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...

Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.

Control of the regenerative properties of urothelial tissue would greatly aid the clinician in the management of urinary tract disease and disorders. Fibroblast growth factor 10 (FGF-10) is a mitogen which is particularly promising as a protein therapy for urothelial injury. The spatial synthesis, transport, targeting, and mechanistic pathway of FGF-10 and its receptor were studied in a human urothelial cell culture model and in fixed sections of lower urinary tract tissue. Synthesis of FGF-10 was restricted to mesenchymal fibroblasts, and secreted FGF-10 exhibited paracrine transport to two proximal sites, transitional epithelium and smooth muscle cell bundles, both of which were also the exclusive sites of FGF-10 receptor synthesis. The addition of recombinant FGF-10 to quiescent urothelial cells in vitro was sufficient to stimulate DNA synthesis. This stimulation was through a pathway independent of the epidermal growth factor receptor pathway. Deconvolution, light and transmission electron microscopic studies captured FGF-10 and its receptor in association with the urothelial cell surface, in cytoplasm, and within nuclei, observations that describe the mechanism that transduces the mitogenic signal in these tissues. Localization of the FGF-10 receptor to the superficial urothelial layer is clinically significant because intravesical administration of FGF-10 may provide the clinician a means to control the turnover of transitional epithelium in bladder disorders such as interstitial cystitis.

The anti-spreading activity of secreted protein acidic and rich in cysteine (SPARC) has been assigned to the C-terminal third domain, a region rich in alpha-helices. This "extracellular calcium-binding" (EC) domain contains two EF-hands that each coordinates one Ca2+ ion, forming a helix-loop-helix structure that not only drives the conformation of the protein but is also necessary for biological activity. Recombinant (r) EC, expressed in E. coli, was fused at the C-terminus to a His hexamer and isolated under denaturing conditions by nickel-chelate affinity chromatography. rEC-His was renatured by procedures that simultaneously (i) removed denaturing conditions, (ii) catalyzed disulfide bond isomerization, and (iii) initiated Ca2+-dependent refolding. Intrinsic tryptophan fluorescence and circular dichroism spectroscopies demonstrated that rEC-His exhibited a Ca2+-dependent conformation that was consistent with the known crystal structure. Spreading assays confirmed that rEC-His was biologically active through its ability to inhibit the spreading of freshly plated human urothelial cells propagated from transitional epithelium. rEC-His and rSPARC-His exhibited highly similar anti-spreading activities when measured as a function of concentration or time. In contrast to the wild-type and EC recombinant proteins, rSPARC(E268F)-His, a point substitution mutant at the Z position of EF-hand 2, failed to exhibit both Ca2+-dependent changes in alpha-helical secondary structure and anti-spreading activity. The collective data provide evidence that the motif of SPARC responsible for anti-spreading activity was dependent on the coordination of Ca2+ by a Glu residue at the Z position of EF-hand 2 and provide insights into how adhesive forces are balanced within the extracellular matrix of urothelial cells. .