Kinetics of the nucleotide binding to the strong (S) and weak (W) nucleotide-binding site of the Escherichia coli PriA helicase have been studied using the fluorescence stopped-flow technique. Experiments were performed with TNP-ADP and TNP-ATP analogues. Binding of the ADP analogue to the strong binding site is a four-step sequential reaction: (PriA)S + D (k1)<-->(k(-1)) + (S)1 (k2)<-->(k(-2)) (S)2 (k3)<-->(k(-3)) (S)3 (k4)<-->(k(-4)) (S)4. Association of TNP-ATP proceeds through an analogous three-step mechanism. The first two steps and intermediates are similar for both cofactors. However, the (S)3 intermediate is dramatically different for ADP and ATP analogues. Its emission is close to the emission of the free TNP-ADP, while it is by a factor of approximately 16 larger than the free TNP-ATP fluorescence. Thus, only the ADP analogue passes through an intermediate where it leaves the hydrophobic cleft of the site. This behavior corroborates with the fact that ADP leaves the ATPase site without undergoing a chemical change. The fast bimolecular step and the sequential mechanism indicate that the site is easily accessible to the cofactor, and it does not undergo an adjustment prior to binding. The subsequent step is also fast and stabilizes the complex. Magnesium profoundly affects the population of intermediates. The data indicate that the dominant (S)2 species is a part of the ATP catalytic cycle. ADP analogue binding to the weak nucleotide-binding site proceeds in a simpler two-step mechanism: (PriA)W + D (k1)<-->(k(-1)) (W)1 (k2)<-->(k(-2)) (W)2 with (W)1 being a dominant intermediate both in the presence and in the absence of Mg2+. The results indicate that the weak site is an allosteric control site in the functioning of the PriA helicase.

The dynamics of the nucleotide binding to a single, noninteracting nucleotide-binding site of the hexameric helicase RepA protein of plasmid RSF1010 has been examined, using the fluorescence stopped-flow method. The experiments have been performed with fluorescent analogues of ATP and ADP, TNP-ATP and TNP-ADP, respectively. In the presence of Mg2+, the association of the cofactors proceeds as a sequential three-step process RepAhexamer+Ntrue⇄k−1k1false(H−Nfalse)1true⇄k−2k2false(H−Nfalse)2true⇄k−3k3false(H−Nfalse)3 The sequential nature of the mechanism indicates the lack of significant conformational equilibria of the helicase prior to nucleotide binding. The major conformational change of the RepA helicase–nucleotide complex occurs in the formation of (H–N)2, which is characterized by a very high value of the partial equilibrium constant and large positive changes in the apparent enthalpy and entropy. Strong stabilizing interactions between subunits of the RepA hexamer contribute to the observed dynamics and energetics of the internal transitions of the formed complexes. Magnesium cations mediate the efficient and fast conformational transitions of the protein, in a manner independent of the structure of the cofactor phosphate group. The ssDNA bound to the enzyme preferentially selects a single intermediate of the RepA–ATP analogue complex, (H–N)2, while the DNA has no effect on the intermediates of the RepA–ADP complex. Allosteric interactions between the nucleotide- and DNA-binding site are established in the initial stages of formation of the complex. Moreover, in the presence of the single-stranded DNA, all the transitions in the nucleotide binding to the helicase become sensitive to the structure of the phosphate group of the cofactor.

With the alarming rise of infected cases and deaths, COVID-19 is a pandemic, affecting 220 countries worldwide. Until now, no specific treatment is available against SARS-CoV-2. The causal virus SARS-CoV-2 primarily infects lung cells, leading to respiratory illness ranging in severity from the common cold to deadly pneumonia. This, with comorbidities, worsens the clinical outcome, particularly for immunosuppressed individuals with COVID-19. Interestingly, the commensal gut microbiota has been shown to improve lung infections by modulating the immune system. Therefore, fine-tuning of the gut microbiome with probiotics could be an alternative strategy for boosting immunity and treating COVID-19. Here, we present a systematic biological network and meta-analysis to provide a rationale for the implementation of probiotics in preventing and/or treating COVID-19. We have identified 90 training genes from the literature analysis (according to PRISMA guidelines) and generated an association network concerning the candidate genes linked with COVID-19 and probiotic treatment. The functional modules and pathway enrichment analysis of the association network clearly show that the application of probiotics could have therapeutic effects on ACE2-mediated virus entry, activation of the systemic immune response, nlrp3 -mediated immunomodulatory pathways, immune cell migration resulting in lung tissue damage and cardiovascular difficulties, and altered glucose/lipid metabolic pathways in the disease prognosis. We also demonstrate the potential mechanistic domains as molecular targets for probiotic applications to combat the viral infection. Our study, therefore, offers probiotics-mediated novel preventive and therapeutic strategies for COVID-19 warfare.

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