Structural characterization of full-length human dehydrodolichyl diphosphate synthase using an integrative computational and experimental approach

Michal Lisnyansky Bar-El, Su Youn Lee, Ah Young Ki, Noa Kapelushnik, Anat Loewenstein, Ka Young Chung, Dina Schneidman-Duhovny, Moshe Giladi, Hadas Newman, Yoni Haitin

Research output: Contribution to journalArticlepeer-review

Abstract

Dehydrodolichyl diphosphate synthase (DHDDS) is the catalytic subunit of the heteromeric human cis-prenyltransferase complex, synthesizing the glycosyl carrier precursor for N-linked protein glycosylation. Consistent with the important role of N-glycosylation in protein biogenesis, DHDDS mutations result in human diseases. Importantly, DHDDS encompasses a C-terminal region, which does not converge with any known conserved domains. Therefore, despite the clinical importance of DHDDS, our understating of its structure–function relations remains poor. Here, we provide a structural model for the full-length human DHDDS using a multidisciplinary experimental and computational approach. Size-exclusion chromatography multi-angle light scattering revealed that DHDDS forms a monodisperse homodimer in solution. Enzyme kinetics assays revealed that it exhibits catalytic activity, although reduced compared to that reported for the intact heteromeric complex. Our model suggests that the DHDDS C-terminus forms a helix–turn–helix motif, tightly packed against the core catalytic domain. This model is consistent with small-angle X-ray scattering data, indicating that the full-length DHDDS maintains a similar conformation in solution. Moreover, hydrogen–deuterium exchange mass-spectrometry experiments show time-dependent deuterium uptake in the C-terminal domain, consistent with its overall folded state. Finally, we provide a model for the DHDDS–NgBR heterodimer, offering a structural framework for future structural and functional studies of the complex.

Original languageEnglish
Article number660
JournalBiomolecules
Volume9
Issue number11
DOIs
StatePublished - Nov 2019

Keywords

  • Cis-prenyltransferase
  • Computational modeling
  • DHDDS
  • Deuterium exchange mass-spectrometry
  • Enzyme kinetics
  • Hydrogen
  • Small-angle X-ray scattering

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry

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