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

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 r...

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Autores principales: Lisnyansky Bar-El, Michal, Lee, Su Youn, Ki, Ah Young, Kapelushnik, Noa, Loewenstein, Anat, Chung, Ka Young, Schneidman-Duhovny, Dina, Giladi, Moshe, Newman, Hadas, Haitin, Yoni
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921004/
https://www.ncbi.nlm.nih.gov/pubmed/31661879
http://dx.doi.org/10.3390/biom9110660
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author Lisnyansky Bar-El, Michal
Lee, Su Youn
Ki, Ah Young
Kapelushnik, Noa
Loewenstein, Anat
Chung, Ka Young
Schneidman-Duhovny, Dina
Giladi, Moshe
Newman, Hadas
Haitin, Yoni
author_facet Lisnyansky Bar-El, Michal
Lee, Su Youn
Ki, Ah Young
Kapelushnik, Noa
Loewenstein, Anat
Chung, Ka Young
Schneidman-Duhovny, Dina
Giladi, Moshe
Newman, Hadas
Haitin, Yoni
author_sort Lisnyansky Bar-El, Michal
collection PubMed
description 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.
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spelling pubmed-69210042019-12-24 Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach Lisnyansky Bar-El, Michal Lee, Su Youn Ki, Ah Young Kapelushnik, Noa Loewenstein, Anat Chung, Ka Young Schneidman-Duhovny, Dina Giladi, Moshe Newman, Hadas Haitin, Yoni Biomolecules Article 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. MDPI 2019-10-28 /pmc/articles/PMC6921004/ /pubmed/31661879 http://dx.doi.org/10.3390/biom9110660 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lisnyansky Bar-El, Michal
Lee, Su Youn
Ki, Ah Young
Kapelushnik, Noa
Loewenstein, Anat
Chung, Ka Young
Schneidman-Duhovny, Dina
Giladi, Moshe
Newman, Hadas
Haitin, Yoni
Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach
title Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach
title_full Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach
title_fullStr Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach
title_full_unstemmed Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach
title_short Structural Characterization of Full-Length Human Dehydrodolichyl Diphosphate Synthase Using an Integrative Computational and Experimental Approach
title_sort structural characterization of full-length human dehydrodolichyl diphosphate synthase using an integrative computational and experimental approach
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6921004/
https://www.ncbi.nlm.nih.gov/pubmed/31661879
http://dx.doi.org/10.3390/biom9110660
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