Structure of a fatty acid-binding protein fromBacillus subtilisdetermined by sulfur-SAD phasing using in-house chromium radiation

Abstract

Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5 A resolution using Cr Kalpha radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family.

Identification of a two-component fatty acid kinase responsible for host fatty acid incorporation by Staphylococcus aureus

“…2D). This conclusion was confirmed by analytical ultracentrifugation (Table S1) and was consistent with the crystal structures of this protein family (7,8). FakA plus a FakB reconstituted Fak activity in vitro (Fig.…”

“…The FakA carboxylterminal domain is also predicted to adopt an EDD-like fold; however, we did not detect FA binding to FakA. The DegV family (32) EDD fold is related to the DhaL Dha-binding protein/domain of Dha kinases (33) and all members of this family characterized to date are FA binding proteins (7,8).…”

“…Gram-positive bacteria express multiple representatives of the DegV protein family (COG1307) that have been structurally characterized as FA binding proteins (7,8). The type of FA modeled into the structures deposited in the PDB depends on the source of the DegV homolog.…”

Biochemical Roles for Conserved Residues in the Bacterial Fatty Acid-binding Protein Family

“…We did not detect any incorporation of [1-14 C]oleate into phospholipid in the ⌬fakB2 strain consistent with the absolute requirement of Fak for fatty acid activation and incorporation (Table 5). Strains expressing FakB2(T61A) or FakB2(H266A) showed 20 -40-fold lower incorporation of [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]oleic acid into membrane phospholipids illustrating these proteins had severely impaired Fak activity in vivo (Table 5), as they had in vitro ( Table 2). The FakB2(R170A) mutant was indistinguishable from the ⌬fakB2 deletion strain with no detectable exogenous fatty acid incorporation into phospholipid (Table 5) corroborating the essential nature of Arg-170 in Fak catalysis (Table 2).…”

“…The FakB proteins belong to the EDD superfamily, which includes the mannose transporter EIIA domain and dihydroxyacetone kinase (6 -8). All crystallized FakB proteins have a fatty acid or fatty acid-like ligand bound (7,8). The discovery of their role in Fak provided a function for these ubiquitous FakB proteins in bacterial physiology (1), but the role(s) of the highly conserved FakB residues that define the protein family in Fak biochemistry are unknown.…”