Cloning, Expression, and Biophysical Investigations of Truncated Inclusion Protein A (IncA) from Chlamydia trachomatis

Chlamydia (C.) trachomatis is an obligate bacterial pathogen of eukaryotic cells that, despite over a century of scientific inquiry, has avoided eradication.1, 2 C. trachomatis replicates within an intracellular vacuole called an inclusion body. Inclusion membrane protein A (IncA), an effector protein expressed by C. trachomatis, is secreted by a type III secretory system into the host-derived membrane of the inclusion. IncA is proposed to mediate homotypic fusion of inclusion bodies. Inclusion proteins are characterized by a bi-lobed trans-membrane segment with a C-terminal domain that extends into the host cytoplasm. Sequence analysis of IncA reveals a “soluble N-ethylmaleimide-sensitive attachment protein receptors” (SNAREs) motif in the C-terminal region which is found in fusion-facilitating proteins.3 A signature of the SNARE motif is the leucine zipper which may initiate the formation of homo-dimers or hetero-dimers. In this study, a truncated construct with an N-terminal histidine tag was generated which yielded the SNARE soluble domain without the transmembrane region. Size exclusion chromatography (SEC) demonstrated the hexa-histidine tag disrupted dimer formation; however, after cleavage of the tag with thrombin, dimer was reformed. Circular dichroism of the construct confirmed the dimer was all α-helical as expected for a SNARE motif. Finally, two single cysteine mutants were prepared and spin-labeled. Continuous wave electron paramagnetic resonance confirmed the sites were spin-labeled and the lineshapes were consistent with moderately immobilized spin labels expected for a tertiary contact. Future double electron-electron resonance experiments will measure the distance between the spin labels, which will elucidate the dimer orientation.