Human manganese superoxide dismutase is a critical oxidoreductase found in the mitochondrial matrix. Concerted proton and electron transfers are used by the enzyme to rid the mitochondria of O2•−. The mechanisms of concerted transfer enzymes are typically unknown due to the difficulties in detecting the protonation states of specific residues and solvent molecules at particular redox states. Here, neutron diffraction of two redox-controlled manganese superoxide dismutase crystals reveal the all-atom structures of Mn3+ and Mn2+ enzyme forms. The structures deliver direct data on protonation changes between oxidation states of the metal. Observations include glutamine deprotonation, the involvement of tyrosine and histidine with altered pKas, and four unusual strong-short hydrogen bonds, including a low barrier hydrogen bond. We report a concerted proton and electron transfer mechanism for human manganese superoxide dismutase from the direct visualization of active site protons in Mn3+ and Mn2+ redox states.

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The COVID‐19 pandemic caused by SARS‐CoV‐2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of a SARS‐CoV‐2 spike protein receptor binding domain (RBD) that includes the SD1 domain. Bacterial cytoplasm is a reductive environment, which is problematic when the recombinant protein of interest requires complicated folding and/or processing. The use of the CyDisCo system (cytoplasmic disulfide bond formation in E. coli) bypasses this issue by pre‐expressing a sulfhydryl oxidase and a disulfide isomerase, allowing the recombinant protein to be correctly folded with disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce an active RBD in bacteria that is capable of recognizing and binding to the ACE2 (angiotensin‐converting enzyme) receptor as well as antibodies in COVID‐19 patient sera.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19.HighlightsBromelain inhibits / cleaves the expression of ACE-2 and TMPRSS2Bromelain cleaves / degrades SARS-CoV-2 spike proteinBromelain inhibits S-Ectodomain binding and SARS-CoV-2 infection

Human mitochondrial manganese superoxide dismutase (MnSOD) is a major player in combating reactive oxygen species in the human body. Methods have been found to control the redox state of the active-site metal of large perdeuterated MnSOD crystals. Neutron diffraction data from these crystals were collected to study the effect of the redox state on proton location. These methods can be applied to other crystal systems where information on the location of protons in specific chemical states is needed.

The COVID-19 pandemic caused by SARS-CoV-2 infection has led to socioeconomic shutdowns and the loss of over 5 million lives worldwide. There is a need for the identification of therapeutic targets to treat COVID-19. SARS-CoV-2 spike is a target of interest for the development of therapeutic targets.We developed a robust SARS-CoV-2 S spike expression and purification protocol from insect cells and studied four recombinant SARS-CoV-2 spike protein constructs based on the original SARS-CoV-2 sequence using a baculovirus expression system: a spike protein receptor-binding domain that includes the SD1 domain (RBD) coupled to a fluorescent tag (S-RBD-eGFP), spike ectodomain coupled to a fluorescent tag (S-Ecto-eGFP), spike ectodomain with six proline mutations and a foldon domain (S-Ecto-HexaPro(+F)), and spike ectodomain with six proline mutations without the foldon domain (S-Ecto-HexaPro(-F)). We tested the yield of purified protein expressed from the insect cell lines Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tni) and compared it to previous research using mammalian cell lines to determine changes in protein yield. We demonstrated quick and inexpensive production of functional glycosylated spike protein of high purity capable of recognizing and binding to the angiotensin converting enzyme 2 (ACE2) receptor. To further confirm functionality, we demonstrate binding of eGFP fused construct of the spike ectodomain (S-Ecto-eGFP) to surface ACE2 receptors on lung epithelial cells by flow cytometry analysis and show that it can be decreased by means of receptor manipulation (blockade or downregulation).

Structurally identifying the enzymatic intermediates of redox proteins has been elusive due to difficulty in resolving the H atoms involved in catalysis and the susceptibility of ligand complexes to photoreduction from X-rays. Cryotrapping ligands for neutron protein crystallography combines two powerful tools that offer the advantage of directly identifying hydrogen positions in redox-enzyme intermediates without radiolytic perturbation of metal-containing active sites. However, translating cryogenic techniques from X-ray to neutron crystallography is not straightforward due to the large crystal volumes and long data-collection times. Here, methods have been developed to visualize the evasive peroxo complex of manganese superoxide dismutase (MnSOD) so that all atoms, including H atoms, could be visualized. The subsequent cryocooling and ligand-trapping methods resulted in neutron data collection to 2.30 Å resolution. The P6122 crystal form of MnSOD is challenging because it has some of the largest unit-cell dimensions (a = b = 77.8, c = 236.8 Å) ever studied using high-resolution cryo-neutron crystallography. The resulting neutron diffraction data permitted the visualization of a dioxygen species bound to the MnSOD active-site metal that was indicative of successful cryotrapping.

The COVID-19 pandemic caused by SARS-CoV-2 has applied significant pressure on overtaxed healthcare around the world, underscoring the urgent need for rapid diagnosis and treatment. We have developed a bacterial strategy for the expression and purification of the SARS-CoV-2 spike protein receptor binding domain using the CyDisCo system to create and maintain the correct disulfide bonds for protein integrity and functionality. We show that it is possible to quickly and inexpensively produce functional, active antigen in bacteria capable of recognizing and binding to the ACE2 (angiotensin-converting enzyme) receptor as well as antibodies in COVID-19 patient sera.