Extended Data Fig. 4: The large VKGC chamber may permit the binding of the entire Glu-rich region and the folding of Gla-rich region into helices.
From: Molecular basis of vitamin-K-driven γ-carboxylation at the membrane interface
a, Model of the 64 AA Prop-Glu region of FIX in an extended conformation within the large chamber of VKGC. The modelling used HADDOCK76, with basic residues in VKGC and Glu residues in FIX Glu region selected for direct interactions. The modelled part of Glu-rich region is coloured in purple, and the propeptide and Glu-rich region observed in cryo-EM structure in orange and red, respectively. b, View of the FIX Prop-Glu model with VKGC shown in electrostatic surface. The lower part of the large chamber is positively charged, encircled by a belt of basic residues that potentially facilitate the binding of negatively charged Glu or Gla residues24,58 during processive γ-carboxylation. c, The cryo-EM model of partially carboxylated FIX in front (left) and side (right) views with the ER membrane modelled by PPM analysis25. The double arrow indicates proximity of the Gla-rich region to the membrane opening of VKGC. d, MS quantification of γ-carboxylation levels of individual Glu residues in partially carboxylated FIX fused with VKGC. Data are mean ± s.d. from n = 3 biological replicates. Glu/Gla residues detectable by ETD fragmentation were shown; ETD was used to optimally preserve γ-carboxylation86,87. The relative carboxylation levels are consistent with HCD fragmentation, but the absolute levels in HCD are relatively lower due to partial loss of this labile post-translational modification. e, MS-MS spectra of γ-carboxylated peptides (used in d) with the ETD fragmentation mode. f, Conformational change of the FIX Glu-rich region from the uncarboxylated state (propeptide-tethered, VKGC bound, and without Ca2+; Fig. 4a–e), the partially carboxylated state (propeptide-tethered, VKGC bound, and with Ca2+, as in c), to the carboxylated, functional state (in absence of propeptide and VKGC, but with Ca2+; PDB 1NL0)88. The uncarboxylated and partially carboxylated structures are superimposed, and the fully carboxylated structure is shown in a similar N to C direction. g, Density maps of the propeptide (left) and partially carboxylated Gla-rich region (right, unsharpened map) of FIX.
