Table 1.
Data collection and refinement statistics for AVCP in apo form and dioxane bound form.
Figure 1.
Structure of Aura Virus Capsid Protease.
(A) Overall structure of the capsid protein with two β-barrels of subdomains I and II colored in blue and green respectively. The catalytic triad residues and Trp267 are shown in sticks; (B) Close-up view of the active site shows catalytic triad composed of Ser218, Asp166 and His144 along with the carboxy-terminal Trp267 approaching the active site.
Figure 2.
Multiple sequence alignment of AVCP with CPs from other alphaviruses.
The conserved residues are shown in red background. The circles under the amino acids indicate the hydrophobic pocket residues interacting with the dioxane molecule while the triangles denote the catalytic triad residues. The residues forming salt bridges are denoted by squares below the residues. The motif responsible for specificity in encapsidation is highlighted in rectangular box 1. Interdomain flexible loop residues are shown in box 2 whereas the flexible loop separating the two pockets for E1 and E2 binding is shown in box 3. Capsid protein sequences used for alignment are: Aura virus (AVCP), Sindbis (SCP), Ross River (RRCP), Semliki Forest (SFCP), Chikungunya (ChikVCP), Venezuelan equine encephalitis (VEEVCP) and Western equine encephalitis (WEEVCP).
Figure 3.
Structural alignment of CPs from different alphaviruses.
Structure alignment of both apo (green) and complex (blue) form of AVCP with SCP (pink), SFCP(yellow) and VEEVCP (cyan) with PDB IDs: 1KXA, 1VCP and 1EP5 respectively showing structural variation. Three circles show loop regions of high variability that include inter-domain flexible loop region (Circle 2) and flexible loop that separate the two pockets responsible for binding to E1 and E2 (Circle 3).
Figure 4.
CP-glycoproteins interaction along with analysis of different amino acids in cdE2.
(A) Representation of the crystal structure of AVCP-dioxane complex and the homology models of E1 and E2 glycoproteins fitted into the cryo-EM electron density map of VEEV (EMDB ID: 5275). AVCP, E1 and E2 are shown in green, yellow and pink respectively; bound dioxane is shown in red while the cryo-EM density map is denoted by gray; (B) Structural alignment of cdE2 predicted structure from Aura virus (pink) and VEEV (blue) showing Tyr401 residue oriented towards other helix away from CP and the disulfide bond (yellow) within the helix-loop-helix motif of E2. The Pro405 interaction with the hydrophobic pocket present on the surface of the AVCP crystal structure is also shown; (C) Cartoon view of the AVCP-glycoproteins interaction. Binding of Aura virus E2 and E1 cytoplasmic tails to different pockets (P1 and P2 respectively) found in close proximity at the surface of AVCP. E2 shows the helix-loop-helix structural architecture and E1 has a helical structure that interacts with AVCP. The loop separating both interacting pockets is shown in red color; (D) The critical polar interactions between E2 and capsid as well as within the helix-loop-helix motif of cdE2 are shown in dotted lines. E2 residues are shown in pink while the AVCP residues are displayed in green. The interacting residues are shown as sticks.
Figure 5.
Multiple sequence alignment of the cytoplasmic domain of E2 (cdE2) from different alphaviruses.
The secondary structure of 33 residues from Aura virus cdE2 displays a helix-loop-helix structure. Highly conserved residues are in red background. The circles under residues denote the disulfide bridge forming Cys residues, squares indicate the residues responsible for polar interactions within the E2 helix-loop-helix motif and the triangles represent the residues of E2 making polar interactions with CP. cdE2 sequences used for alignment are: Sindbis (SinVcdE2), Western equine encephalitis (WEEVcdE2), Aura (AuraVcdE2), Chikungunya (ChikVcdE2), Venezuelan equine encephalitis (VEEVcdE2), Ross River (RRVcdE2) and Semliki Forest (SFVcdE2).
Figure 6.
AVCP-dioxane complex structure showing the dioxane binding pocket and the position of Pro405 from cdE2.
(A) Hydrophobic pocket showing a bound dioxane molecule on the surface of AVCP crystal structure with 2Fobs-Fcalc electron density contoured at 1σ level. The residues present in the hydrophobic pocket are shown in violet and the dioxane molecule in blue. The electron density of the pocket is shown in gray; (B) The hydrophobic pocket of the AVCP complexed with dioxane as revealed by crystal structural studies. The interacting residues (violet) and dioxane molecule (blue) are shown as sticks. The hydrophobic interactions shown in dashed lines were analyzed by ligand explorer of RCSB PDB (ligpro.sdsc.edu); (C) Superimposition of dioxane bound forms of AVCP (violet) and SCP (1WYK) (green) crystal structures highlighting the hydrophobic pocket residues; (D) The dioxane molecule (blue) in the crystal structure of AVCP-dioxane complex occupies the same position as Pro405 (pink) present in the helix-loop-helix motif of cdE2. A homology model of cdE2 was combined with the AVCP crystal structure. The residues in AVCP that are involved in the interaction are shown as gray sticks.