Expression, purification, crystallization and preliminary crystallographic study of FtsA from methicillin-resistantStaphylococcus aureus

Abstract

FtsA from methicillin-resistant Staphylococcus aureus (MRSA) was cloned, overexpressed and purified. The protein was crystallized using the sitting-drop vapour-diffusion technique. A cocrystal with β-γ-imidoadenosine 5'-phosphate (AMPPNP; a nonhydrolysable ATP analogue) was grown using PEG 3350 as a precipitant at 293 K. X-ray diffraction data were collected to a resolution of 2.3 Å at 100 K. The crystal belonged to the monoclinic space group P2₁, with unit-cell parameters a = 75.31, b = 102.78, c = 105.90 Å, β = 96.54°. The calculated Matthews coefficient suggested that the asymmetric unit contained three or four monomers.

Crystal structure of FtsA from Staphylococcus aureus

“…SaFtsA (UniProt ID: Q6GHQ0) was expressed and purified as described [20]. SaFtsZ (UniProt ID: Q6GHP9) was cloned, expressed, and purified as reported for SaFtsA except that the cloning primers were: ftsZ ‐for (5′‐GCCATATGTTAGAATTTGAACAAGGATTTAATC‐3′) and ftsZ ‐rev (5′‐GCGGATCCTTAACGTCTTGTTCTTCTTGAACG‐3′).…”

“…SaFtsA was crystallized in the presence of AMPPNP as reported [20]. SaFtsA was also crystallized in the presence of ADP by mixing an equal volume (1 μl) of protein solution (10 mg/ml) containing 1 mM ADP and 2 mM MgCl 2 with 0.2 M lithium nitrate, 20% (w/v) PEG3350, 0.1 M sodium cacodylate, pH 7.0 (reservoir solution) and equilibrating against 1 ml of the reservoir solution.…”

“…We previously reported that full‐length FtsA from mesophilic methicillin‐resistant S . aureus (SaFtsA) could be purified in a tractable form and crystallized [20]. Herein, we report the crystal structure of SaFtsA‐β‐γ‐imidoadenosine 5′‐phosphate (AMPPNP; a non‐hydrolyzable ATP analog) complex.…”