Table 1.
Crystallographic data collection and refinement statistics.
Statistics for the highest-resolution shell are shown in parentheses.
Fig 1.
The oligomeric state of the purified B4GalT1 catalytic domain.
(a). Gel filtration and SDS-PAGE profiles of the purified B4GalT1 catalytic domain. Molecular weight calculated from the sequence of the construct is 36.35 kDa. Elution volumes of the calibration proteins of the gel filtration column formed a standard curve with R2 value of 0.9993. Elution volumes, as seen from the scale in milliliters, are 14.7 ml for peak 1 and 17.6 ml for peak 2, and they correspond to molecular weights (indicated by arrows and figures in kDa) of 72.6 (dimer) and 36.3 kDa (monomer), respectively. PageRuler Prestained Protein Ladder (Thermo Fisher) was used as the molecular weight standard. (b). The two leftmost lanes show BSA (bovine serum albumin) and B4GalT1 as run on a native blue gel, while the rightmost lane shows a western blot analysis of the B4GalT1 sample detected with the B4GalT1 antibody. BSA fraction V (monomer size 66 kDa) and its oligomers (dimer, 133 kDa; trimer 200 kDa; tetramer 266 kDa) were used as a reference for molecular weight, as indicated in the figure in kDa.
Fig 2.
Structures of the human wild-type B4GalT1 in the open and closed conformation (left and right, PDB codes 6FWT and 6FWU, respectively). (a). Cartoon view of the structures. The highlighted areas are loop1 (blue), Trp loop (orange) and the lid (red). For loop1, the changes in secondary structure between open and closed structures are indicated with small arrows with “a” indicating residues 274–277 and “b” indicating residues 280–284, respectively. Trp310 is highlighted in orange sticks. Inset: Omit electron density map (2mFo-DFc) for Trp310. In the closed conformation structure the position of the closed lid (absent from the electron density), inferred by superimposition with the structure of the human mutated closed structure (PDB code 4EE4), is indicated as dashed black circles. (b, c). Close-up view of key amino acids in the vicinity of Trp310 and loop1, respectively. A faded image shows the corresponding structure in closed or open conformation or vice versa for easier comparison. One-letter code of amino acid residues is used for clarity due to space limitation in the figure.
Fig 3.
View of the dimer interface of human wild-type B4GalT1 as observed in the asymmetric unit of 6FWU (a): View of the dimer in a cartoon representation. The dimer interaction surface is highlighted in sticks in YRB coloring [54]: yellow (hydrophobic), red (negative), blue (positive), white (polar uncharged). (b, c): Detailed view of the interaction surface. Side chains of amino acids participating in the dimer interaction are shown in sticks and labeled. In panel (c), extra amino acids from the reconstructed lid (F356, I359, A360) involved in the interaction are depicted with gray background.
Table 2.
Summary of the analysis of the dimer interface of the human wild-type B4GalT1 closed conformation crystal structure, which shows metrics in accordance with biological relevance.
Metrics calculated by the jsPISA server are interface area (IA), solvation energy (DG) and total binding energy (BE). jsPISA score is a weighted average of each of the jsPISA radar metrics, for which a value higher than 50% depicts good chances of the interface being biologically relevant. DiMoVO score values below 0.5 predict crystallographic dimers, while values above 0.5 predict biological dimers.
Fig 4.
Dimer formation of full-length B4GalT1 type II membrane protein in vivo.
(a). Localization of the human wild-type B4GalT1 and its variants in COS-7 cells. Green: B4GalT1-mVenus. Red: GM130 Golgi marker antibody as detected by Alexa Fluor 594 conjugated secondary antibody. Yellow: the previous two merged in the same figure. Scale bar, 10 μM. (b). FRET inhibition assays in COS-7 cells. Note the almost complete loss of homodimers with the M340H and H343A constructs. Error bars are calculated from triplicate experiments.
Fig 5.
Structural differences between the wild-type and mutant B4GalT1 structures.
(a). Details of the Trp loop and lid regions in the open wild-type structure (orange and red, respectively, PDB code 6FWT) and in the open mutated structure (gray, PDB code 2FY7). (b). Details of the Trp loop and loop1 regions in the closed wild-type structure (orange and blue, respectively, PDB code 6FWU) and closed mutated structure (gray, PDB code 4EE4). (c, d). Root mean square deviation (RMSD) of atomic positions in pairwise comparisons plotted by residue number between 6FWT and 2FY7 (c), and 6FWU and 4EE4 (d). Differences (RMSD > 2 Å) are highlighted by arrows. Residues that could not be traced in the crystal structures were omitted in the RMSD plot (343–351 in 2FY7, 345–363 in 6FWU).
Fig 6.
Functional relevance of the B4GalT1 homodimers.
Monomers within the dimer are displayed with the surface representation and colored gray. Key residues and regions are highlighted as follows: Lid (red), loop1 (blue), Trp310 (orange), Asp315 (purple), Met340 (yellow), His343 (green). Darker color tints are used for the second monomer in the dimer. (a). Relative positions of the active site positions (black arrows) in the B4GalT1 homodimer structure (PDB code 6FWU). (b). Model of a B4GalT1 dimer with one chain in an active conformation (including the donor UDP-Gal, acceptor GlcNAc and Mn2+ ion from PDB codes 1O0R and 4EE4, respectively) on the B4GalT1 homodimer structure (PDB code 6FWU) with reconstructed lid.