TY - JOUR
T1 - Structural characterization of full-length human dehydrodolichyl diphosphate synthase using an integrative computational and experimental approach
AU - Bar-El, Michal Lisnyansky
AU - Lee, Su Youn
AU - Ki, Ah Young
AU - Kapelushnik, Noa
AU - Loewenstein, Anat
AU - Chung, Ka Young
AU - Schneidman-Duhovny, Dina
AU - Giladi, Moshe
AU - Newman, Hadas
AU - Haitin, Yoni
N1 - Funding 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).
PY - 2019/11
Y1 - 2019/11
N2 - 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.
AB - 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.
KW - Cis-prenyltransferase
KW - Computational modeling
KW - DHDDS
KW - Deuterium exchange mass-spectrometry
KW - Enzyme kinetics
KW - Hydrogen
KW - Small-angle X-ray scattering
UR - http://www.scopus.com/inward/record.url?scp=85074252100&partnerID=8YFLogxK
U2 - https://doi.org/10.3390/biom9110660
DO - https://doi.org/10.3390/biom9110660
M3 - مقالة
C2 - 31661879
SN - 2218-273X
VL - 9
JO - Biomolecules
JF - Biomolecules
IS - 11
M1 - 660
ER -