Homotropic Cooperativity from the Activation Pathway
of the Allosteric Ligand-Responsive Regulatory <i>trp</i> RNA-Binding Attenuation Protein
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Abstract
The <i>trp</i> RNA-binding attenuation protein (TRAP) assembles into
an 11-fold symmetric ring that regulates transcription and translation
of <i>trp</i>-mRNA in bacilli via heterotropic allosteric
activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic
resonance studies have revealed that Trp-induced activation coincides
with both microsecond to millisecond rigidification and local structural
changes in TRAP, the pathway of binding of the 11 Trp ligands to the
TRAP ring remains unclear. Moreover, because each of 11 bound Trp
molecules is completely surrounded by protein, its release requires
flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence
to study the kinetics of Trp binding by <i>Bacillus stearothermophilus</i> TRAP over a range of temperatures and observed well-separated kinetic
steps. These data were analyzed using nonlinear least-squares fitting
of several two- and three-step models. We found that a model with
two binding steps best describes the data, although the structural
equivalence of the binding sites in TRAP implies a fundamental change
in the time-dependent structure of the TRAP rings upon Trp binding.
Application of the two-binding step model reveals that Trp binding
is much slower than the diffusion limit, suggesting a gating mechanism
that depends on the dynamics of apo TRAP. These data also reveal that
dissociation of Trp from the second binding mode is much slower than
after the first Trp binding mode, revealing insight into the mechanism
for positive homotropic allostery, or cooperativity. Temperature-dependent
analyses reveal that both binding modes imbue increases in bondedness
and order toward a more compressed active state. These results provide
insight into mechanisms of cooperative TRAP activation and underscore
the importance of protein dynamics for ligand binding, ligand release,
protein activation, and allostery