9NBN

Serial synchrotron X-ray diffraction structure of papain microcrystals soaked with a mixture of E-64, E-64C, and E-64D

This is a non-PDB format compatible entry.

Summary for 9NBN

• Entry DOI: 10.2210/pdb9nbn/pdb
• Related: 9NBF 9NBJ 9NBK
• Descriptor: Papain, (3S)-4-{[(2S)-1-amino-4-methyl-1-oxopentan-2-yl]amino}-3-hydroxy-4-oxobutanoic acid (3 entities in total)
• Functional Keywords: inhibitor, complex, cocktail, serial, hydrolase
• Biological source: Carica papaya (papaya)
• Total number of polymer chains: 1
• Total formula weight: 23698.56

Authors

Vlahakis, N.W.,Summers, J.A.,Wakatsuki, S.,Rodriguez, J.A. (deposition date: 2025-02-14, release date: 2025-03-26)

Primary citation

Vlahakis, N.W.,Flowers, C.W.,Liu, M.,Agdanowski, M.,Johnson, S.,Summers, J.A.,Keyser, C.,Russell, P.,Rose, S.,Orlans, J.,Adhami, N.,Chen, Y.,Sawaya, M.R.,Basu, S.,de Sanctis, D.,Wakatsuki, S.,Nelson, H.M.,Loo, J.A.,Tang, Y.,Rodriguez, J.A.
Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes.
Biorxiv, 2025

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 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.

PubMed: 40060639
DOI: 10.1101/2025.02.20.638743

Experimental method

X-RAY DIFFRACTION (1.8 Å)