The two actin-related subunits of the Arp2/3 complex, Arp2 and Arp3, are proposed to form a pseudo actin dimer that nucleates actin polymerization. However, in the crystal structure of the inactive complex, they are too far apart to form such a nucleus. Here, we show using EM that yeast and bovine Arp2/3 complexes exist in a distribution among open, intermediate and closed conformations. The crystal structure docks well into the open conformation. The activator WASp binds at the cleft between Arp2 and Arp3, and all WASp-bound complexes are closed. The inhibitor coronin binds near the p35 subunit, and all coronin-bound complexes are open. Activating and loss-of-function mutations in the p35 subunit skew conformational distribution in opposite directions, closed and open, respectively. We conclude that WASp stabilizes p35-dependent closure of the complex, holding Arp2 and Arp3 closer together to nucleate an actin filament.

Interleukin-6 (IL-6) has been implicated in the pathogenesis of inflammatory events including those seen with COVID-19 patients. Positive clinical responses to monoclonal antibodies directed against IL-6 receptors (IL-6Rs) suggest that interference with IL-6-dependent activation of pro-inflammatory pathways offers a useful approach to therapy. We exposed IL-6 to hypochlorous acid (HOCl) in vitro at concentrations reported to develop in vivo . After HOCl treatment, binding of IL-6 to IL-6R was reduced in a dose-dependent manner using a bioassay with human cells engineered to provide a luminescence response to signal transduction upon receptor activation. Similar results followed the exposure of IL-6 to N -chlorotaurine (NCT) and hypobromous acid (HOBr), two other reactive species produced in vivo . SDS-PAGE analysis of HOCl-treated IL-6 showed little to no fragmentation or aggregation up to 1.75 mM HOCl, suggesting that the modifications induced at concentrations below 1.75 mM took place on the intact protein. Mass spectrometry of trypsin-digested fragments identified oxidative changes to two amino acid residues, methionine 161 and tryptophan 157, both of which have been implicated in receptor binding of the cytokine. Our findings suggest that exogenous HOCl and NCT might bring about beneficial effects in the treatment of COVID-19. Further studies on how HOCl and HOBr and their halogenated amine derivatives interact with IL-6 and related cytokines in vivo may open up alternative therapeutic interventions with these compounds in COVID-19 and other hyperinflammatory diseases.

Interleukin-6 (IL-6) has been implicated in the pathogenesis of acute inflammatory events in COVID-19 patients. We exposed IL-6 to hypochlorous acid (HOCl) in vitro at concentrations reported to develop in vivo. After HOCl treatment the cytokine failed to bind to IL-6 receptors in a bioassay using engineered human cells. Similar results followed exposure of IL-6 to N-chlorotaurine (NCT) and hypobromous acid (HOBr). SDS-PAGE analysis of HOCl-treated IL-6 showed neither fragmentation nor aggregation, suggesting that the modifications induced by these agents occurred on the intact protein. Mass spectrometry of intact and trypsin-digested fragments identified oxidative changes limited to two amino acid residues, methionine 161 and tryptophan 157, both of which have been implicated in receptor binding of the cytokine. Further studies on the effects of hypohalous acids and their halogenated amine derivatives on IL-6 and related cytokines is needed.