Structural elucidation of the heterodimericcis-prenyltransferase NgBR/DHDDS complex reveals novel insights in regulation of protein glycosylation

Abstract

Cis-prenyltransferase (cis-PTase) catalyzes the rate-limiting step in the synthesis of glycosyl carrier lipids required for protein glycosylation in the lumen of endoplasmic reticulum. Here we report the crystal structure of the human NgBR/DHDDS complex, which represents the first atomic resolution structure for any heterodimeric cis-PTase. The crystal structure sheds light on how NgBR stabilizes DHDDS through dimerization, participates in the enzyme's active site through its C-terminal -RXG-motif, and how phospholipids markedly stimulate cis-PTase activity. Comparison of NgBR/DHDDS with homodimeric cis-PTase structures leads to a model where the elongating isoprene chain extends beyond the enzyme's active site tunnel, and an insert within the 3 helix helps to stabilize this energetically unfavorable state to enable long chain synthesis to occur. These data provide unique insights into how heterodimeric cis-PTases have evolved from their ancestral, homodimeric forms to fulfill their function in long chain polyprenol synthesis.

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

“…These measurements rely on the increased inherent MANT-O-GPP emission at F 420 upon chain elongation ( 33 ). Consistent with the ability of phospholipids to enhance sh cis -PT activity ( 13 ), inclusion of nanodiscs at increasing nanodisc/enzyme ratios resulted in increased fluorescence amplitude and slope over the reaction time course (Fig. 7A).…”

“…Long-chain cis -prenyltransferases and rubber synthases are membrane delimited, and the presence of detergents or phospholipids was shown to markedly enhance their catalytic activity ( 6 , 13 , 28 ). As our structural studies suggest product elongation through a novel outlet, possibly into the adjacent membrane, we sought to monitor the chemical environment and position of the elongating isoprene moiety.…”

“…Together, our results depict a product elongation mechanism involving its dynamic interplay with the N-terminal tail of DHDDS, where expulsion of W3 results in the formation of an outlet through which product immersion into the adjacent lipid bilayer can be achieved, circumventing the need for housing long-chain products. Moreover, this mechanism may also explain the activity enhancement of h cis -PT observed in the presence of detergents or lipids ( 13 , 28 ), as well as the molecular basis for natural rubber synthesis ( 6 ).…”

“…DHDDS and NgBR are colored blue and yellow, respectively. Surface representations of the FPP and IPP [placed by superposition with PDB 6W2L ( 13 )] molecules are colored pink and green, respectively. The residue W3, at the DHDDS N terminus, is shown as spheres.…”

“…Dol-P serves as a lipidic glycosyl carrier for N-glycosylation, residing in the ER membrane ( 14 , 24 ). The structures of the soluble regions of h cis -PT (sh cis -PT), devoid of the transmembrane region of NgBR ( 12 , 13 ), revealed that h cis -PT is a heterotetramer, formed by two DHDDS-NgBR heterodimers (fig. S1).…”