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

• Main Authors: 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
• Format: Article
• Language: English
• Published: MDPI AG 2019-10-01
• Series: Biomolecules
• Subjects: <i>cis</i>-prenyltransferase; computational modeling; hydrogen–deuterium exchange mass-spectrometry; small-angle x-ray scattering; enzyme kinetics; dhdds
• Online Access: https://www.mdpi.com/2218-273X/9/11/660

Dehydrodolichyl diphosphate synthase (DHDDS) is the catalytic subunit of the heteromeric human <i>cis</i>-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&#8722;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&#8722;turn&#8722;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&#8722;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&#8722;NgBR heterodimer, offering a structural framework for future structural and functional studies of the complex.