Physicochemical evidence that Treponema pallidum TroA is a zinc-containing metalloprotein that lacks porin-like structure

Abstract

Although TroA (Tromp1) was initially reported to be a Treponema pallidum outer membrane protein with porin-like properties, subsequent studies have suggested that it actually is a periplasmic substrate-binding protein involved in the transport of metals across the treponemal cytoplasmic membrane. Here we conducted additional physicochemical studies to address the divergent viewpoints concerning this protein. Triton X-114 phase partitioning of recombinant TroA constructs with or without a signal sequence corroborated our prior contention that the native protein's amphiphilic behavior is due to its uncleaved leader peptide. Whereas typical porins are trimers with extensive beta-barrel structure, size exclusion chromatography and circular dichroism spectroscopy revealed that TroA was a monomer and predominantly alpha-helical. Neutron activation, atomic absorption spectroscopy, and anomalous X-ray scattering all demonstrated that TroA binds zinc in a 1:1 molar stoichiometric ratio. TroA does not appear to possess structural features consistent with those of bacterial porins.

FIG. 1

An uncleaved signal sequence confers amphiphilicity on TroA. Detergent-enriched (D) and aqueous (A) phases were collected following Triton X-114 phase partitioning of T. pallidum, and recombinant constructs possessed (L-TroA) or lacked (S-TroA) the native polypeptide’s 22-amino-acid leader peptide. Proteins were visualized by immunoblot analysis by using a TroA-specific murine monoclonal antibody. Note that both L-TroA and S-TroA contain 28-amino-acid N-terminal extensions derived from the pProEx1 expression vector. Numbers at the left correspond to molecular mass markers.

FIG. 2

Large hexagonal rod S-TroA crystals used for the anomalous zinc scattering experiment.

FIG. 3

A zinc anomalous difference Patterson map of TroA at section Z = 1/6. The Zn anomalous dispersion data at resolutions from 20.0 to 2.7 Å were used. Two Zn sites (A and B), reflecting two protein molecules in one asymmetric unit, are shown. Each contour represents a standard deviation from the mean.