Fig 1.
Biochemical characterization of ALSV MTase.
(A) A schematic view of the protein-engineering strategy used to yield ALSV MTase. The putative signal peptide (SP), the methyltransferase domain, and the RNA-dependent RNA polymerase domain are individually marked with the boundary-residue numbers. ALSV MTase is initially expressed as a GST-fusion protein, which is then enzymatically cleaved to remove the GST tag. (B and C) The upper panel, solution behavior of native (B) or refolded (C) ALSV MTase protein on a Superdex 200 Increase 10/300 GL column. The inset figure shows the SDS-PAGE analyses of the pooled samples. The bottom panel, affinity determination between native (B) or refolded (C) ALSV MTase and SAM or SAH using ITC.
Fig 2.
(A) Overall structure of ALSV MTase with three subdomains being presented in violet (N-terminal extension region), cyan (core region) and lemon (C-terminal extension region) colors, respectively. The secondary structural elements are labeled with nomenclatures described for DENV2 MTase [22]. The regions that are not visible in the structure are indicated with dashed lines. The terminal residues that are density-traceable are also labeled. (B) The upper panel, superposition of the ALSV MTase structure onto the previously reported DENV3 MTase structure (PDB code: 4R8R) [32]. The color scheme for our structure is the same as in (A), and the DENV3 MTase structure is shown in gray. The bottom panel, comparison of the individual ALSV MTase subdomains with those of DENV3 MTase. (C) Topological plots of ALSV MTase [shown in upper panel, subdomains are individually colored in violet (N-terminal extension region), cyan (core region) and yellow (C-terminal extension region)] and DENV3 MTase [shown in bottom panel, subdomains are individually colored in green (N-terminal extension region), gray (core region) and orange (C-terminal extension region). Cylinders and arrows represent helices and strands, respectively. Compared with ALSV MTase, the additional secondary structural elements in DENV3 MTase are highlighted by oval boxes.
Fig 3.
Complex structures of ALSV MTase with SAM and SAH.
(A and D) An electrostatic-surface representation of ALSV MTase in the SAM-bound (A) and SAH-bound (D) forms. The SAM and SAH molecules are shown as yellow and green sticks, respectively. The SAM/SAH-binding pocket is indicated with black dotted rectangle. The bound SAM and SAH molecules, whose electron densities are contoured at 2.8 σ using the |Fo|-|Fc| map, are shown in the black rectangle. (B and E) A cartoon representation of ALSV MTase in the SAM-bound (B) and SAH-bound (E) forms. Those structural elements involved in the formation of the ligand-binding pocket are labeled. (C and F) Detailed interactions between ALSV MTase and SAM (C) or SAH (F). Dashed lines indicate hydrogen bonds. (G, H and I) Overview of the SAM-binding pocket in DENV3 MTase (based on PDB code: 5E9Q). (G) An electrostatic-surface representation of DENV3 MTase bound with SAM. The SAM molecule is shown as orange sticks. (H) A cartoon representation of DENV3 MTase bound with SAM. Those structural elements involved in the formation of the ligand-binding pocket are labeled. (I) Detailed interactions between DENV3 MTase and SAM. Dashed lines indicate hydrogen bonds. Those SAM-binding residues in DENV3 MTase that are equivalent to those responsible for ALSV-MTase/SAM interactions are shown as cyan sticks, while the other two residues in DENV3 MTase that form additional hydrogen bonds with SAM are highlighted and shown as gray sticks.
Fig 4.
Comparison of the SAM binding pockets between ALSV MTase and the canonical flavivirus MTase.
(A) Superposition of the ALSV MTase/SAM structure onto the previously reported DENV3 MTase/SAM structure (PDB code: 5E9Q) [33]. Electrostatic-surface representations of SAM binding pockets are shown on the right panel. (B) Comparison of the β2/β3 interloops between ALSV MTase and DENV3 MTase. Interactions between β2/β3 interloops and SAM molecules are shown on the right panel. Dashed lines indicate hydrogen bonds. (C) Structure-based multiple sequence alignment of methyltransferases from Jingmenvirus group and canonical flaviviruses. The secondary structural elements of ALSV MTase and DENV3 MTase are labeled above and below the sequences, respectively. The residues that interact with SAM molecules are labeled with blue boxes (for ALSV MTase) and grey triangles (for DENV3 MTase). The β2/β3 interloops are highlighted with violet rectangle.
Fig 5.
Affinity determination between MTase mutants and SAM using ITC.
(A) Binding of SAM to native or refolded DENV3 MTase mutants (H110A, E111A, E112A, H110A/E111A or H110A/E111A/E112A). (B) Binding of SAM to native ALSV MTase mutants (K153A, E154A or E155A). (C) Summary of affinity-fold change between mutant MTase and wild-type MTase [Kd (mutant/wild type)]. The Kd values for wild-type DENV3 MTase are shown in S1C Fig (native protein) and S1D Fig (refolded protein). The Kd value for wild-type ALSV MTase is shown in Fig 1B (native protein).
Fig 6.
Binding capacity of SIN towards ALSV MTase and the molecular basis of complex formation.
(A) The chemical formula of sinefungin (SIN), which includes a C-NH2 group to substitute the S-CH3 group of methyl donor SAM. (B) Affinity determination between ALSV MTase and SIN using ITC. (C) The overall electrostatic-surface representation of ALSV MTase in the SIN-bound form. The bound SIN molecule, whose electron density is contoured at 2.8 σ using the |Fo|-|Fc| map, is shown in the black rectangle. (D) Detailed interactions between ALSV MTase and SIN. Dashed lines indicate hydrogen bonds.