Combining MicroED and native mass spectrometry for structural discovery of enzyme-small molecule complexes

Area of Science:

• Structural Biology
• Biochemistry
• Drug Discovery

Background:

• Accelerating the discovery of small molecule-protein complexes is crucial for drug development.
• Current methods for structure determination can be time-consuming and require purified samples.

Purpose of the Study:

• To develop and validate a novel method, electron diffraction with native mass spectrometry (ED-MS), for rapid identification and structural characterization of small molecule-protein complexes.
• To demonstrate the utility of ED-MS in discovering natural product ligands and screening small molecule binders.

Main Methods:

• Utilized fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection.
• Integrated MicroED with native mass spectrometry (MS).
• Applied ED-MS to crystal slurries soaked with known inhibitors, crude biosynthetic reactions, and printed ligand libraries.

Main Results:

• Successfully resolved the structures of E-64 and its analogs bound to papain.
• Identified preferred natural product binding to papain and detected complexes from crude biosynthetic reactions without purification.
• Determined the binding of avibactam to CTX-M-14 β-lactamase, including in a mixture of compounds.

Conclusions:

• ED-MS is a powerful technique for accelerating the discovery of small molecule-protein complexes.
• The method enables structure-based screening of ligands against macromolecular targets using crude samples.
• ED-MS shows significant promise for advancing drug discovery efforts.