Fig 1.
Atomic structure of CASV L-Cterm.
A) The structure of the protein dimer in the asymmetric unit is shown as a ribbon diagram in front and side view. Chain A is colored in blue and green, chain B is colored in dark and light grey. N- and C-termini are labelled. B) Chain A is shown as a ribbon diagram. N- and C-termini are labelled. Domain 1 is shown in blue, domain 2 in green. C) Superimposition of SAXS-derived molecular shape with the crystal structure (ribbon diagram) confirms the dimeric conformation of the protein at 1 mg/ml in solution.
Fig 2.
Comparison of CASV L-Cterm structure with influenza PB2 (PDB ID 5FMM) structure.
A) Comparison of domain arrangements within PB2 and L-Cterm. Identifiers of the areas within the protein are shown in the bars. Domain 1 (D1) of L-Cterm is separated into three parts (D1-I, D1-II, and D1-III). Linkers to domain 2 or the cap-binding domain are colored in yellow. Residue numbers of the differently colored areas are given below the bars. N- and C-termini are labelled. C-terminal parts of PB2 missing in the figure are indicated by dashed lines. B) Structures of parts I and II of L-Cterm domain 1 (left panel) and the mid-link domains of PB2 (right panel) are shown as ribbon diagrams. Colors are coded as presented in A). Linkers to domain 2 and the cap-binding domain are shown in yellow. N-termini are labelled. C) Comparison of L-Cterm domain 2 (left panel) with PB2 cap-binding domain (right panel) with structures presented as ribbon diagrams. Structurally similar elements have similar colors. Linkers to other domains are colored in yellow. D) Structural comparison of parts II and III of L-Cterm domain 1 (left panel) and link-627 domains of PB2 (right panel). Structures are shown as ribbon diagrams. Colors are coded as in A). β-strands of part III of L-Cterm domain 1 and 627-domain of PB2 are colored in red. C-termini are labelled.
Fig 3.
Topological overview of CASV L-Cterm and influenza virus PB2 structures.
A schematic representation of the general domain architecture of A) CASV L-Cterm domain 1 –parts I and II (colored in blue and teal, respectively) and domain 2 (colored in greens) as well as of B) influenza virus PB2 mid-domain (blue), cap-binding-domain (greens) and linker-domain (teal). α-Helices are shown as cylinders, β-strands as large arrows. The N-termini are labelled and the protein parts that follow the represented domains are indicated by a small arrow with names given.
Fig 4.
Examination of CASV L-Cterm cap-binding in comparison to influenza virus PB2.
A) Comparison of CASV L-Cterm domain 2 (left panel) with PB2 cap-binding domain (PDB ID 2VQZ, right panel) with structures presented as ribbon diagrams. Structurally similar elements have the same color. Potential cap-binding aromatic residues in CASV L-Cterm and actual cap-binding residues in PB2 are shown as sticks and colored in orange. Bound m7GTP molecules are shown as sticks. B) The figure shows binding of m7GTP to the CASV L-Cterm dimer in the crystal after soaking experiments. An overview (left) and close-up (right) are shown. CASV L-Cterm is presented as a ribbon diagram with the residues relevant for binding shown as sticks. m7GTP is shown as sticks and the surrounding electron density (2|Fo|-|Fc| map at 2σ) as blue mesh. C) Interaction of W1818 with P1810 from a neighboring loop. CASV L-Cterm domain 2 is shown as green ribbon diagram, potential cap-binding residue Y1872 and W1818 are shown as orange sticks, and P1810 as blue sticks.
Fig 5.
Atomic structure of isolated CASV L-Cterm domain 2.
A) Ribbon diagram of CASV L-Cterm domain 2 structure. Chain A is shown in palegreen, chain B in grey. The N- and C-termini are marked and potential cap-binding aromatic sidechains Y1872 and W1818 are shown as sticks and colored in orange. B) Superimposition of SAXS derived molecular shape of L-Cterm at a concentration of 4.5 mg/ml and ribbon diagram of crystal structure. C) Superimposition of ribbon diagrams of chain A and B from isolated CASV L-Cterm domain 2 crystal structure (magenta and yellow, respectively) and L-Cterm crystal structure (green). Potential cap-binding aromatic sidechains are highlighted with saturate colors. D) Representation of chain B of L-Cterm domain 2 colored by B-factor with the highest observed B being 106 (orange) and the lowest 22 (dark blue).
Fig 6.
Structure and thermal stabilization of CASV endonuclease.
A) Ribbon diagram of the CASV endonuclease crystal structure. N- and C-termini are labelled and active site residues are shown as sticks. The conserved β-sheet and the long α-helix are colored in orange, a conserved helix-bundle domain in green and the remaining part with loops and α-helices in yellow. B) Endonuclease structures of LASV (PDB ID 5J1P), LCMV (PDB ID 3JSB) and PICV (PDB ID 4I1T) are shown as ribbon diagrams and colored according to CASV endonuclease in A). Manganese ions of LASV structure 5J1P are shown as red spheres, active site residues are shown as sticks, and N- and C-termini are marked. C) Electrostatic surface potential of the endonuclease structures shown in A) and B). The surface potential is shown from -5 KT/e in red to +5 KT/e in blue and was calculated using PDB2PQR and the APBS-tool of PyMOL. D) Thermal stability of CASV endonuclease depending on Mn2+ concentration. Melting temperatures are presented as mean and standard deviations of three independent measurements. Stability of the protein was tested in presence of different concentrations of Mn2+ and in presence of 10 mM EDTA. E) Close-up of the superimposed endonuclease active sites of the structures shown in A) and B). Conserved active site residues are shown as sticks, and manganese ions of LASV structure 5J1P are shown as red spheres.