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

• 成果类型: Article
• 署名作者: He, Wenguang; Seabrook, Genevieve M. C. Gasmi; Ikura, Mitsuhiko; Lee, Jeffrey E.; Ohh, Michael
• 署名单位: University of Toronto; University of Toronto; University Health Network Toronto; Princess Margaret Cancer Centre; University of Toronto; University of Toronto
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10438
• DOI: 10.1073/pnas.2408104121
• 发表日期: 2024-09-10
• 关键词: hypoxia-inducible factor; structural basis; alpha-synuclein; hif-alpha; protein; oxygen; binding; phosphorylation; vhl; cyclophilin

摘要

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) alpha 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 1 alpha 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.