(A) Structures of C-4 methylated sterols synthesized by Methylococcus capsulatus. (B) Operon of sterol biosynthesis and transport genes and (top) and schematic description of their localization. OM: outer membrane; CM: cytoplasmic membrane.

Bioinformatic analysis of Bst proteins. (A-C): Protein sequence similarity networks (SSN) of BstA, BstB and BstC with the cutoff of 35%, 40%, and 40% sequence identity, respectively. Taxonomic distribution of Bst protein families showing their presence in bacterial (green), eukaryotic (purple), and archaeal (orange) genomes. (D) Genome neighborhood network of Bst proteins. Shown in circles and ovals are genes within 10 genes of BstC; colored in orange are those with identified or predicted roles in sterol synthesis and trafficking.

Binding of C-4 methylated sterols to transporter proteins.

(A) GC chromatograms of sterols bound to purified (40 μM) M. capsulatus BstAPD, BstB, and BstC during protein-lipid pull-down assays. The proteins preferentially bind 4-monomethyl (I) and 4,4-dimethyl (II) sterols when incubated with the total lipid extract (TLE) and hydroxy lipid (HS) fraction from M. capsulatus. (B) MST analysis of the proteins (50 nM) binding to C-4 methylated sterols (3.8 nM -1125 mM). Equilibrium dissociation constants (Kd) and Hill coefficients (nH) are reported on the plots.

Structure of BstB.

(A) Cartoon representation of BstB with the αhelixes and β-strands colored in red and magenta, respectively. (B) Hydrophobicity representations of BstB show a cleft in the middle and a cavity in domain A. The cavity is dominatingly hydrophobic and open only to one side. (C) Comparison of the cleft between the two domains of BstB, apo-PhnD (3S4U), and liganded PhnD (3P7I). BstB displays a similar but narrower opening (∼10 Å) compared to non-ligand PhnD (∼22 Å). (D) Docking model of the structures of BstB with 4-monomethyl sterol. Left, the docking model is shown in surface representation with 80% transparency. Sterol is shown in sphere and colored in green with the hydroxyl group is colored in red; Right, docking complex of MonoA. The surrounding residues are shown in stick and ball. Hydrogen bonds between the hydroxyl group of the sterol and Tyr120 is shown in the red dashed line.

Structure of BstC.

(A) Cartoon representation of BstC. The two αhelixes at the N-terminus are colored in green. Two monomers (RMSD of 0.656 A for all Cαs) with 180° rotational symmetry exist in the asymmetric unit. (B) A representation of cavities BstC (left, with Tunnel 1 colored in green, Cavity 1 in red, and Cavity 2 in blue) alongside hydrophobicity representation of the cavities (right). (C) Docking model of the structures of BstC with 4-monomethyl sterol. Left, the docking model is shown in surface representation with 80% transparency. Sterol is shown in sphere and colored in green with the hydroxyl group is colored in red; Right, docking complex before and after MD simulation. The surrounding residues are shown in stick and ball. The sterol prior to the MD simulation is colored in grey and while green shows its position after the simulation. A hydrogen bond between the hydroxyl group of the sterol and Glu86 is shown as a red dashed line.