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Protein phosphorylation is a pivotal post-translational modification modulating various cellular processes. In Gram-positive bacteria, the protein arginine kinase McsB, along with its activator McsA, has a key role in labeling misfolded and damaged proteins during stress. However, the activation mechanism of McsB by McsA remains elusive. Here we report the cryo-electron microscopy structure of a tetrameric McsA-McsB complex at 3.41 Å resolution. Biochemical analysis indicates that the homotetrameric assembly is essential for McsB's kinase activity. The conserved C-terminal zinc finger of McsA interacts with an extended loop in McsB, optimally orienting a critical catalytic cysteine residue. In addition, McsA binding decreases the CtsR's affinity for McsB, enhancing McsB's kinase activity and accelerating the turnover rate of CtsR phosphorylation. Furthermore, McsA binding also increases McsB's thermostability, ensuring its activity under heat stress. These findings elucidate the structural basis and activation mechanism of McsB in stress response., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Despite advancements in antiretroviral therapy (ART) suppressing HIV-1 replication, existing antiviral drugs pose limitations, including lifelong medication, frequent administration, side effects and viral resistance, necessitating novel HIV-1 treatment approaches. CD4, pivotal for HIV-1 entry, poses challenges for drug development due to neutralization and cytotoxicity concerns. Nevertheless, Ibalizumab, the sole approved CD4-specific antibody for HIV-1 treatment, reignites interest in exploring alternative anti-HIV targets, emphasizing CD4's potential value for effective drug development. Here, we explore anti-CD4 nanobodies, particularly Nb457 from a CD4-immunized alpaca. Nb457 displays high potency and broad-spectrum activity against HIV-1, surpassing Ibalizumab's efficacy. Strikingly, engineered trimeric Nb457 nanobodies achieve complete inhibition against live HIV-1, outperforming Ibalizumab and parental Nb457. Structural analysis unveils Nb457-induced CD4 conformational changes impeding viral entry. Notably, Nb457 demonstrates therapeutic efficacy in humanized female mouse models. Our findings highlight anti-CD4 nanobodies as promising HIV-1 therapeutics, with potential implications for advancing clinical treatment against this global health challenge., (© 2024. The Author(s).)