Extended Data Fig. 1: Mutational tolerance and location of position 110 within T4 bacteriophage clamp loader AAA+ ATPase domains.
From: Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM
a, We used a focused mutagenesis library in which only the identity of the residue at position 110 was varied and measured the fitness of the variants. The fitness assay was conducted as described in Fig. 1b. Fitness of each substitution of position 110 is plotted. The fitness of synonymous codons was also measured to insure internal reproducibility of fitness measurements. Data are presented as mean values +/− standard deviations (SD), n = 5 biologically independent experiments b, The crystal structures of the remodeled wild-type clamp loader (translucent) and D110C mutant clamp loaders are aligned on residues 1-120 (domain 1) of subunit C (purple). At each of the ATP-binding sites in the wild-type complex, the sidechain of Asp 110, in the DEAD-box motif, forms hydrogen bonds with an arginine sidechain (Arg 122) provided by the adjacent subunit, and also with the backbone of the catalytically important sensor 1 residue, Asn 13927. In the D110C complex, the cysteine residue at position 110 interacts with only one of these residues at a time. When the cysteine sidechain does interact closely with that of Arg 122, the termini of the sidechains are within 3.0 Å of each other. This suggests that the cysteine is deprotonated and negatively charged at those sites, explaining the mild phenotype of the D110C mutant. The inability of the cysteine sidechain to engage Arg 122 and Asn 139 simultaneously results in a subtle change in the interfacial geometry between subunits that is distributed throughout the structure. This leads to a slight change in the overall engagement with DNA, which might result in the weakened DNA affinity.
