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Enterovirus 71 (EV71) is the causative agent of hand, foot and mouth disease and can spread its infections to the central nervous and other systems with severe consequences. The replication of EV71 depends on its 3C proteinase (3Cpro), a significant drug target. By X-ray crystallography and functional assays, the interactions between inhibitors and EV71 3Cpro were evaluated. It was shown that improved interactions at S4 for the substrate binding could significantly enhance the potency. A new series of potent inhibitors with high ligand efficiency was generated for developing antivirals to treat and control the EV71-associated diseases. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Pyrazinamide ( PZA) is a first-line drug for tuberculosis ( TB) treatment and is responsible for shortening the duration of TB therapy. The mode of action of PZA remains elusive. RpsA, the ribosomal protein S1 of M ycobacterium tuberculosis ( M tb), was recently identified as a target of PZA based on its binding activity to pyrazinoic acid ( POA), the active form of PZA. POA binding to RpsA led to the inhibition of trans-translation. However, the nature of the RpsA- POA interaction remains unknown. Key questions include why POA exhibits an exquisite specificity to RpsA of M tb and how RpsA mutations confer PZA resistance. Here, we report the crystal structures of the C-terminal domain of RpsA of M tb and its complex with POA, as well as the corresponding domains of two RpsA variants that are associated with PZA resistance. Structural analysis reveals that POA binds to RpsA through hydrogen bonds and hydrophobic interactions, mediated mainly by residues ( Lys303, Phe307, Phe310 and Arg357) that are essential for tmRNA binding. Conformational changes induced by mutation or sequence variation at the C-terminus of RpsA abolish the POA binding activity. Our findings provide insights into the mode of action of PZA and molecular basis of PZA resistance associated with RpsA mutations. [ABSTRACT FROM AUTHOR]

The crystal structure of 3C proteinase (3Cpro) from Enterovirus 71 (EV71) was determined in space group C2221 to 2.2 Å resolution. The fold was similar to that of 3Cpro from other picornaviruses, but the difference in the β-ribbon reported in a previous structure was not observed. This β-ribbon was folded over the substrate-binding cleft and constituted part of the essential binding sites for interaction with the substrate. The structure of its complex with rupintrivir (AG7088), a peptidomimetic inhibitor, was also characterized in space group P212121 to 1.96 Å resolution. The inhibitor was accommodated without any spatial hindrance despite the more constricted binding site; this was confirmed by functional assays, in which the inhibitor showed comparable potency towards EV71 3Cpro and human rhinovirus 3Cpro, which is the target that rupintrivir was designed against. [ABSTRACT FROM AUTHOR]

Data acquisition from crystals of an icosahedral virus, cowpea mosaic virus (CPMV), was carried out to 2.8 Å resolution under an elevated hydrostatic pressure of 330 Mpa. This was the first example of a complex macromolecular assembly to be studied by high-pressure crystallography. The data were obtained from he ESRF ID30 beamline using a quasi-plane wave of ultrashort wavelength with a diamond anvil cell and an imaging-plate detector. The results of the high-pressure data analysis are given and are compared with those obtained under standard conditions, showing that the experimental procedures implemented are very efficient in terms of diffraction information collected per unit volume of crystal. These results suggest that the use of a quasi-parallel sychrotron radiation beam of ultrashort wavelength should also be considered for conventional macromolecular crystallography data collection. [ABSTRACT FROM AUTHOR]

A previous study showed that the diffraction from cubic crystals of an icosahedral virus, cowpea mosaic virus (CPMV), was dramatically improved under elevated hydrostatic pressure. This use of pressure may have a significant impact on structural biology if it is found to be generally applicable. There were two types of cubic crystals assigned in either an I23 or P23 space group. They show the same rhombic dodecahedral morphology at atmospheric pressure. The crystals assigned to the I23 space group diffracted X-rays to higher resolution than those with P23 space group. The assignment of P23 space group was owing to the presence of reflections with indices of h + k + l = (2n + 1) (odd reflections), which are forbidden in space group I23. Analysis of the odd reflections from the P23 crystals at atmospheric pressure showed that they can originate from a rotational disorder in the I23 crystals. The odd reflections were eliminated with the application of 3.5 kbar of pressure, which transformed the crystals from the apparently primitive cell to the body-centered I23 space group with dramatic improvement in diffraction. A mechanistic model is proposed to describe the induction of order by rectifying the imperfection, which is consistent with the experimental data.