3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase
Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.
Authors
- Adriana Coricello (ORCID: https://orcid.org/0000-0002-3306-9261)
- Stefano Alcaro (ORCID: https://orcid.org/0000-0002-0437-358X)
- Yuichiro Takagi (ORCID: https://orcid.org/0000-0002-0988-6798)
- Vincent Chaptal (ORCID: https://orcid.org/0000-0003-2603-0294)
- Nigel G. J. Richards (ORCID: https://orcid.org/0000-0002-0375-0881)
- Antonio Lupia (ORCID: https://orcid.org/0000-0003-0870-2376)
- Wen Zhu (ORCID: https://orcid.org/0000-0003-3190-0071)
- Matthijn Vos (ORCID: https://orcid.org/0000-0002-6046-9949)
- Carmen Gratteri (ORCID: https://orcid.org/0009-0007-3527-2080)
- Alanya J. Nardone
Institutions
- Université Claude Bernard Lyon 1 (FR)
- Florida State University (US)
- Centre National de la Recherche Scientifique (FR)
- Florida A&M University - Florida State University College of Engineering (US)
- Institut Pasteur (FR)
- University of Cagliari (IT)
- University of Urbino (IT)
- Magna Graecia University (IT)
- Laboratoire de Biologie Structurale de la Cellule (FR)
- Foundation for Applied Molecular Evolution (US)
- Indiana University School of Medicine
- Indiana University – Purdue University Indianapolis (US)
- Cardiff University (GB)
Publication Details
- Journal
- Nature Communications
- Published
- 2024-12-03
- DOI
- https://doi.org/10.1038/s41467-024-54912-9
- Citations
- 5
- Primary Topic
- Enzyme Structure and Function
- Type
- article
- Field-Weighted Citation Impact
- 0.59
Funders
- Florida State University
- Agence Nationale de la Recherche
- Centre National de la Recherche Scientifique
- Regione Calabria
- National Institutes of Health
- Directorate for Biological Sciences
- School of Medicine, Indiana University
- European Regional Development Fund
- National Institute of General Medical Sciences