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

Abstract

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.

Complexation and evolution of cis‐prenyltransferase homologues in Cinnamomum kanehirae deduced from kinetic and functional characterizations

“…SDS‐PAGE analysis of the complexes revealed the two protein components in roughly 1:1 ratio (Figure 3a). His‐tagged RWR78 alone expressed in S. cerevisiae and purified via Ni‐NTA resin and size‐exclusion chromatography was a homodimer (83.6 kDa) (Figure 3a), the same as the reported human DHDDS conformation (Lisnyansky et al, 2019) (we also confirmed that E. coli ‐expressed human DHDDS formed a homodimer as shown in Figure S4b). Besides, while using StrepTrap XT column and size‐exclusion chromatography to purify the S. cerevisiae ‐expressed sRWR71 or sRWR93, they formed a homodimer (67.3 and 61.4 kDa, respectively), similar to yeast Nus1 (Ma et al, 2019), but unlike the human sNgBR that forms a monomer (Holcomb et al, 2018).…”

“…Yeast Nus1, a NgBR homologue, forms a homodimer as revealed by its crystal structure (Ma et al, 2019). This Nus1 protein was modeled for binding with Rer2 to form a heterodimer (Lisnyansky et al, 2019), which is one of the two DHDDS‐like enzymes Rer2 and Srt1 in yeast (Shimizu et al, 1998), but the complexed structure and whether Nus1 could stimulate Rer2 activity were not investigated.…”

Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59

“…While such speculations may turn out to be true, there is no direct empirical evidence extant to support this hypothesis. It is also entirely possible, however, that mutations (whether K42E or others) in DHDDS may affect its interactions with its enzymatic partner, Nogo-B receptor (NgBR, encoded by the Nus1 gene) [8,9], with concomitant alterations in dolichol synthesis and protein N-glycosylation [28,29]. At present, nothing is known about the expression of Nogo-B receptor or its interactions with DHDDS, specifically in the retina.…”

“…DHDDS catalyzes cis-prenyl chain elongation in the synthesis of dolichyl diphosphate (Dol-PP), which is required for protein N-glycosylation [6,7]. DHDDS catalyzes the condensation of multiple units of isopentenyl pyrophosphate (IPP, also called isopentenyl diphosphate) 2 of 11 to farnesyl pyrophosphate (FPP, also called farnesyl diphosphate) to produce Dol-PP [8,9]. This is used as the "lipid carrier" onto which oligosaccharide chains are built that are ultimately transferred to specific asparagine (N) residues on nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER) to form N-linked glycoproteins [10].…”