Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew Agdanowski / Samuel Johnson / Jacob A Summers / Catherine Keyser / Phoebe Russell / Samuel Rose / Julien Orlans / Nima Adhami / Yu Chen / Michael R Sawaya / Shibom Basu / Daniele de Sanctis / Soichi Wakatsuki / Hosea M Nelson / Joseph A Loo / Yi Tang / Jose A Rodriguez /
PubMed Abstract
With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection ...With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.
PDB-9n9d: MicroED structure of papain co-crystallized with E-64C Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.2 Å
PDB-9nae: MicroED structure of papain co-crystallized with E-64 Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.3 Å
PDB-9nag: MicroED structure of the apo-form of papain Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.5 Å
PDB-9nao: MicroED structure of papain complexed with natural product E64-A65 Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.5 Å
PDB-9nar: MicroED structure of papain microcrystals soaked with E-64 for 10 minutes Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.5 Å
PDB-9nat: X-ray diffraction structure of papain co-crystallized with leupeptin Method: X-RAY DIFFRACTION / Resolution: 1.6 Å
PDB-9nax: MicroED structure of the papain-E-64 complex from microcrystals soaked with crude biosynthetic reaction mixture Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.3 Å
PDB-9nay: MicroED structure of papain complexed with natural product E-64-A65 from microcrystals soaked in crude biosynthetic reaction mixture Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.5 Å
PDB-9nb2: X-ray diffraction structure of papain soaked with E-64 Method: X-RAY DIFFRACTION / Resolution: 1.5 Å
PDB-9nb4: Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with natural product E-64-A65 Method: X-RAY DIFFRACTION / Resolution: 1.8 Å
PDB-9nb7: Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with natural product E405 Method: X-RAY DIFFRACTION / Resolution: 1.8 Å
PDB-9nbf: Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with E-64 Method: X-RAY DIFFRACTION / Resolution: 1.8 Å
PDB-9nbj: Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with E-64C Method: X-RAY DIFFRACTION / Resolution: 1.8 Å
PDB-9nbk: Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with E-64D Method: X-RAY DIFFRACTION / Resolution: 1.8 Å
PDB-9nbn: Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with a mixture of E-64, E-64C, and E-64D Method: X-RAY DIFFRACTION / Resolution: 1.8 Å
PDB-9nbp: MicroED structure of the papain-E-64 complex from microcrystals mixed on-grid with microarrayed ligand Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.8 Å
PDB-9nbq: MicroED structure of papain co-crystallized with E-64D Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.3 Å
PDB-9nc1: MicroED structure of papain-E-64 complex from microcrystals soaked with protease inhibitor cocktail Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.4 Å
PDB-9nca: MicroED structure of microcrystals soaked with a mixture of E-64, E-64C, and E-64D Method: ELECTRON CRYSTALLOGRAPHY / Resolution: 2.5 Å
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Keywords
carica papaya (papaya)