Correspondence: Samantha Gildenhuys (gildes@unisa.ac.za) Coronavirus are the causative agents in many globally concerning respiratory disease outbreaks such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and coronavirus disease-2019 . It is therefore important that we improve our understanding of how the molecular components of the virus facilitate the viral life cycle. These details will allow for the design of effective interventions. Krichel and coauthors in their article in the Biochemical Journal provide molecular details of how the viral polyprotein (nsp7-10) produced from the positive single stranded RNA genome, is cleaved to form proteins that are part of the replication/transcription complex. The authors highlight the impact the polyprotein conformation has on the cleavage efficiency of the main protease (M pro ) and hence the order of release of non-structural proteins 7-10 (nsp7-10) of the SARS-CoV. Cleavage order is important in controlling viral processes and seems to have relevance in terms of the protein-protein complexes formed. The authors made use of mass spectrometry to advance our understanding of the mechanism by which coronaviruses control nsp 7, 8, 9 and 10 production in the virus life cycle.

A series of 2-aryl-3-hydroxy-6-iodo-4H-chromen-4-ones substituted at the 7-position with a halogen atom (X = F, Cl and Br) or methoxy group and their corresponding 4-substituted 2-hydroxy-5-iodochalcone precursors were evaluated in vitro for inhibitory effect against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and β-secretase (BACE1) activities. Although moderate inhibitory effect was observed for the chalcones against AChE, derivatives 2h, 2j and 2n exhibited significant inhibitory effect against BChE and BACE-1. The 2-aryl-7-fluoro-8-iodoflavonols 3b and 3c, on the other hand, exhibited increased activity and selectivity against AChE and reduced effect on BACE-1. The flavonols 3h, 3i, 3k, 3l and 3p exhibited moderate inhibitory effect against AChE, but significant inhibition against BChE. Compounds 2j and 3l exhibited non-competitive mode of inhibition against BACE-1. Molecular docking predicted strong interactions with the protein residues in the active site of BACE-1 implying these compounds bind with the substrate. Similarly docking studies predicted interaction of the most active compounds with both CAS and PAS of either AChE or BChE with mixed type of enzyme inhibition confirmed by kinetic studies.

Cytosolic class pi glutathione transferase P1-1 (GSTP1-1) is associated with drug resistance and proliferative pathways because of its catalytic detoxification properties and ability to bind and regulate protein kinases. The native wild-type protein is homodimeric, and whereas the dimeric structure is required for catalytic functionality, a monomeric and not dimeric form of class pi GST is reported to mediate its interaction with and inhibit the activity of the pro-apoptotic enzyme c-Jun N-terminal kinase (JNK) [Adler, V., et al. (1999) EMBO J. 18, 1321-1334]. Thus, the existence of a stable monomeric form of wild-type class pi GST appears to have physiological relevance. However, there are conflicting accounts of the subunit's intrinsic stability since it has been reported to be either unstable [Dirr, H., and Reinemer, P. (1991) Biochem. Biophys. Res. Commun. 180, 294-300] or stable [Aceto, A., et al. (1992) Biochem. J. 285, 241-245]. In this study, the conformational stability of GSTP1-1 was re-examined by equilibrium folding and unfolding kinetics experiments. The data do not demonstrate the existence of a stable monomer but that unfolding of hGSTP1-1 proceeds via an inactive, nativelike dimeric intermediate in which the highly dynamic helix 2 is unfolded. Furthermore, molecular modeling results indicate that a dimeric GSTP1-1 can bind JNK. According to the available evidence with regard to the stability of the monomeric and dimeric forms of GSTP1-1 and the modality of the GST-JNK interaction, formation of a complex between GSTP1-1 and JNK most likely involves the dimeric form of the GST and not its monomer as is commonly reported.

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Glutaredoxin 2 (Grx2) from Escherichia coli is monomeric and an atypical glutaredoxin that takes part in the monothiol deglutathionylation of proteins. Unlike its orthologs, Grx2 is a larger molecule with a canonical glutathione transferase (GST) fold that consists of two structurally distinct domains, an N-terminal glutaredoxin domain and a C-terminal alpha-helical domain. While GSTs are dimeric proteins, the conformational stability and unfolding kinetics of Grx2 were investigated to establish the contribution made by the domain interface to the stability of the tertiary structure of GST-like proteins without any influence from quaternary interactions. Equilibrium unfolding transitions for Grx2, using urea as a denaturant, are monophasic and exhibit coincidence of the fluorescence and CD data indicative of a concerted loss or formation of tertiary and secondary structure. The data fit well to a two-state N <--> U model with no evidence that an intermediate is being formed. The experimental m value [2.7 kcal mol (-1) (M urea) (-1)] is in excellent agreement with a predicted value of 2.5 kcal mol (-1) (M urea) (-1) based on the amount of surface area expected to become exposed during unfolding. These findings provide evidence that the two structurally distinct domains of Grx2 behave as a single cooperative folding unit. The unfolding kinetics are complex which, as a result of native-state heterogeneity, are characterized by two observable unfolding reactions that occur in parallel. A major population representing one distinct nativelike form unfolds on a fast track to denatured Grx2 with cis-Pro49. This is followed by a spectroscopically silent cis-trans proline isomerization reaction as determined by interrupted unfolding experiments. A minor population representing the other distinct nativelike form unfolds slowly with its rate being limited by an undetermined structural isomerization reaction. Further, there is no evidence indicating that unfolding proceeds via a high-energy intermediate that might suggest independent unfolding of the two nonidentical domains in Grx2. The kinetics data are, therefore, consistent with the existence of cooperativity between the domains, in agreement with the equilibrium data.