Structures of apo- and ssDNA-bound YdbC from Lactococcus lactis uncover the function of protein domain family DUF2128 and expand the single-stranded DNA-binding domain proteome

Abstract

Single-stranded DNA (ssDNA) binding proteins are important in basal metabolic pathways for gene transcription, recombination, DNA repair and replication in all domains of life. Their main cellular role is to stabilize melted duplex DNA and protect genomic DNA from degradation. We have uncovered the molecular function of protein domain family domain of unknown function DUF2128 (PF09901) as a novel ssDNA binding domain. This bacterial domain strongly associates into a dimer and presents a highly positively charged surface that is consistent with its function in non-specific ssDNA binding. Lactococcus lactis YdbC is a representative of DUF2128. The solution NMR structures of the 20 kDa apo-YdbC dimer and YdbC:dT19G1 complex were determined. The ssDNA-binding energetics to YdbC were characterized by isothermal titration calorimetry. YdbC shows comparable nanomolar affinities for pyrimidine and mixed oligonucleotides, and the affinity is sufficiently strong to disrupt duplex DNA. In addition, YdbC binds with lower affinity to ssRNA, making it a versatile nucleic acid-binding domain. The DUF2128 family is related to the eukaryotic nuclear protein positive cofactor 4 (PC4) family and to the PUR family both by fold similarity and molecular function.

The large family of PC4-like domains – similar folds and functions throughout all kingdoms of life

“…Each structure is depicted in two different orientations. The coordinates are taken from: (A) Solanum tuberosum P24 subunit of PBF-2 (PDB ID: 1l3a) [ 20 ], (B) Trypanosoma brucei MRP1 (PDB ID: 2gje) [ 21 ], (C) The human replication and transcription cofactor PC4 (PDB ID: 1pcf) [ 18 ], (D) Bacteriophage T5 homologue of PC4 (PDB ID: 4bg7) [ 36 ], (E) Lactococcus lactis DUF2128 family member YdbC (PDB ID: 2ltt) [ 37 ], (F) Pur-α from Borrelia burgdorferi (PDB ID: 3n8b) [ 13 ], (G) Streptococcus pneumonia SP_0782 protein (PDB ID: 5zkl) [ 39 ], (H) The bacteriophage coat protein PP7 (PDB ID: 2qux) [ 22 ]. The structures of type I PC4-like fold have been represented as cyan (repeat I) and navy blue (repeat II), whereas in the structures of type II PC4-like fold individual chains have been shown in green and grey.…”

“… DNA/RNA binding by type II PC4-like fold proteins. The coordinates are taken from: (A) hs PC4 CTD in complex with ssDNA (U mode; PDB ID: 2c62) [ 64 ], (B) Pyricularia oryzae MoSub1 in complex with ssDNA (Straight mode; PDB ID: 4bhm) [ 66 ], (C) Magnaporthe oryzae MoSub1 in complex with ssDNA (L mode; PDB ID: 5zg9) [ 65 ], (D) Lactococcus lactis Ydbc in complex with ssDNA (PDB ID: 2ltt) [ 37 ], (E) Streptococcus pneumonia SP_0782 protein in complex with ssDNA dT12 (PDB ID: 5zkl) [ 39 ], (F) Pseudomonas phage protein PP7 in complex with RNA (PDB ID: 2qux) [ 22 ]. The figure has been prepared in PyMol v 1.3 (pymol.org).…”

“…YdbC is an ssDNA-and RNA-binding protein from the bacterium Lactococcus lactis and a member of the DUF2128 family, which also folds into a type II domain structure (PDB ID: 2ltd and 2ltt) [37] ( Fig. 2E and Supplementary Fig.…”

“…Also YdbC from Lactococcus lactis, which is a multifunctional nucleic acid-binding protein of the DUF2128 family, binds ssDNA in a straight mode (PDB ID: 2ltt) [37] ( Fig. 5D and Supplementary Fig.…”

Substitution of tryptophan 89 with tyrosine switches the DNA binding mode of PC4

“…The DNA binding domains in Sub1 and PC4, the human orthologue, have been characterized using biochemical methods 3 4 . Structures of the DNA binding domains of PC4 as well as MoSub1 from the rice blast fungus Magnaporthe oryzae have been determined 9 10 11 and more recently, structures of two other PC4 orthologues from Lactococcus lactis and bacteriophage T5, have also been solved 12 13 . In PC4, the dimeric DNA binding domain is located in a C-terminal region spanning amino acids 63–127 (PC4-CTD), and is comprised of a curved five strand anti-parallel β-sheet followed by one α-helix.…”