Poster abstracts

Poster number 9 submitted by Elihu Ihms

Detection and characterization of large TRAP/Anti-TRAP heterocomplexes via SAXS

Elihu Ihms (Biochemistry, The Ohio State University), Mark Foster (Biochemistry, The Ohio State University), Paul Gollnick (Biological Sciences, SUNY Buffalo), Gregory Hura (Physical Biosciences, Lawrence Berkeley National Laboratory)

Abstract:
In many Bacilli species, the donut-shaped regulatory protein TRAP is allosterically activated by tryptophan to bind to specific mRNAs, attenuating their transcription. The protein Anti-TRAP (AT) serves as an additional level of regulation, as it is expressed at high levels of uncharged Trp-tRNA and antagonizes the TRAP/RNA interaction by occluding the RNA binding site of TRAP. Previous biochemical and in vivo data indicate that only 1-2 AT trimers are required for inhibition of the TRAP/RNA complex. Solution-state studies of the complexes via NMR and analytical ultracentrifugation indicate that structures at these ratios are large and heterogenous in configuration. Interestingly, crystallographic studies of TRAP + AT result in a stoichiometry of six AT trimers symmetrically arranged around the circumference of a 12-mer TRAP, but this may be the result of selective crystallization and packing forces. We have acquired SAXS data that at 1-3 AT trimers per TRAP donut, large (>250A diameter) heterocomplexes are formed, consistent with multiple TRAP donuts bound to one or more AT trimers. Interestingly, as the ratio of AT3:TRAP11 is increased, the scatterer population condenses to form structures less than 100A in diameter, despite the overall increase in protein concentration. We performed ab initio calculations on a dataset in which the contribution of unbound AT trimers was removed from profiles acquired at a ratio of 8 AT3 per TRAP11. The resulting averaged envelope suggests a novel model in which two AT trimers are bound to opposite sides of a TRAP donut. This model explains the structural mechanism by which the extended TRAP/AT heterocomplexes are formed (TRAP donuts linked by AT trimers), as well as ITC data indicating 1-2 strong AT3 binding modes per TRAP11. We hypothesize that the ability of individual AT trimers to inactivate multiple TRAP oligomers is an example of regulatory amplification, and is achieved through the formation of TRAP/AT repeats arranged into soluble fibrils

Keywords: trap, regulation, saxs