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

Abstract

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

Fig. 1.. Overview of hcis-PT structure and reaction scheme.

(A) Cartoon representation of a single DHDDS-NgBR heterodimer in complex with FPP [Protein Data Bank (PDB) 6Z1N] (12). 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. (B) Condensation reaction scheme. At the first cycle, the allylic diphosphate primer, FPP (C₁₅, pink), undergoes a condensation with IPP (C₅, green) to produce geranylgeranyl-diphosphate (GGPP) (C₂₀). The cycle repeats with further condensations (14–17) of the allylic diphosphate at S₁, ultimately leading to a final product length of C_85–100.

Fig. 2.. Structural analysis of shcis-PT_cryst reveals active site reorganization along the first condensation cycle.

(A to D) Cartoon representation of a single DHDDS-NgBR heterodimer (left column) and zoomed-in perspectives of the S₁ and S₂ inlet (right column) in complex with FsPP and IPP (A), GGsPP and sulfate (B), GGsPP and IsPP (C), or GGPP and IsPP (D). DHDDS and NgBR are colored blue and yellow, respectively. Coordinating residues, bound substrates, and/or products are shown as sticks. Mg²⁺ and water molecules are shown as purple and red spheres, respectively. A difference map (DF_o − mF_c) is shown as a gray mesh (contoured at σ = 2.5). Polar interactions are highlighted as dashed black lines, and R290 is highlighted by spheres.

Fig. 3.. The N terminus of DHDDS translocates from the active site outlet during the catalytic cycle.

(A to D) Cartoon representations of DHDDS in complex with FsPP and IPP (A), GGsPP and sulfate (B), GGsPP and IsPP (C), and GGPP and IsPP (D). Bound substrates, W3, serving as a stopcock for the active site outlet, and F154, stabilizing the bent conformation of the C₂₀ product within the active site, are shown as sticks. The terminal carbons of the substrate at S₁ are framed. (E) Superposition of shcis-PT_cryst in complex with GGsPP (cyan) and GGPP-IsPP (blue). The N terminus of DHDDS, GGsPP, and GGPP are shown as sticks. (F) 2F_o − F_c electron density maps of the N terminus of DHDDS, contoured at σ = 1, are shown as green mesh for shcis-PT_cryst in complex with GGsPP (left) and GGPP-IsPP (right). No electron density could be identified upstream of residue G7. (G) SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of shcis-PT following limited proteolysis by the indicated proteases, in the presence or absence of substrates, as described in Materials and Methods. The DHDDS cleavage product is indicated by an arrow. (H) KG405 cells were cotransformed with hcis-PT harboring the indicated DHDDS subunits. Transformants were plated in serial dilutions with or without 5-fluoroorotic acid (FOA), as indicated (left). W3R failed to produce functional complementation. Glcis-PT served as a positive control, and transformations with either subunit (DHDDS or NgBR) or empty vectors were used as negative controls (right). WT, wild type.

Fig. 4.. HDX-MS profile of shcis-PT active site supports separated and dynamic inlet and outlet.

(A) Cartoon representations of a single DHDDS-NgBR heterodimer. Heatmap coloring represents the HDX level following 20-s incubation in D₂O, with highest and lowest exchange levels indicated in red and blue, respectively. In each orientation, an inset provides a DHDDS (blue) and NgBR (yellow) heterodimer surface representation, for orientation purpose. The N terminus, α2, and α3 of DHDDS and the C terminus of NgBR are indicated. (B) Deuteration levels at the indicated time points for DHDDS (top) and NgBR (bottom). The secondary structures elements are indicated above the heatmaps for each protein.

Fig. 5.. MANT-O-GPP binds at S₁ and is a viable substrate of shcis-PT.

(A) MANT-O-GPP–dependent shcis-PT activity in the presence of 100 μM IPP, measured as ¹⁴C-IPP incorporation (n = 3, mean ± SEM). (B) Tryptophan emission spectrum (left) and MANT-O-GPP excitation (purple) and emission (pink) spectra (right). The chemical schemes of tryptophan and MANT-O-GPP are depicted. (C) Cartoon representation of shcis-PT_cryst in complex with FPP (PDB 6Z1N), highlighting the close proximity between FPP (green sticks) and W3 (blue sticks). (D to F) Average fluorescence emission spectra following excitation at F₂₈₀. Increasing concentrations of MANT-O-GPP resulted in concomitant decrease of F₃₄₀ and increase of F₄₂₀ in shcis-PT–WT (n = 3) (D) or W3L mutant (n = 3) (F). Increasing FPP concentrations, in the presence of constant MANT-O-GPP (10 μM) resulted in concomitant increase of F₃₄₀ and decrease of F₄₂₀, indicating MANT-O-GPP displacement (n = 3) (E). (G) F₄₂₀/F₃₄₀ as a function of MANT-O-GPP concentration, measured using shcis-PT–WT or W3L (n = 3, mean ± SEM).

Fig. 6.. W3 is involved in determining the rate of the first condensation by shcis-PT.

(A) FPP-dependent activity was measured as ¹⁴C-IPP incorporation in the presence of 100 μM IPP. Data are presented as means ± SEM, n = 3. (B) Averaged stopped-flow kinetics of a single condensation reaction measured using FRET as described in Materials and Methods. shcis-PT (2 μM; WT or W3L) preincubated with 2 μM MANT-O-GPP was mixed in a 1:1 ratio with 10 μM IsPP. Data were fit to a single-exponential equation. (C) Rate constants determined from individual stopped-flow kinetics traces presented as means ± SEM (n = 14 and 20 for WT and W3L, respectively; unpaired t test with Welch’s correction; ****P < 0.0001).

Fig. 7.. Direct product-lipid interactions enhance long-chain isoprenoid synthesis by shcis-PT.

(A) Average time-dependent increase in MANT-O-GPP fluorescence (F₄₂₀) in the presence of increasing MSP1D1E3 nanodisc molar ratios (n = 3 for each molar ratio). A.U., arbitrary units. (B) TLC analysis of the reaction products formed in the absence (−) or presence (+) of nanodiscs (2:1 nanodiscs:shcis-PT ratio), with MANT-O-GPP as a substrate, detected using ultraviolet illumination (λ = 365 nm). (C) MANT-O-GPP emission spectrum (left) and PE-NBD excitation (blue) and emission (green) spectra (right). The experimental scheme is depicted above spectra. (D) Averaged fluorescence emission spectra following excitation at F₃₅₂. Incubation of shcis-PT in the presence of MANT-O-GPP, IPP, Mg²⁺, and PE-NBD–labeled nanodiscs (“all components”) resulted in a fluorescence peak corresponding to PE-NBD emission (F₅₃₂) due to FRET (green) (n = 3 for each condition). No FRET was observed upon omitting any of the reaction constituents. (E) Average time-dependent MANT-O-GPP fluorescence (F₄₂₀) and PE-NBD FRET (F₅₃₂) traces reveal nearly identical time dependence (n = 3). Em, emission. (F) Comparison of the averaged fluorescence emission spectra following excitation at F₃₅₂ and incubation of shcis-PT in the presence of “all components” with IsPP instead of IPP (black) (n = 3). The emission spectra in the presence of IPP is shown as dashed gray curve.

Fig. 8.. Proposed model for long-chain isoprenoid synthesis.

(A) Before the initial condensation reaction, FPP (pink) and IPP (green) occupy the DHDDS active site. The active site outlet is occluded by the DHDDS N terminus. (B) Following the condensation reaction, the N terminus of DHDDS dislodges from the outlet, allowing extension of the elongating product to the vicinity of the ER membrane. Further product lengthening is enabled by its insertion into the bilayer. Created with BioRender.com.