Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Friday Abstracts
Why are Phytochromes Knotted?
Katrina T. Forest
, Shyamosree Bhattacharya, Anna W. Baker.
University of Wisconsin-Madison, Madison, USA.
Members of the phytochrome family of photoreceptors contain a deep figure-of-eight knot. We
have hypothesized that signal transduction by phytochromes is efficient because of this unusual
topology. Absorption of a 700 nm photon by the chromophore directs 41 kcal/mol into the
protein; the knot may rigidify the photosensory core of phytochrome so that work is done to
reposition the effector domain appropriately in the transition from dark to lit state, rather than
permitting energy losses to random motions. To test this model, we are studying the
biochemistry and structural biology of the signal transduction pathway of the bacterial
phytochromes of Deinococcus radiodurans and Ramlibacter tatouinensis. Each contains a
histidine kinase effector domain, and each is predicted to be a light-regulated enzyme that
phosphorylates across a homodimeric interface and subsequently transfers phosphate to a
response regulator protein. We have established a robust in vitro kinase assay and our
preliminary results suggest R. tatouinensis bacterial phytochrome is an autokinase that interacts
specifically with its bacterial phytochrome response regulator (Brr), with lower activity under
red light. We have used recombineering to generate a knotless bacterial phytochrome and will
test its kinase activity and the light-dependence of that activity. In addition, we have refined the
X-ray crystal structure of the R. tatouinensis Brr protein. In an unusual topology, a C-terminal
extension of the canonical response regulator fold wraps around its symmetry mate and returns to
the originating monomer, forming an alpha helix that packs against the globular domain. Thus, to
our surprise, this single domain response regulator dimerizes to form a light-independent,
noncovalent catenane. The coincidence that a knotted photoreceptor is paired with a linked
response regulator dimer is an intriguing phenomenon whose biological relevance has yet to be
investigated.
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