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

Cellular production of tryptophan is metabolically expensive and tightly regulated. The small zinc binding Anti-TRAP protein (AT), which is the product of the gene, is upregulated in response to accumulating levels of uncharged tRNA through a T-box antitermination mechanism. AT binds to the undecameric axially symmetric ring-shaped protein TRAP ( RNA Binding Attenuation Protein), thereby preventing it from binding to the leader RNA. This reverses the inhibitory effect of TRAP on transcription and translation of the operon. AT principally adopts two symmetric oligomeric states, a trimer (AT) featuring three-fold axial symmetry or a dodecamer (AT) comprising a tetrahedral assembly of trimers, whereas only the trimeric form binds and inhibits TRAP. We apply native mass spectrometry (nMS) and small-angle x-ray scattering (SAXS), together with analytical ultracentrifugation (AUC) to monitor the pH and concentration-dependent equilibrium between the trimeric and dodecameric structural forms of AT. In addition, we use solution nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AT, while heteronuclear N relaxation measurements on both oligomeric forms of AT provide insights into the dynamic properties of binding-active AT and binding-inactive AT, with implications for TRAP binding and inhibition.