The DUF328 family member YaaA is a DNA-binding protein with a novel fold

Abstract

DUF328 family proteins are present in many prokaryotes; however, their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Cofitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to dsDNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200-300 nm YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65-Å resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix DNA-binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.

Keywords: DNA binding; DNA-binding protein; UPF0246; X-ray crystallography; bacterial stress response; bioinformatics; comparative genomics; oxidative stress; stress response.

Figure 1.

Phylogenetic distribution of DUF328 proteins and representative genomes in bacteria. A, results of an AnnoTree query using Pfam family PF03883 (DUF328) in bacteria with resolution at the genus level. Branches are highlighted in blue for phyla that harbor members of the DUF328 family. The trees were generated in AnnoTree with the e-value cutoff of 0.00001 and adapted for visualization (RRID:SCR_018980). B, representative genome neighborhood diagrams of PF03883 (DUF328) family (red arrows). YaaJ: PF01235 (sodium:alanine symporter family), QueF: PF14489 (QueF-like protein), 2OG: PF13640 (2-oxoglutarate-Fe(II) oxygenase superfamily), DEAD: PF00270 (DEAD/DEAH box helicase), helicase: PF00271 (helicase conserved C-terminal domain), RecQ-like: PF16124 (RecQ zinc-binding), adh: PF00106 (short-chain dehydrogenase), AhpC: PF00578 (alkyl hydroperoxide reductase subunit C/ thiol-specific antioxidant family), Nif: PF01784 (NIF3 (NGG1p interacting factor 3)), zf: PF02591 (C4-type zinc ribbon domain).

Figure 2.

Schematics of examples of PF03883/IPR005583/DUF328 fusion proteins. Representatives were retrieved from pfam and Conserved Domain Architecture Retrieval Tool. H2O2_YaaD (DUF328): PF03883; GIY-YIG: PF01541; SpoVK: COG0464; Ser_Rec: Ser_Recombinase superfamily, cd00338; Rec: recombinase, pfam07508; Zn: recombinase zinc β ribbon domain, pfam13408; AlbA_2: putative DNA-binding domain, PF04326; HTH: helix-turn-helix domains. aa, amino acids.

Figure 3.

YaaA_Ec binds to diverse DNA constructs with nanomolar affinity. A–C, EMSA of recombinant YaaA_Ec with various DNA constructs illustrated below each panel. The concentration of YaaA_Ec is shown at the top of each panel and the fraction of the total DNA signal that is shifted from the free position is quantified beneath each lane. D, the binding of YaaA_Ec to the 12 nt bubble duplex DNA from EMSA is fitted to a single-site, positively cooperative binding model. Data were measured in triplicate and verified for multiple preparations of YaaA_Ec. The Hill coefficient was fitted as 3.1.

Figure 4.

Three-dimensional structure of YaaA_Ec defines a new fold. A and B, two views of the ribbon diagram of YaaA_Ec with α-helices lettered and β-strands numbered. The views in (A) and (B) are related by the 90° rotation indicated by the arrow. YaaA_Ec defines a new protein fold class. C, the electrostatic potential surface for YaaA_Ec looking down into the apical cleft as calculated by APBS (see “Experimental procedures”). Electrostatic potential is colored from blue (+10 kT/e) to red (−10 kT/e), where T is 298 K. D, the same view into the apical cleft as (C) with key residues labeled. The green and gold residues compose two groups related by an approximate dyad axis that passes between residue pairs Glu¹³⁰, Tyr¹⁷⁵ and Lys⁹, Lys²⁰⁹.

Figure 5.

YaaA_Ec possesses long stretches of nonstandard structures extending from the core to the periphery of the protein. A, a ribbon diagram of YaaA_Ec with the conserved SGXYG motif in yellow and the surrounding stretches of peptides adopting structures that are neither α-helix or β-strand in magenta. B, a close-up view of this region, where the dotted lines show a network of hydrogen bonds that mediate interactions between the conserved SGXYG motif and the surrounding residues. Tyrosine residues are highly represented in this region.

Figure 6.

YaaA_Ec has a HhH DNA-binding motif. A, a ribbon diagram of YaaA_Ec with the region that is structurally conserved in other DNA-binding proteins colored magenta. The classical HhH motif is defined by αB and αC (labeled). B, a superposition of YaaA_Ec (gray, blue) with the HhH motif (yellow) and bound DNA from XPF-ERCC1 endonuclease (PDB 6SXB). This illustrates one potential DNA-binding mode for YaaA_Ec and other DUF328 proteins.