Table 1.
Data collection and refinement statistics for the MtL structure.
Fig 1.
A) SDS-PAGE reveals a pure protein with an apparent molecular mass of 83 kDa. B) IEF analysis shows a highly homogeneous sample with a pI around 4.2. C) Capillary electrophoresis profiles of MtL under native conditions, 2.2 mg/ml in 20 mM Na/K-phosphate buffers at pH 5 (solid line) and 8 (dashed line), respectively. In both cases a single peak is observed. D) MALDI-TOF spectrum of MtL revealed that the mass of the intact protein is only around 74.7 kDa.
Fig 2.
Size-exclusion chromatography analysis of MtL.
Analytical size-exclusion on a Superdex 200 HR 10/30 column equilibrated with 50 mM NaH2PO4, 150 mM NaCl, pH 7. Molecular weight protein standards: bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa) and horse myoglobulin (17 kDa). The size estimate for MtL is 128 kDa. The figure is adapted from [30].
Fig 3.
MtL structure with the three cupredoxin-like domains color-coded as follows: domain A (1–161) blue, domain B (162–341) green and domain (342–559) yellow. The four catalytic Cu ions are shown as orange spheres. Ligands from the PDB entries 3FU7 (MaL:2,6-dimethoxyphenol; 2,6-DMP displayed as black spheres) and 4YVN (CotA:ABTS; ABTS displayed as cyan spheres) have been superimposed on the MtL structure to illustrate the location of the T1-substrate binding pocket and an alternative substrate binding site, respectively.
Fig 4.
Structure-based alignment of MtL, MaL (2Q9O), TaL (3PPS), BaL (3QSR) and AnL (5LM8).
Structural features of particular interest in this study, such as glycosylation, dimer interface and MtL variants, are indicated above the alignment. For MtL, the putative consensus sites for N-glycosylation (N-x-T/S) are shown as orange triangles. The triangles are filled at the positions where glycan was observed in the structure. Residues located at the interface of the MtL dimer are indicated with an “i”. MtL variants discussed in the paper are denoted with blue triangles. Conserved secondary structure elements, domains, Cu-ligands and the residues forming the T1-pocket are shown below the alignment. β-Strands are color-coded corresponding to the three domains (A residues 1–161 blue, B residues 162–341 green and C residues 342–559 yellow) as in the Fig 3. Cu-ligands are represented with orange circles. Open circles indicate hydrophobic ligands like Leu513. Hydrophobic residues that line the T1-pocket, based on the MaL:2,6-DMP complex [8], are indicated with black dots while His508 and Glu235, which form the polar recognition motif, are shown as blue and red dots, respectively. The extra polar residue, Glu497, in the T1-pocket of BaL (and AnL) is indicated with a green dot. Note that the N-terminal region of the AnL structure is disordered and the residue numbers for AnL in the MSA must be corrected (+27) to match the numbering in the mature enzyme sequence.
Fig 5.
Phenolic substrate binding pockets in asco-type laccases.
A) Superposition of MtL (blue) and TaL (3PPS, green) with the MaL:2,6-DMP complex (3FU7, grey). MtL residues lining the pocket are labelled. Glu235 and His508 that comprise the polar recognition motif are highlighted with red and blue bars, respectively. B) Superposition of BaL (3SQR, yellow) and AnL (5LM8, orange). The polar recognition motif and the extra polar residue, Glu497 (BaL numbering), are indicated. C)—G) Surface representations illustrating the topology of the T1-pockets and mapping of polar residues. Color-coding as in A)—B). H) Structural superposition shown as Cα-traces illustrating variations in loops around the T1-pocket. The ligand 2,6-DMP (black spheres) from 3FU7 indicates the location of the T1-pocket.
Fig 6.
Dimeric MtL assembly observed in the crystal.
A) Surface representation with protomers shown in black and white, respectively. In this dimeric arrangement, the only entrance to the T1-pocket is through the conserved, (putative) substrate entrance channels [8] indicated in yellow. B) Zoom in on the dimer interface near the T1-pocket. Cartoon representation with selected residues shown as sticks. To mimick the position of a putative ligand, 2,6-DMP (dotted surface) from the MaL complex (3FU7) was superimposed on each monomer separately. In the MtL structure, the Phe427 docks into the T1-pocket of the other protomer and thereby hampering substrate binding.
Table 2.
Dimer interface analysis (PISA server).
Fig 7.
Conservation of glycosylation and surface residues between asco-laccases.
A)- D) Surface representations of MtL color-coded according to domains and showing the distribution of glycans around the protein. Glycosylation seen in the MtL crystal structure are shown as orange sticks together with the numbering of the anchoring Asn residues. E)- H) Surface representations of MtL with mapping of sequence conservation according to the MSA in Fig 4. Residues that are fully conserved among all five asco-laccases with known structures (MtL, MaL, TaL, BaL and AnL) are shown in dark blue (including conservative substitutions Arg/Lys and Asp/Glu). Additional residues that are only conserved within the MtL-MaL-TaL subgroup, but not in BaL and/or AnL, are mapped in light blue. Ligands from 3FU7 (2,6-DMP black) and 4YVN (ABTS cyan) are shown as space-filling molecules to indicate the location of the T1-substrate binding pocket and putative ABTS-site, respectively. Surface areas corresponding to the MtL dimer interface and the entry/exit of the T2-solvent channel are indicated with dashed green lines.
Fig 8.
A) Location of the Tyr residues mutated to Ile in this study. The MtL structure is shown in cartoon representation and Tyr residues as blue sticks. B) Tyr17 stabilizes loops in the N-terminal extension through hydrogen bonds and stacking interactions with Pro28. C) Tyr36 connects two β-sheets within domain A. D) Tyr416 packs in a hydrophobic environment and helps to stabilize a neighbouring loop via a hydrogen bond to the carbonyl oxygen of Pro347. E) The aromatic side chain of Tyr552 stacks between the glycan attached to Asn88 and Pro551 near the entry site of the C-terminal plug, shown to be essential for asco-laccase activity [29].
Table 3.
Activity of MtL variants in SGZ-assay.
Table 4.
N-glycosylation observed in crystal structures of asco-laccases.