Abstract
Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved ‘DEAD-box’ motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.
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Data availability
Deep mutagenesis data generated during this study have been deposited in the Sequence Read repository with BioProject accession number PRJNA1025274. Deep mutagenesis data of the wild-type clamp loader can be found in our previous publication20. Crystal structures of the p.D110C mutant bacteriophage clamp loader–clamp–DNA complex and reprocessed wild-type bacteriophage clamp loader–clamp–DNA complex have been deposited in the PDB with accession codes 8UK9 and 8UH7. The cryo-EM density maps of T4 bacteriophage clamp loader–clamp DNA-bound complex and T4 bacteriophage clamp loader–clamp DNA-free complex been deposited to the Electron Microscopy Data Bank under the accession codes EMD-42399 and EMD-42402. The associated coordinates have been deposited to the PDB under accession codes 8UNF and 8UNH. Biochemical data are supplied as Excel spreadsheets, with relevant figures listed in the filename. Source data are provided with this paper.
Code availability
Bash scripts used to extract counts per allele from the raw sequencing data have been deposited in Github: https://github.com/KuriyanLab/DMS-D110C-NSMB2023
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Acknowledgements
We thank T. Eisen for assistance in the review of this manuscript and helpful discussions about improving our data analysis. We thank the Berkeley Center for Structural Biology beamline staff at the Advanced Light Source, Lawrence Berkeley National Laboratory. We also acknowledge the Berkeley Bay Area Cryo-EM Facility for cryo-EM data collection. Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. Funding for this work was provided by the Howard Hughes Medical Institute and NIH R01-GM144512.
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Contributions
K.M., Y.H., and J.K. conceived the project; K.M. and S. Subramanian designed the high-throughput experiments; K.M. performed and analyzed the high-throughput experiments; Y.H. and S. Subramaniam performed and analyzed the EM experiments; K.M., K.G., S.G.-K, X.L.R., and L.Z. designed and performed the biochemical assays; K.M., C.G., and S.G-K. performed and analyzed the X-ray crystallography experiments; J.K. supervised the project; K.M., Y.H., and J.K. drafted the manuscript; K.M., Y.H., S. Subramanian, and M.O.’D. edited the manuscript. All authors commented on the manuscript.
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J.K. is a cofounder of Nurix Therapeutics and is on the scientific advisory boards of Carmot and Revolution Medicine. S. Subramanian is founder and chief executive officer of Gandeeva Therapeutics. The remaining authors declare no competing interests.
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Nature Structural & Molecular Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Dimitris Typas was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Extended data
Extended Data Fig. 1 Mutational tolerance and location of position 110 within T4 bacteriophage clamp loader AAA+ ATPase domains.
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.
Extended Data Fig. 2 Biochemical assessment of D110C mutant clamp loader.
a, Purified wild-type T4 clamp-loader complex and the D110C variant are assessed for DNA-binding affinity by using a fluorescence polarization anisotropy assay. In previous work, a fluorescent tag on a DNA duplex with a 5′ overhang (representing primed DNA) was used to measure the change in fluorescence anisotropy of the labeled DNA after adding the clamp loader22,53,54. In the present experiments, we used primed DNA (50 nM) consisting of a 20 base-pair DNA duplex with a 10-nucleotide overhang labeled with TAMRA dye. The labeled DNA was added to a reaction mixture containing the T4 sliding clamp (7 µM) and the ATP analog ADP·BeF3 (1 mM). Purified clamp loader (0–5 µM) was then titrated into the sliding-clamp:primed DNA:nucleotide analog mixture and allowed to equilibrate for 3 hours at room temperature before fluorescence anisotropy measurement (see Methods). The anisotropy per clamp loader concentration is plotted for wild-type D110C mutant clamp loader. Data are presented as mean values +/− standard deviations (SD), n = 3 biological replicates. b, We measured the kinetics of ATP hydrolysis by using an assay in which the consumption of ATP by the clamp loader is coupled to the oxidation of NADH12,40,54. Kinetic measurements were made for both the wild-type clamp loader and the D110C mutant (0.5 µM) bound to clamp (2.5 µM) and the same primed DNA construct used for the fluorescence anisotropy assay (2.5 µM), but without the fluorophore label. ATP hydrolysis rate of the D110C mutant is ~33% of the rate exhibited by the wild-type clamp loader. The DNA concentration used for this assay is ~5-fold higher than the KD value for the wild-type clamp loader, which presumably accounts for the partial stimulation of ATP hydrolysis by the D110C mutant. Example traces of these kinetic measurements are shown.
Extended Data Fig. 3 Electron density of wild-type and D110C T4 bacteriophage crystal structures.
a, The electron density map (2FoFc) of the re-refined wild-type clamp loader structure is shown centered around the active site of subunit D. The map is rendered at a contour level of 0.6176 e/Å3 (2.09 RMSD). Two view angles of the active site are shown. The nucleotide is the central axis of the view. b, The electron density map (2FoFc) of the D110C clamp loader mutant structure is shown centered around the active site of subunit D. The map is rendered at a contour level of 0.2253 e/Å3 (2.07 RMSD). Two view angles of the active site are shown. The nucleotide is the central axis of the view. C, The crystal structure of D110C mutated clamp loader (molecule A) is aligned against the wild-type clamp loader structure (gray). The alignment is performed on subunits C and D of both structures. The A, B, C, and E, subunits of the D110C structure are colored green, blue, magenta, yellow, and pink, respectively. The clamp subunits of the D110C structure are colored light blue, purple, and orange.
Extended Data Fig. 4 Statistics of D110C clamp loader deep mutagenesis screen.
a, Agreement between fitness measurements from three replicate experiments per assay pool (residues 2-115 assay pool n=1130, and residues 115-230 assay pool n=2076) is shown in scatter plots. Each point in the scatter plot represents the fitness measurements made from the two trials. b, The histogram shows the spread of relative fitness values for the AAA+ module of the ATPase subunit in the background of the D110C mutant clamp loader. c, Small-scale libraries of recovery mutations in the background of D110C were constructed and subjected to the phage replication assay. The fitness of this small library per substitution (x-axis) is plotted against the fitness scores of same mutations as assayed in the full-scale deep mutagenesis screen (y-axis). Data are presented as mean values +/− standard deviations (SD), n=3 biological replicates.
Extended Data Fig. 5 Biochemical characterization of D110C rescue mutations.
a, Using TAMRA-labeled double-stranded DNA, fluorescence anisotropy measurements are taken to assess the DNA-binding capabilities of four recovery mutations in the background of the D110C mutant clamp loader (denoted by a star *). These four representative mutants were chosen to span both strong (P50K*) and weaker recovery (D98W*) activity. Data are presented as mean values +/− standard deviations (SD), n=3 biologically independent experiments b, The ATP hydrolysis recovery of four rescue mutations in the background of D110C mutant clamp loader is measured via use of the coupled kinase assay. We determined the ATP hydrolysis rates of each clamp loader variant alone, in the presence of DNA alone, in the presence of clamp alone, and an activated clamp loader complex containing both clamp and duplexed DNA. Data are presented as mean values +/− standard deviations (SD), n = 2 to 6 technical replicates, with at least two biological replicates showing the same hydrolysis trends per clamp loader variant studied.
Extended Data Fig. 7 Representative Cryo-EM micrographs.
a. Representative Cryo-EM micrographs of wild-type T4 clamp-loader:clamp with DNA b. Representative Cryo-EM micrographs of wild-type T4 clamp-loader:clamp without DNA.
Extended Data Fig. 8 Mechanistic evaluation of rescue hotspot residues.
a, An expanded view from Figure 7b is taken of residue Pro 50 (red spheres). The DNA-unbound structure (wheat) features greater distance between Pro 50 and Asn 139. The distance between ATPyS and Asn 139 in this structure is also greater (red dashes, 5.6 angstroms). When DNA is bound (purple), Pro 50 moves closer to Asn 139, while Asn 139 moves closer to ADP·BeF3 (black dashes, 4.7 angstroms) b, The interfacial surface seen in Fig. 7a is expanded to show five distinct rescue hotspots (red spheres) around the hydrophobic plug residue, Phe 28. The DNA-bound conformation of chains C (purple) and B (blue) is aligned with the DNA-unbound conformation of chain C (wheat) along the central coupler region. The hydrophobic plug is formed by residues Tyr 214, Ile 224, and Leu 227.
Extended Data Fig. 9 Kinetic measurement of fluorescence anisotropy over a range of clamp loader concentrations.
The fluorescence anisotropy of clamp loader to fluorescently labeled primer-template DNA is measured over time and across clamp loader concentration. This complex was also bound to the non-hydrolysable analog ADP*BeF3. Very slow equilibration of the anisotropy readout is seen at low clamp loader concentration (between 0.009 uM and 0.039 uM). Clamp loader at a concentration of around 0.08 uM generates consistent anisotropy values after approximately 10,000 seconds (~2.5 hours).
Supplementary information
Source data
Source Data Fig. 1
Enrichment frequencies per codon in D110C recovery selection experiments
Source Data Fig. 2
Raw NSG counts, calculated enrichment values of large library D110C selections
Source Data Fig. 4
DNA-binding anisotropy data per ATP analog
Source Data Extended Data Fig. 1
Raw NSG counts, calculated enrichment values of focused D110NNS selection
Source Data Extended Data Fig. 2
A. DNA binding anisotropy data of wild-type and D110C mutant clamp loader B. ATP hydrolysis traces of wild-type and D110C mutant clamp loader
Source Data Extended Data Fig. 4
Compiled count and enrichment statistics per large library selections
Source Data Extended Data Fig. 5
A. DNA binding anisotropy data per purified D110C rescue mutant B. ATP hydrolysis slope data per D110C rescue mutant (in triplicate)
Source Data Extended Data Fig. 9
DNA-binding anisotropy values as a function of clamp loader concentration
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Marcus, K., Huang, Y., Subramanian, S. et al. Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM. Nat Struct Mol Biol 31, 424–435 (2024). https://doi.org/10.1038/s41594-023-01177-3
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DOI: https://doi.org/10.1038/s41594-023-01177-3
