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The 70 kDa heat-shock proteins (Hsp70s) are ATP-dependent molecular chaperones that contain an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain. Hsp70s bind to misfolded/unfolded proteins and thereby prevent their aggregation. The ATP hydrolysis reaction in the NBD plays a key role in allosteric control of the binding of substrate proteins. In the present study, the neutron crystal structure of the NBD of Hsp72, a heat-inducible Hsp70 family member, was solved in complex with ADP in order to study the structure–function relationship with a focus on hydrogens. ADP bound to Hsp72 was fully deprotonated, and the catalytically important residues, including Asp10, Asp199 and Asp206, are also deprotonated. Neutron analysis also enabled the characterization of the water clusters in the NBD. Enzymatic assays and X-ray crystallographic analysis revealed that the Y149A mutation exhibited a higher ATPase activity and caused disruption of the water cluster and incorporation of an additional magnesium ion. Tyr149 was suggested to contribute to the low intrinsic ATPase activity and to stabilize the water cluster. Collectively, these structural studies will help to elucidate the molecular basis of the function of Hsp72.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is activated by binding to DNA. Activated cGAS produces 2'3'-cGAMP, which subsequently binds to the adaptor protein STING (stimulator of interferon genes). This interaction triggers the cGAS/STING signaling pathway, leading to the production of type I interferons. Three types of DNA, namely double-stranded DNA longer than 40 base pairs, a 70-nucleotide single-stranded HIV-1 DNA known as SL2, and Y-form DNA with unpaired guanosine trimers (G3 Y-form DNA), induce interferon production by activating cGAS/STING signaling. However, the extent of cGAS activation by each specific DNA type remains unclear. The comparison of cGAS stimulation by various DNAs is crucial for understanding the mechanisms underlying cGAS-mediated type I interferon production in the innate immune response. Here, we revealed that cGAS produces 2'3'-cGAMP at a significantly lower rate in the presence of single-stranded SL2 DNA than in the presence of double-stranded DNA or G3 Y-form DNA. Furthermore, the guanine-to-cytosine mutations and the deletion of unpaired guanosine trimers significantly reduced the 2'3'-cGAMP production rate and the binding of cGAS to Y-form DNA. These studies will provide new insights into the cGAS-mediated DNA-sensing in immune response. [ABSTRACT FROM AUTHOR]