Solution structure, dynamics and tetrahedral assembly of Anti-TRAP, a homo-trimeric triskelion-shaped regulator of tryptophan biosynthesis inBacillus subtilis

Abstract

AbstractCellular production of tryptophan is metabolically expensive and tightly regulated. The smallBacillus subtiliszinc binding Anti-TRAP protein (AT), which is the product of theyczA/rtpAgene, is upregulated in response to accumulating levels of uncharged tRNATrpthrough a T-box antitermination mechanism. AT binds to the undecameric ring-shaped protein TRAP (trpRNA Binding Attenuation Protein), thereby preventing it from binding to thetrpleader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of thetrpoperon. AT principally adopts two symmetric oligomeric states, a trimer (AT3) featuring a three-helix bundle, or a dodecamer (AT12) comprising a tetrahedral assembly of trimers, whereas only the trimeric form has been shown to bind and inhibit TRAP. We demonstrate the utility of native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) for monitoring the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we report the use of solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT3, while heteronuclear15N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT3and binding-inactive AT12, with implications for TRAP inhibition.