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...
