Abstract
The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. Here, we reveal that hcis-PT exhibits a heterotetrameric assembly in solution, consisting of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and inactive Nogo-B receptor (NgBR) heterodimers. Importantly, the 2.3 Å crystal structure reveals that the tetramer assembles via the DHDDS C-termini as a dimer-of-heterodimers. Moreover, the distal C-terminus of NgBR transverses across the interface with DHDDS, directly participating in active-site formation and the functional coupling between the subunits. Finally, we explored the functional consequences of disease mutations clustered around the active-site, and in combination with molecular dynamics simulations, we propose a mechanism for hcis-PT dysfunction in retinitis pigmentosa. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.
Highlights
The human cis-prenyltransferase is an enzymatic complex essential for protein Nglycosylation
dehydrodolichyl diphosphate synthase (DHDDS) missense mutations were shown to result in phenotypes ranging from autosomal recessive retinitis pigmentosa[12,13], through developmental epileptic encephalopathies[14], to a case of fatal congenital disorder of glycosylation reported in a patient heterozygous for both a splice site and a nonsense mutation[15]
Sequence and biochemical analyses of Nogo-B receptor (NgBR) revealed that it interacts with DHDDS via its cytosolic C-terminal pseudo cis-prenyltransferase homology domain (Supplementary Fig. 1)[10]
Summary
The human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein Nglycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, mutations in hcis-PT cause severe human diseases. While shortand medium-chain cis-prenyltransferase complexes are homodimeric, long-chain cis-prenyltransferases and rubber synthases are formed by a heteromeric subunit assembly of unknown stoichiometry[1,4,5]. The human cis-prenyltransferase (hcis-PT) complex catalyzes the formation of dehydrodolichyl diphosphate (DHDD, C85–100), a long-chain isoprenoid, by chain elongation of farnesyl diphosphate (FPP, C15) via multiple condensations with IPP (Fig. 1a)[10]. Localized to the endoplasmic reticulum, hcis-PT is composed of two structurally and functionally distinct subunit types These include the catalytically active DHDD synthase (DHDDS) and the quiescent Nogo-B receptor (NgBR) subunits[10]. In line with the crucial significance of N-linked glycosylation for proper cellular function, mutations in both hcis-PT subunits were associated with human diseases. The interplay between the genotype and cellular phenotype may be more complex than originally thought and awaits further exploration
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