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.
Bibliographical noteFunding Information:
Funding: This research was funded by the Israel Science Foundation, grant numbers 1721/16, 1775/12 (Y.H.), 1466/18 (D.S.), Israel Cancer Research Foundation, grant number 01214 (Y.H.), German–Israeli Foundation, grant number I-2425-418.13/2016 (Y.H.), Stoltz Foundation (H.N.), Tel-Aviv Sourasky Medical Center (A.L. and M.G.), the Clair and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel-Aviv University (Y.H. and M.G.), and the Korean government (NRF-2018R1A2B6001554) (K.Y.C).
© 2019 by the authors. Licensee MDPI, Basel, Switzerland.
- Computational modeling
- Deuterium exchange mass-spectrometry
- Enzyme kinetics
- Small-angle X-ray scattering