TY - JOUR

T1 - Structural landscape of mutant form NEIL2 DNA glycosylase obtained by HDX-MS

AU - Голышкин, Арсений Антонович

AU - Аксенова, Лилия Владимировна

AU - Chernonosov, Alexander A.

AU - Коваль, Владимир Васильевич

AU - Лукина, Мария Владимировна

PY - 2025

Y1 - 2025

N2 - DNA base damage, particularly oxidation, represents an inevitable yet mutagenic process requiring timely recognition and repair by the base excision repair (BER) system. The bifunctional DNA glycosylase NEIL2 (Nei Endonuclease VIII-like 2) is one of 11 human glycosylases responsible for initiating the removal of oxidized purines and pyrimidines, exhibiting broad substrate specificity. A unique structural feature of NEIL2 is the intrinsically disordered proline-rich motif (Leu58–Arg128) adjacent to Lys50, a critical catalytic residue. To investigate the role of Lys50 in different steps of lesion recognition, binding, and excision, we decided to study mutant NEIL2 form with K50A substitution. NEIL2 conformational domain flexibility and interdomain disordered region complicate its structural resolution via X-ray crystallography. To study its structural dynamics during DNA interactions, we employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), a solution-phase method that reveals protein conformational dynamics by measuring exchange rates between amide protons and solvent deuterium. We optimized HDX-MS experimental conditions and generated deuterium uptake time courses. Peptide mapping for the NEIL2 K50A mutant achieved > 85% sequence coverage. Challenges arose in analyzing the unstructured loop (residues 59–115), which contains 10 proline residues that hindered pepsin cleavage. Deuterium uptake rates varied across protein regions, correlating with secondary and tertiary structural features characteristic of NEIL2 glycosylase. The study highlights HDX-MS as a powerful tool for probing NEIL2 dynamic architecture and provides new information about its repair mechanism.

AB - DNA base damage, particularly oxidation, represents an inevitable yet mutagenic process requiring timely recognition and repair by the base excision repair (BER) system. The bifunctional DNA glycosylase NEIL2 (Nei Endonuclease VIII-like 2) is one of 11 human glycosylases responsible for initiating the removal of oxidized purines and pyrimidines, exhibiting broad substrate specificity. A unique structural feature of NEIL2 is the intrinsically disordered proline-rich motif (Leu58–Arg128) adjacent to Lys50, a critical catalytic residue. To investigate the role of Lys50 in different steps of lesion recognition, binding, and excision, we decided to study mutant NEIL2 form with K50A substitution. NEIL2 conformational domain flexibility and interdomain disordered region complicate its structural resolution via X-ray crystallography. To study its structural dynamics during DNA interactions, we employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), a solution-phase method that reveals protein conformational dynamics by measuring exchange rates between amide protons and solvent deuterium. We optimized HDX-MS experimental conditions and generated deuterium uptake time courses. Peptide mapping for the NEIL2 K50A mutant achieved > 85% sequence coverage. Challenges arose in analyzing the unstructured loop (residues 59–115), which contains 10 proline residues that hindered pepsin cleavage. Deuterium uptake rates varied across protein regions, correlating with secondary and tertiary structural features characteristic of NEIL2 glycosylase. The study highlights HDX-MS as a powerful tool for probing NEIL2 dynamic architecture and provides new information about its repair mechanism.

UR - https://febs.onlinelibrary.wiley.com/doi/10.1002/2211-5463.70071

M3 - Conference article

VL - 15

SP - 151

JO - FEBS Open Bio

JF - FEBS Open Bio

SN - 2211-5463

IS - S2

M1 - P-18-040

ER -