TY - JOUR
T1 - Solution Structure of a Cyanobacterial Phytochrome GAF Domain in the Red-Light-Absorbing Ground State
AU - Cornilescu, Gabriel
AU - Ulijasz, Andrew T.
AU - Cornilescu, Claudia C.
AU - Markley, John L.
AU - Vierstra, Richard D.
N1 - Funding Information:
We thank Dr. Mario Rivera for providing the 13 C-isotopically labeled α-aminolevulinic acid, and Drs. Katrina Forest and William M. Westler for helpful advice. This work was supported by a grant from the National Science Foundation (MCB 07191530 to R.D.V.) and an American Heart Association postdoctoral fellowship (to A.T.U.). C.C.C. was supported by National Institutes of Health (NIH) grant 1U54 GM074901 (National Institute of General Medical Sciences). This study was a collaboration with the National Magnetic Resonance Facility at Madison, which was supported by NIH grants P41 RR02301 (Biomedical Research Training Program/National Center for Research Resources) and 1P41 GM66326 (National Institute of General Medical Sciences). Additional equipment was purchased with funds from the University of Wisconsin-Madison, the NIH (RR02781 and RR08438), the National Science Foundation (DMB-8415048, OIA-9977486, and BIR-9214394), and the United States Department of Agriculture.
PY - 2008/11/7
Y1 - 2008/11/7
N2 - The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three-dimensional NMR structure of Synechococcus OS-B′ cyanobacterial Phy 1 in the red-light-absorbing state of Phy (Pr) revealed that PCB is bound to Cys138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted by approximately 80° relative to the B/C rings by contacts with Lys52 and His169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg101 and Arg133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles, with Arg101 and Arg133 helping stabilize and destabilize the far-red-light-absorbing state of Phy (Pfr), respectively. Given the fact that the Synechococcus OS-B′ GAF domain can, by itself, complete the Pr → Pfr photocycle, it should now be possible to determine the solution structure of the Pfr chromophore and surrounding pocket using this Pr structure as a framework.
AB - The unique photochromic absorption behavior of phytochromes (Phys) depends on numerous reversible interactions between the bilin chromophore and the associated polypeptide. To help define these dynamic interactions, we determined by NMR spectroscopy the first solution structure of the chromophore-binding cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domain from a cyanobacterial Phy assembled with phycocyanobilin (PCB). The three-dimensional NMR structure of Synechococcus OS-B′ cyanobacterial Phy 1 in the red-light-absorbing state of Phy (Pr) revealed that PCB is bound to Cys138 of the GAF domain via the A-ring ethylidene side chain and is buried within the GAF domain in a ZZZsyn,syn,anti configuration. The D ring of the chromophore sits within a hydrophobic pocket and is tilted by approximately 80° relative to the B/C rings by contacts with Lys52 and His169. The solution structure revealed remarkable flexibility for PCB and several adjacent amino acids, indicating that the Pr chromophore has more freedom in the binding pocket than anticipated. The propionic acid side chains of rings B and C and Arg101 and Arg133 nearby are especially mobile and can assume several distinct and energetically favorable conformations. Mutagenic studies on these arginines, which are conserved within the Phy superfamily, revealed that they have opposing roles, with Arg101 and Arg133 helping stabilize and destabilize the far-red-light-absorbing state of Phy (Pfr), respectively. Given the fact that the Synechococcus OS-B′ GAF domain can, by itself, complete the Pr → Pfr photocycle, it should now be possible to determine the solution structure of the Pfr chromophore and surrounding pocket using this Pr structure as a framework.
KW - bilin chromophore
KW - GAF domain
KW - NMR
KW - phytochrome
KW - red-light-absorbing form
UR - http://www.scopus.com/inward/record.url?scp=52949096101&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2008.08.034
DO - 10.1016/j.jmb.2008.08.034
M3 - Article
C2 - 18762196
AN - SCOPUS:52949096101
SN - 0022-2836
VL - 383
SP - 403
EP - 413
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -