Structural Characterization of a Mutant form of the Chlamydial Protein Pgp3

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Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide. Untreated infection of the human ocular or urogenital tract can lead to blindness, ectopic pregnancy, pelvic inflammation, and infertility. The C. trachomatis protein Pgp3 is a homotrimer believed to play a prominent role in chlamydial pathogenesis. Pgp3 is one of the most immunodominant antigens found in infected people, and it stimulates macrophages into releasing inflammatory cytokines.We have recently determined the structure of Pgp3. The protein is comprised of three domains: a C‐terminal domain that resembles the TNF family of cytokines, an N‐terminal domain with structural motifs similar to those found in trimeric viral proteins, and a central triple‐helical coiled‐coil (THCC) that forms a rare right‐handed superhelical twist.The structure determination of Pgp3 was challenging due to the strong diffuse scattering observed in the diffraction pattern of the crystals. The diffuse scatter is believed to be caused by the flexibility of the THCC domain as a result of a sharp turn at glycine 85.To improve the model and produce better diffracting crystals, we mutated glycine 85 into alanine to remove the sharp turn, thus producing better diffracting crystals to build a better model of the THCC domain.The G85A protein was expressed, purified, and, crystallized, and the structure was determined. The crystal shares the same space group with WT Pgp3 and the diffuse scattering persisted. The G85A mutation did not alter the twist in the THCC domain.