Structure of a protein–DNA complex essential for DNA protection in spores of Bacillus species

Abstract

The DNA-binding ␣/␤-type small acid-soluble proteins (SASPs) are a major factor in the resistance and long-term survival of spores of Bacillus species by protecting spore DNA against damage due to desiccation, heat, toxic chemicals, enzymes, and UV radiation. We now report the crystal structure at 2.1 Å resolution of an ␣/␤-type SASP bound to a 10-bp DNA duplex. In the complex, the ␣/␤-type SASP adopt a helix-turn-helix motif, interact with DNA through minor groove contacts, bind to Ϸ6 bp of DNA as a dimer, and the DNA is in an A-B type conformation. The structure of the complex provides important insights into the molecular details of both DNA and ␣/␤-type SASP protection in the complex and thus also in spores.␣/␤-type SASP ͉ A-type DNA ͉ DNA damage ͉ molecular modeling ͉ spore resistance S pores of Bacillus and Clostridium species are extremely resistant to desiccation, heat, toxic chemicals, enzymes, and radiation. This high resistance is the major reason that spores of some species are agents of food spoilage and food poisoning and that B. anthracis spores are a biological weapon. Spore resistance is due to many factors, a major one being the protection of spore DNA against damage by the binding of small, acid-soluble proteins (SASPs) of the ␣/␤-type (1, 2). These 59-75 residue proteins: (i) are encoded by multiple genes, (ii) comprise a protein family whose amino acid sequences are very highly conserved within and between species, (iii) are nonspecific DNA-binding proteins with apparent binding constants for random sequence DNA of 15-100 mM, (iv) are synthesized only within the developing forespore during sporulation; and (v) comprise 3-5% of total spore protein. The high levels of ␣/␤-type SASPs in spores are sufficient to saturate the spore DNA, and the DNA within this nucleoprotein complex is protected from a variety of environmental insults. Indeed, the binding of ␣/␤-type SASPs provides primary protection of spore DNA against damage by desiccation, heat, many genotoxic chemicals, enzymes, and UV radiation (1,3).In this study, we used x-ray crystallography to determine a 2.1 Å resolution structure of a complex between a 10-bp DNA duplex and an engineered ␣/␤-type SASP originally obtained from B. subtilis. Analysis of this crystal structure provides important insight into the molecular mechanisms underlying the protection of both the protein and DNA components of the complex and the molecular details of their interactions. These results explain a great deal about the extreme resistance of the DNA in bacterial spores and thus explain much of bacterial spore resistance in atomic detail. Results and DiscussionOverall Structure and Protection of the Protein in the Complex. The ␣/␤-type SASP chosen to form the complex with DNA for crystallization was B. subtilis SspC ⌬N11-D13K-C3, a 64-aa derivative engineered to bind tightly to DNA (4), which was an oligo(dG)⅐oligo(dC) 10-mer with single 3Ј-dA overhangs (5) The preparation and crystallization of the protein-DNA complex are described in ref. 6, and the struct...

Nuclear Tau, a Key Player in Neuronal DNA Protection

“…Spores of Bacillus are extremely resistant to a wide array of extreme insults, including heat, desiccation, toxic chemicals, enzymes, and radiation. This spore resistance is mainly due to protection against DNA damage by the binding of small acid-soluble proteins to the DNA minor groove, inducing protective changes in the structure of DNA (47,48). Interestingly, as small acid-soluble proteins, Tau is an acid-soluble molecule.…”

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

“…Generally, SASPs appear to be conserved within and between species (Setlow, 2007), especially with respect to their N-and C-terminal consensus regions, which form a helix-turn-helix DNA-binding motif (Lee et al, 2008). Based on our refined similarity searches, we suggest precise renaming of the SASPs of C. acetobutylicum based on BLAST identities (Table 1; Table S3) and the degree of conserved amino acids against the SASPs consensus motifs (Fig.…”

“…For SspH, DNA protection was rather weak, probably due to the reduced degree of amino acid conservation to the SASP consensus consequently weakening the formation of a helix-turn-helix motif (Lee et al, 2008). Whether, as in C. perfringens (Li et al, 2009), different preferences of the respective SASPs for DNA exist remains to be examined.…”

“…The a/b-type SASPs bind DNA non-specifically at high density, leading to its protection against enzymic degradation Lee et al, 2008). To analyse DNA protection by SASPs of C. acetobutylicum, each ssp gene was cloned into pT-catP (Fig.…”

“…The latter are not able to bind DNA and are expected to be restricted to the genus Bacillus (Francesconi et al, 1988;Vyas et al, 2011). Usually, most SASPs are encoded by monocistronic genes termed ssp, which are expressed only in the developing forespore compartment, and immediately after protein biosynthesis a/btype SASPs bind DNA up to saturation with the effect of DNA protection by dense packaging (Lee et al, 2008;Ge et al, 2011). During spore outgrowth, SASPs serve as an important source of energy and building blocks for the de novo protein biosynthesis (Setlow, 1988;Driks, 2002).…”