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.

Blavatnik Center for Drug Discovery Tel Aviv University Tel Aviv 6997801 Israel

Department of Biochemistry Faculty of Science Charles University Hlavova 2030 8 128 43 Prague 2 Czech Republic

Department of Physiology and Pharmacology Sackler Faculty of Medicine Tel Aviv University Tel Aviv 6997801 Israel

Institute of Microbiology of the Czech Academy of Sciences Division BioCeV Prumyslova 595 252 50 Vestec Czech Republic

Sagol School of Neuroscience Tel Aviv University Tel Aviv 6997801 Israel

Tel Aviv Sourasky Medical Center Tel Aviv 6423906 Israel

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Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases

De novo DHDDS variants cause a neurodevelopmental and neurodegenerative disorder with myoclonus