Structural Analysis of Papain-Like NlpC/P60 Superfamily Enzymes with a Circularly Permuted Topology Reveals Potential Lipid Binding Sites

NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, “closed” conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes

ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping

Objectives ST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. Methods Vaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. Results Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. Conclusions Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved

Summary of histologic findings for placebo treated NHPs that met study endpoint before end of study.

Summary of histologic findings for placebo treated NHPs that met study endpoint before end of study.</p

Transmission electron micrographs of VARV infected, placebo-treated NHP haired skin.

Panels A, B, C all reveal flaking epidermis and disorganized dermal skin layers in three euthanized variola infected, placebo-treated non-human primates. Panels A’, B’, and C’ are higher magnification images of the boxed regions in panels A, B, and C, respectively, revealing the presence of VARV. Immature (5C’, white asterisk) and mature virions are evident. Brick-shaped, enveloped mature (black asterisk) and mature virions with bar-bell shaped cores (arrowheads) are readily visible. Inset in A’ shows the dimensions (~ 139nm X 246nm) of one mature virion. Superficial bacteria are also evident (arrows) in panels A and C’ (arrows). A’, B’, C’ scalebar = 500nm.</p

Variola-infected, placebo treated NHP x days post infection.

Haired skin, pox lesions. A: (H&E, 4x) Proliferative and necrotizing dermatitis. B: (IHC, 4x) Viral antigen is present within epithelial cells at the edge of the pox lesion. C: (H&E 20x) Higher magnification of A, Necrotic debris and superficial serocellular crust. D: (IHC, 40x) Viral antigen in epithelial cells. E: (40x/100x) Higher magnification of A, Pyknotic and karyorrhectic (necrotic) debris with intracytoplasmic inclusions (arrowhead; inset, higher magnification of black box) and ballooning degeneration (arrow) as well as surface colonies of bacteria. F: (IHC, 60x) Viral antigen in epithelial cells.</p