000},"page":"11226-11245","source":"Crossref","is-referenced-by-count":1,"title":["Mutation-Guided Recovery of Ligand-Compatible Holo-Like\nConformations in Proteins with Cryptic Pockets"],"prefix":"10.1021","volume":"66","author":[{"given":"Reshob","family":"Routh","sequence":"first","affiliation":[{"name":"Indian Institute of Technology Gandhinagar , , , ,","place":["Gandhinagar, Gujarat, India, 382055"]},{"name":"Indian Institute of Technology Gandhinagar , , , ,","place":["Gandhinagar, Gujarat, India, 382055"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7127-4744","authenticated-orcid":true,"given":"Mithun","family":"Radhakrishna","sequence":"additional","affiliation":[{"name":"Indian Institute of Technology Gandhinagar , , , ,","place":["Gandhinagar, Gujarat, India, 382055"]},{"name":"Indian Institute of Technology Gandhinagar , , , ,","place":["Gandhinagar, Gujarat, India, 382055"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,25]]},"reference":[{"key":"2026091409072122100_cit1","doi-asserted-by":"publisher","first-page":"1598","DOI":"10.1126\/science.1749933","article-title":"The Energy Landscapes and Motions\nof Proteins","volume":"254","author":"Frauenfelder","year":"1991","journal-title":"Science"},{"key":"2026091409072122100_cit2","doi-asserted-by":"publisher","first-page":"964","DOI":"10.1038\/nature06522","article-title":"Dynamic Personalities of Proteins","volume":"450","author":"Henzler-Wildman","year":"2007","journal-title":"Nature"},{"key":"2026091409072122100_cit3","doi-asserted-by":"publisher","first-page":"789","DOI":"10.1038\/nchembio.232","article-title":"The Role of Dynamic\nConformational\nEnsembles in Biomolecular Recognition","volume":"5","author":"Boehr","year":"2009","journal-title":"Nat.\nChem. Biol."},{"key":"2026091409072122100_cit4","doi-asserted-by":"publisher","first-page":"6679","DOI":"10.1073\/pnas.0408930102","article-title":"Molecular Dynamics\nand Protein Function","volume":"102","author":"Karplus","year":"2005","journal-title":"Proc. Natl. Acad. Sci.\nU. S. A."},{"key":"2026091409072122100_cit5","doi-asserted-by":"publisher","first-page":"227","DOI":"10.1146\/annurev-biophys-061824-104900","article-title":"Statistical\nThermodynamics of the Protein Ensemble: Mediating Function and Evolution","volume":"54","author":"Hilser","year":"2025","journal-title":"Annu. Rev. Biophys."},{"key":"2026091409072122100_cit6","doi-asserted-by":"publisher","first-page":"11681","DOI":"10.1073\/pnas.1209309109","article-title":"Equilibrium Fluctuations\nof a Single Folded Protein\nReveal a Multitude of Potential Cryptic Allosteric Sites","volume":"109","author":"Bowman","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026091409072122100_cit7","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.cbpa.2018.05.003","article-title":"Cryptic Binding\nSites on Proteins: Definition, Detection, and Druggability","volume":"44","author":"Vajda","year":"2018","journal-title":"Curr. Opin. Chem. Biol."},{"key":"2026091409072122100_cit8","doi-asserted-by":"publisher","first-page":"102975","DOI":"10.1016\/j.sbi.2024.102975","article-title":"Computational\nAdvances in Discovering Cryptic Pockets for Drug Discovery","volume":"90","author":"Bemelmans","year":"2025","journal-title":"Curr. Opin. Struct. Biol."},{"key":"2026091409072122100_cit9","doi-asserted-by":"publisher","first-page":"1177","DOI":"10.1038\/s41467-023-36699-3","article-title":"Predicting\nLocations of Cryptic Pockets from Single Protein Structures Using\nthe PocketMiner Graph Neural Network","volume":"14","author":"Meller","year":"2023","journal-title":"Nat. Commun."},{"key":"2026091409072122100_cit10","doi-asserted-by":"publisher","first-page":"548","DOI":"10.1038\/nature12796","article-title":"K-Ras(G12C)\nInhibitors Allosterically Control GTP Affinity and Effector Interactions","volume":"503","author":"Ostrem","year":"2013","journal-title":"Nature"},{"key":"2026091409072122100_cit11","doi-asserted-by":"publisher","first-page":"1818","DOI":"10.1002\/med.70001","article-title":"The Discovery\nof Cryptic Pockets Increases the Druggability of \u201cUndruggable\u201d\nProteins","volume":"45","author":"Mou","year":"2025","journal-title":"Med. Res. Rev."},{"key":"2026091409072122100_cit12","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1016\/S0022-2836(65)80285-6","article-title":"On the Nature of Allosteric Transitions:\nA Plausible Model","volume":"12","author":"Monod","year":"1965","journal-title":"J. Mol. Biol."},{"key":"2026091409072122100_cit13","doi-asserted-by":"publisher","first-page":"539","DOI":"10.1016\/j.tibs.2010.04.009","article-title":"Induced Fit, Conformational\nSelection\nand Independent Dynamic Segments: An Extended View of Binding Events","volume":"35","author":"Csermely","year":"2010","journal-title":"Trends Biochem. Sci."},{"key":"2026091409072122100_cit14","doi-asserted-by":"publisher","first-page":"98","DOI":"10.1073\/pnas.44.2.98","article-title":"Application\nof a Theory of Enzyme Specificity to Protein Synthesis","volume":"44","author":"Koshland","year":"1958","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026091409072122100_cit15","doi-asserted-by":"publisher","first-page":"14257","DOI":"10.1021\/jacs.6b05425","article-title":"Understanding Cryptic\nPocket Formation in Protein Targets by Enhanced Sampling Simulations","volume":"138","author":"Oleinikovas","year":"2016","journal-title":"J. Am. Chem. Soc."},{"key":"2026091409072122100_cit16","doi-asserted-by":"publisher","first-page":"E3416","DOI":"10.1073\/pnas.1711490115","article-title":"Exploring the structural\norigins of cryptic sites on proteins","volume":"115","author":"Beglov","year":"2018","journal-title":"Proc.\nNatl. Acad. Sci. U. S. A."},{"key":"2026091409072122100_cit17","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1146\/annurev-physchem-040215-112229","article-title":"Enhancing Important Fluctuations:\nRare Events and Metadynamics from a Conceptual Viewpoint","volume":"67","author":"Valsson","year":"2016","journal-title":"Annu. Rev. Phys. Chem."},{"key":"2026091409072122100_cit18","doi-asserted-by":"publisher","first-page":"10383","DOI":"10.1021\/acs.jmedchem.6b00399","article-title":"Driving Structure-Based\nDrug Discovery through Cosolvent\nMolecular Dynamics","volume":"59","author":"Ghanakota","year":"2016","journal-title":"J. Med. Chem."},{"key":"2026091409072122100_cit19","doi-asserted-by":"publisher","first-page":"3331","DOI":"10.1021\/acs.jctc.8b01295","article-title":"Cosolvent-Enhanced\nSampling and Unbiased Identification of Cryptic Pockets Suitable for\nStructure-Based Drug Design","volume":"15","author":"Schmidt","year":"2019","journal-title":"J. Chem. Theory\nComput."},{"key":"2026091409072122100_cit20","doi-asserted-by":"publisher","first-page":"2734","DOI":"10.1073\/pnas.1417811112","article-title":"Discovery\nof Multiple Hidden Allosteric Sites by Combining Markov State Models\nand Experiments","volume":"112","author":"Bowman","year":"2015","journal-title":"Proc. Natl. Acad. Sci. U. S.\nA."},{"key":"2026091409072122100_cit21","doi-asserted-by":"publisher","first-page":"709","DOI":"10.1016\/j.jmb.2016.01.029","article-title":"CryptoSite:\nExpanding the Druggable Proteome by Characterization and Prediction\nof Cryptic Binding Sites","volume":"428","author":"Cimermancic","year":"2016","journal-title":"J. Mol. Biol."},{"key":"2026091409072122100_cit22","doi-asserted-by":"publisher","first-page":"2026.05.21.726899","DOI":"10.64898\/2026.05.21.726899","article-title":"AE-PocketMiner\nUses Attention to Simultaneously Predict Cryptic Pockets and Their\nAllosteric Coupling","author":"Zhang","year":"2026","journal-title":"bioRxiv"},{"key":"2026091409072122100_cit23","doi-asserted-by":"publisher","first-page":"4355","DOI":"10.1021\/acs.jctc.2c01189","article-title":"Accelerating Cryptic\nPocket Discovery Using AlphaFold","volume":"19","author":"Meller","year":"2023","journal-title":"J. Chem. Theory\nComput."},{"key":"2026091409072122100_cit24","doi-asserted-by":"publisher","first-page":"832","DOI":"10.1038\/s41586-023-06832-9","article-title":"Predicting\nMultiple Conformations via Sequence Clustering and AlphaFold2","volume":"625","author":"Wayment-Steele","year":"2024","journal-title":"Nature"},{"key":"2026091409072122100_cit25","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.abd0480","article-title":"Targeting the Cryptic\nSites: NMR-Based Strategy to\nImprove Protein Druggability by Controlling the Conformational Equilibrium","volume":"6","author":"Mizukoshi","year":"2020","journal-title":"Sci. Adv."},{"key":"2026091409072122100_cit26","doi-asserted-by":"publisher","first-page":"12965","DOI":"10.1038\/ncomms12965","article-title":"Modelling\nproteins\u2019 hidden conformations to predict antibiotic resistance","volume":"7","author":"Hart","year":"2016","journal-title":"Nat. Commun."},{"key":"2026091409072122100_cit27","doi-asserted-by":"publisher","first-page":"1515","DOI":"10.1021\/acs.jcim.8b00730","article-title":"Holo-like and Druggable Protein Conformations\nfrom\nEnhanced Sampling of Binding Pocket Volume and Shape","volume":"59","author":"Basciu","year":"2019","journal-title":"J. Chem. Inf. Model."},{"key":"2026091409072122100_cit28","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic\nAcids Res."},{"key":"2026091409072122100_cit29","doi-asserted-by":"publisher","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2500A\nVisualization\nSystem for Exploratory Research and Analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"2026091409072122100_cit30","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High\nPerformance\nMolecular Simulations through Multi-Level Parallelism from Laptops\nto Supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"2026091409072122100_cit31","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1038\/nmeth.4067","article-title":"CHARMM36m:\nAn Improved Force Field for Folded and Intrinsically Disordered Proteins","volume":"14","author":"Huang","year":"2017","journal-title":"Nat. Methods"},{"key":"2026091409072122100_cit32","doi-asserted-by":"publisher","first-page":"926","DOI":"10.1063\/1.445869","article-title":"Comparison\nof Simple Potential Functions for Simulating Liquid Water","volume":"79","author":"Jorgensen","year":"1983","journal-title":"J. Chem. Phys."},{"key":"2026091409072122100_cit33","doi-asserted-by":"publisher","first-page":"10089","DOI":"10.1063\/1.464397","article-title":"Particle Mesh\nEwald: An N \u00b7 log(N) Method\nfor Ewald Sums in Large Systems","volume":"98","author":"Darden","year":"1993","journal-title":"J. Chem. Phys."},{"key":"2026091409072122100_cit34","doi-asserted-by":"publisher","first-page":"014101","DOI":"10.1063\/1.2408420","article-title":"Canonical Sampling through Velocity\nRescaling","volume":"126","author":"Bussi","year":"2007","journal-title":"J. Chem. Phys."},{"key":"2026091409072122100_cit35","doi-asserted-by":"publisher","first-page":"3684","DOI":"10.1063\/1.448118","article-title":"Molecular\nDynamics with Coupling to an External Bath","volume":"81","author":"Berendsen","year":"1984","journal-title":"J. Chem. Phys."},{"key":"2026091409072122100_cit36","doi-asserted-by":"publisher","first-page":"7182","DOI":"10.1063\/1.328693","article-title":"Polymorphic Transitions\nin Single Crystals: A New Molecular\nDynamics Method","volume":"52","author":"Parrinello","year":"1981","journal-title":"J. Appl. Phys."},{"key":"2026091409072122100_cit37","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1002\/jcc.21367","article-title":"CHARMM General Force Field: A Force\nField for Drug-Like Molecules Compatible with the CHARMM All-Atom\nAdditive Biological Force Fields","volume":"31","author":"Vanommeslaeghe","year":"2010","journal-title":"J. Comput.\nChem."},{"key":"2026091409072122100_cit38","doi-asserted-by":"publisher","first-page":"3155","DOI":"10.1021\/ci3003649","article-title":"Automation\nof the CHARMM General\nForce Field (CGenFF) II: Assignment of Bonded Parameters and Partial\nAtomic Charges","volume":"52","author":"Vanommeslaeghe","year":"2012","journal-title":"J. Chem. Inf. Model."},{"key":"2026091409072122100_cit39","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.ady6364","article-title":"CryptoBank: A resource\nfor the identification and prediction of cryptic sites in proteins","volume":"12","author":"Martinez","year":"2026","journal-title":"Sci. Adv."},{"key":"2026091409072122100_cit40","doi-asserted-by":"publisher","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold: Making Protein Folding Accessible to All","volume":"19","author":"Mirdita","year":"2022","journal-title":"Nat. Methods"},{"key":"2026091409072122100_cit41","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly Accurate Protein\nStructure Prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"2026091409072122100_cit42","doi-asserted-by":"publisher","first-page":"168","DOI":"10.1186\/1471-2105-10-168","article-title":"Fpocket: An Open Source\nPlatform for Ligand Pocket Detection","volume":"10","author":"Le Guilloux","year":"2009","journal-title":"BMC Bioinf."},{"key":"2026091409072122100_cit43","doi-asserted-by":"publisher","first-page":"3276","DOI":"10.1093\/bioinformatics\/btr550","article-title":"MDpocket:\nOpen-Source\nCavity Detection and Characterization on Molecular Dynamics Trajectories","volume":"27","author":"Schmidtke","year":"2011","journal-title":"Bioinformatics"},{"key":"2026091409072122100_cit44","doi-asserted-by":"publisher","first-page":"455","DOI":"10.1002\/jcc.21334","article-title":"AutoDock Vina: Improving the Speed and Accuracy of\nDocking with a New Scoring Function, Efficient Optimization, and Multithreading","volume":"31","author":"Trott","year":"2010","journal-title":"J. Comput. Chem."},{"key":"2026091409072122100_cit45","doi-asserted-by":"publisher","first-page":"1283","DOI":"10.1016\/j.jmb.2003.12.068","article-title":"Allosteric Inhibition\nthrough Core Disruption","volume":"336","author":"Horn","year":"2004","journal-title":"J. Mol. Biol."},{"key":"2026091409072122100_cit46","doi-asserted-by":"publisher","first-page":"3335","DOI":"10.1021\/acs.jctc.3c01318","article-title":"an Expanded\nApproach to Detect Cryptic Pockets in Proteins and at\nProtein\u2013Protein Interfaces","volume":"20","author":"Borsatto","year":"2024","journal-title":"J. Chem.\nTheory Comput."},{"key":"2026091409072122100_cit47","doi-asserted-by":"publisher","first-page":"7531","DOI":"10.1128\/JB.00631-09","article-title":"Structural plasticity\nand distinct drug-binding modes\nof LfrR, a mycobacterial efflux pump regulator","volume":"191","author":"Bellinzoni","year":"2009","journal-title":"J. Bacteriol."},{"key":"2026091409072122100_cit48","doi-asserted-by":"publisher","first-page":"143","DOI":"10.1016\/S1074-5521(00)00078-8","article-title":"Sexual attraction in\nthe silkworm moth: structure of the pheromone-binding-protein\u2013bombykol\ncomplex","volume":"7","author":"Sandler","year":"2000","journal-title":"Chem. Biol."},{"key":"2026091409072122100_cit49","doi-asserted-by":"publisher","first-page":"1044","DOI":"10.1016\/j.bbrc.2005.07.176","article-title":"Coil-to-helix\ntransition and ligand\nrelease of Bombyx mori pheromone-binding protein","volume":"335","author":"Lautenschla\u0308ger","year":"2005","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"2026091409072122100_cit50","doi-asserted-by":"publisher","first-page":"1243","DOI":"10.1038\/nsmb855","article-title":"Structural\nInsights into FtsZ Protofilament\nFormation","volume":"11","author":"Oliva","year":"2004","journal-title":"Nat. Struct. Mol. Biol."},{"key":"2026091409072122100_cit51","doi-asserted-by":"publisher","first-page":"1947","DOI":"10.1021\/acschembio.7b00323","article-title":"Structural\nFlexibility of an Inhibitor Overcomes Drug Resistance Mutations in Staphylococcus aureus FtsZ","volume":"12","author":"Fujita","year":"2017","journal-title":"ACS Chem.\nBiol."}],"container-title":["Journal of Chemical Information and Modeling"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jcim.6c01260","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jcisd8\/article-pdf\/66\/17\/11226\/67394004\/acs.jcim.6c01260.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jcisd8\/article-pdf\/66\/17\/11226\/67394004\/acs.jcim.6c01260.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:20:10Z","timestamp":1789392010000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jcisd8\/article\/66\/17\/11226\/5328301\/Mutation-Guided-Recovery-of-Ligand-Compatible-Holo"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,25]]},"references-count":51,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,8,25]]},"published-print":{"date-parts":[[2026,9,14]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jcim.6c01260","relation":{},"ISSN":["1549-9596","1549-960X"],"issn-type":[{"value":"1549-9596","type":"print"},{"value":"1549-960X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,25]]}}}