Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145–His41 catalytic dyad. We here characterized the catalytic roles of the cysteine–histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine–histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

The high proliferation rate of tumor cells demands high energy and metabolites that are sustained by a high glycolytic flux known as the ‘Warburg effect’. The activation and further metabolism of glucose is initiated by hexokinase, a focal point of metabolic regulation. The human hexokinase 2 (HK2) is overexpressed in all aggressive tumors and predominantly found on the outer mitochondrial membrane, where interactions through its N-terminus initiates and maintains tumorigenesis. Here, we report the structure of HK2 in complex with glucose and glucose-6-phosphate (G6P). Structural and biochemical characterization of the mitochondrial conformation reveals higher conformational stability and slow protein unfolding rate (ku) compared with the cytosolic conformation. Despite the active site similarity of all human hexokinases, the N-domain of HK2 is catalytically active but not in hexokinase 1 and 3. Helix-α13 that protrudes out of the N-domain to link it to the C-domain of HK2 is found to be important in maintaining the catalytic activity of the N-half. In addition, the N-domain of HK2 regulates the stability of the whole enzyme in contrast with the C-domain. Glucose binding enhanced the stability of the wild-type (WT) enzyme and the single mutant D657A of the C-domain, but it did not increase the stability of the D209A mutant of the N-domain. The interaction of HK2 with the mitochondria through its N-half is proposed to facilitate higher stability on the mitochondria. The identification of structural and biochemical differences between HK2 and other human hexokinase isozymes could potentially be used in the development of new anticancer therapies.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here may improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the discovery of drugs and design of antiviral therapeutics against COVID-19.

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Fluorescence quenching of lipid-bound pyrene was used to assess the binding of cytochrome c (cyt c) to liposomes that mimic the inner mitochondrial membrane (IMM) POPC/DOPE/TOCL, with the conditions that it did or did not contain oxidized phosphatidylcholine molecules, i.e., 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), or a mixture of two hydroperoxide isomers derived from POPC (POPCOX). The binding isotherms reveal two dissociation constants, K(D)(1) and K(D)(2), representing, respectively, the low- and high-affinity states of the membrane. These dissociation constants probably are due to the lipid reorganization promoted by cyt c, as observed in giant unilamellar vesicles that contain fluorescent cardiolipin (CL). The presence of PazePC, which has a nonreactive carboxylic group, increased the K(D)(1) and K(D)(2) values 1.2- and 4.5-fold, respectively. The presence of POPCOX which has a reactive peroxide group, decreased the K(D)(1) value 1.5-fold, increased the K(D)(2) value 10-fold, and significantly reduced the salt-induced detachment of cyt c. MALDI-TOF spectrometry analysis of cyt c incubated with liposomes containing POPCox demonstrated a mass increase corresponding to the formation of nonenal adducts as hydrophobic anchors. Electronic absorption spectroscopy, circular dichroism, and magnetic circular dichroism demonstrated that all of the lipids studied promoted changes in the cyt c coordination sphere. Therefore, in the presence of CL, the oxidation of zwitterionic lipids also promotes changes in the cyt c structure and in the affinity for lipid bilayers.

New crystal structures of red- and green blue–shifted beetle luciferases reveal that the color emission mechanism is dependent on the active site microenvironment affected by the conformation of loop regions.

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