Integrated Solid/Solution NMR Assignment Enables Mapping of Dynamics and Ligand Binding in a 134 kDa Enzyme.
- 1Institute of Science and Technology Austria , Am Campus 1, A-3400Klosterneuburg, Austria.
- 2Bruker Switzerland AG, CH-8117Fällanden, Switzerland.
Abstract
Understanding enzyme function requires characterizing not only the static structure but also dynamics and ligand interactions. NMR spectroscopy provides this insight at atomic resolution, yet for large proteins, the difficulty of resonance assignment has largely confined such studies to systems below ∼50 kDa, or to observing only methyl groups. Here, we present an integrated magic-angle spinning (MAS) and solution NMR study of the 134 kDa tetrameric malate dehydrogenase from Ignicoccus islandicus (IiMDH), an enzyme of particular interest as an evolutionary intermediate between allosteric lactate dehydrogenases and nonallosteric malate dehydrogenases. By combining high-dimensional (up to 4D) MAS NMR experiments on sedimented protein with solution NMR, we achieved 92% backbone heavy-atom assignment and 91% assignment of all Ile-δ1, Leu-δ1/-δ2, Val-γ1/-γ2, Met-ε, and Thr-γ methyl groups. Building on these assignments, we used various probes of backbone and side-chain dynamics: elevated MAS NMR 15N rotating-frame relaxation (R1ρ) points to slow motions in functionally critical regions, including the catalytic loop and the mobile surface loop. Complementary methyl-axis order parameters from solution NMR identified additional flexible sites in the hydrophobic core. Chemical shift perturbation experiments upon addition of the substrate analogue oxamate, monitored via backbone 1H-15N TROSY, revealed both active-site contacts and responses in helices α2F and α3G, regions associated with signal transmission in allosteric homologues. The integrated approach demonstrated here exploits the distinct strengths of MAS and solution NMR and provides a comprehensive view of structure, dynamics, and substrate interactions in a large oligomeric enzyme that neither technique could provide alone.
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BibTeX
@article{napoli2026integrated,
title = {{Integrated Solid/Solution NMR Assignment Enables Mapping of Dynamics and Ligand Binding in a 134 kDa Enzyme.}},
author = {Federico Napoli and Rajkumar Singh and Anna Kapitonova and Virgil Aitenbichler and Giorgia Toscano and Barbara Perrone and Paul Schanda},
journal = {Journal of the American Chemical Society},
year = {2026},
month = oct,
doi = {10.1021/jacs.6c14423},
url = {https://europepmc.org/article/MED/42954526},
note = {CC BY},
}
RIS
TY - JOUR
TI - Integrated Solid/Solution NMR Assignment Enables Mapping of Dynamics and Ligand Binding in a 134 kDa Enzyme.
AU - Federico Napoli
AU - Rajkumar Singh
AU - Anna Kapitonova
AU - Virgil Aitenbichler
AU - Giorgia Toscano
AU - Barbara Perrone
AU - Paul Schanda
PY - 2026
DA - 2026/10/01
JO - Journal of the American Chemical Society
DO - 10.1021/jacs.6c14423
UR - https://europepmc.org/article/MED/42954526
AB - Understanding enzyme function requires characterizing not only the static structure but also dynamics and ligand interactions. NMR spectroscopy provides this insight at atomic resolution, yet for large proteins, the difficulty of resonance assignment has largely confined such studies to systems below ∼50 kDa, or to observing only methyl groups. Here, we present an integrated magic-angle spinning (MAS) and solution NMR study of the 134 kDa tetrameric malate dehydrogenase from Ignicoccus islandicus (IiMDH), an enzyme of particular interest as an evolutionary intermediate between allosteric lactate dehydrogenases and nonallosteric malate dehydrogenases. By combining high-dimensional (up to 4D) MAS NMR experiments on sedimented protein with solution NMR, we achieved 92% backbone heavy-atom assignment and 91% assignment of all Ile-δ1, Leu-δ1/-δ2, Val-γ1/-γ2, Met-ε, and Thr-γ methyl groups. Building on these assignments, we used various probes of backbone and side-chain dynamics: elevated MAS NMR 15N rotating-frame relaxation (R1ρ) points to slow motions in functionally critical regions, including the catalytic loop and the mobile surface loop. Complementary methyl-axis order parameters from solution NMR identified additional flexible sites in the hydrophobic core. Chemical shift perturbation experiments upon addition of the substrate analogue oxamate, monitored via backbone 1H-15N TROSY, revealed both active-site contacts and responses in helices α2F and α3G, regions associated with signal transmission in allosteric homologues. The integrated approach demonstrated here exploits the distinct strengths of MAS and solution NMR and provides a comprehensive view of structure, dynamics, and substrate interactions in a large oligomeric enzyme that neither technique could provide alone.
SN - 0002-7863
SN - 1520-5126
ER -