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Open AccessArticle

Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach

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Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv 6997801, Israel
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School of Pharmacy, Sungkyunkwan University, Jangan-gu, Suwon 16419, Korea
3
Department of Ophthalmology, Sheba Medical Center, Ramat Gan 5265601, Israel
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Department of Ophthalmology, Tel-Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel
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Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
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School of Computer Science and Engineering, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel
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Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Biomolecules 2019, 9(11), 660; https://doi.org/10.3390/biom9110660
Received: 5 October 2019 / Revised: 25 October 2019 / Accepted: 26 October 2019 / Published: 28 October 2019
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. View Full-Text
Keywords: cis-prenyltransferase; computational modeling; hydrogen–deuterium exchange mass-spectrometry; small-angle X-ray scattering; enzyme kinetics; DHDDS cis-prenyltransferase; computational modeling; hydrogen–deuterium exchange mass-spectrometry; small-angle X-ray scattering; enzyme kinetics; DHDDS
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Lisnyansky Bar-El, M.; Lee, S.Y.; Ki, A.Y.; Kapelushnik, N.; Loewenstein, A.; Chung, K.Y.; Schneidman-Duhovny, D.; Giladi, M.; Newman, H.; Haitin, Y. Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach. Biomolecules 2019, 9, 660.

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