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Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry
Published on: October 15, 2018
MLAC: MicroED-assisted ligand structure analysis in complexes and its application to hERG-ligand complexes.
Keita Tanaka1, Yasuomi Miyashita2, Kenji Iwasaki3
1Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan; Degree Programs in Pure and Applied Sciences, Graduate School of Science and Technology, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan.
This study introduces MLAC, a framework combining MicroED and cryo-EM for accurate small-molecule ligand structure determination in protein complexes. MLAC uses MicroED-derived structures as initial models, improving ligand fitting in cryo-EM maps for drug discovery.
Area of Science:
- Structural Biology
- Biochemistry
- Drug Discovery
Background:
- Accurate ligand structure determination is crucial for understanding molecular recognition and advancing structure-based drug discovery.
- Cryogenic electron microscopy (cryo-EM) is powerful for macromolecular structures, but small-molecule ligand identification from cryo-EM maps is challenging without accurate initial models.
Purpose of the Study:
- To present MLAC (MicroED-assisted Ligand structure Analysis in Complexes), an integrative framework combining microcrystal electron diffraction (MicroED) and cryo-EM single-particle analysis (SPA).
- To evaluate the utility of MicroED-determined ligand structures as initial models for fitting into cryo-EM maps of protein-ligand complexes.
Main Methods:
- Developed MLAC, integrating MicroED for high-resolution ligand structure determination from small crystals with cryo-EM SPA for protein complex analysis.
- Applied MLAC to reanalyze known hERG-ligand complexes, using MicroED structures of astemizole, pimozide, and E-4031 for model fitting and refinement in cryo-EM maps.
Main Results:
- MicroED-derived ligand structures facilitated modeling for astemizole and, to a lesser extent, pimozide in hERG complexes.
- MicroED revealed structural polymorphs, offering alternative ligand conformations and resolving modeling ambiguities like piperidine ring variability and ligand placement.
- No clear advantage was observed for E-4031 ligand modeling using this approach.
Conclusions:
- MLAC serves as a proof-of-concept framework providing experimentally determined starting models to enhance computational ligand-generation approaches.
- The integration of MicroED and cryo-EM offers a valuable strategy for improving the accuracy of ligand structure determination in protein complexes.
- This approach has the potential to advance structure-based drug discovery by providing more reliable structural information on protein-ligand interactions.
