Time-resolved NMR detection of prolyl-hydroxylation in intrinsically disordered region of HIF-1α

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 121; no. 37; p. e2408104121
• Main Authors: He, Wenguang, Gasmi-Seabrook, Geneviève M. C., Ikura, Mitsuhiko, Lee, Jeffrey E., Ohh, Michael
• Format: Journal Article
• Language: English
• Published: 10.09.2024
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2408104121

Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time 13 C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the “proton-less” nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.