Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

Abstract

In Bacillus subtilis the anti-TRAP protein (AT) is produced in response to the accumulation of uncharged tRNA Trp . AT regulates expression of genes involved in tryptophan biosynthesis and transport by binding to the tryptophan-activated trp RNA-binding attenuation protein (TRAP) and preventing its interaction with several mRNAs. Here, we report the x-ray structure of AT at 2.8 Å resolution, showing that the protein subunits assemble into tight trimers. Four such trimers are further associated into a 12-subunit particle in which individual trimers are related by twofold and threefold symmetry axes. Twelve DnaJ-like, cysteine-rich zincbinding domains form spikes on the surface of the dodecamer. Available data suggest several possible ways for AT to interact with the 11-subunit TRAP. Interaction between the two symmetrymismatching molecules could be assisted by the flexible nature of AT zinc-binding domains.DnaJ ͉ trp RNA-binding attenuation protein ͉ tryptophan biosynthesis ͉ protein-protein interactions

The nature of the TRAP–Anti-TRAP complex

“…The R32 crystal structure is essentially identical to the model in P6, even though the TRAP used was clearly purified as an 11-mer ring. Both crystal structures described here are entirely different from previously proposed models of the TRAP-AT interaction based on analytical centrifugation (12) and crystallography (13).…”

“…Experimental tests of the current model (13) are, however, highly desirable, because there is no ready explanation for the ability of AT to compete with RNA binding to TRAP. This lack of mechanistic understanding is rather unusual in such a well-studied system, and we have addressed this issue with a variety of biophysical methods including crystallography.…”

“…Analytical ultracentrifugation data have been interpreted to indicate that the AT dodecamer is the active (TRAP-binding) form, giving an AT 12 -TRAP 11 complex, although it was noted that the data did not rule out alternative stoichiometry (12). Antson and colleagues (13) further proposed, on the basis of their X-ray structure of AT, a model in which the AT dodecamer may bind up to 4 TRAP 11-mer rings by aligning their 11-fold symmetry axes with its 3-fold axes. In this model, AT would not directly block RNA binding, or contact TRAP residues Lys-37 and Arg-58 that are known to be required for TRAP-RNA (15) and TRAP-AT interaction (10).…”

“…In Bacillus subtilis, when levels of charged tRNA Trp fall, the protein Anti-TRAP (AT) is expressed, which blocks RNA binding to Trp-bound TRAP and relieves expression of the trp operon (10,11). AT forms a stable trimer, 4 copies of which can further associate into a dodecamer form (12,13). Each 53-residue polypeptide chain carries a zincbinding site made of 4 cysteine residues, and chemical crosslinking studies suggested that the active form of AT may be a hexamer (14).…”

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti- trp RNA-binding attenuation protein

“…Crystallographic studies of Bsu AT revealed a dodecamer (AT 12 ) comprising a tetrameric arrangement of trimers (AT 3 ). Each trimer is constructed around a three-helix bundle involving the C-terminal residues, and four trimers assemble to form the dodecamer through interactions in the zinc-binding and N-terminal regions (21). Notably, neither oligomeric form of AT, trimer or dodecamer, shares symmetry elements with its physiological target: undecameric TRAP.…”