Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed.
KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a water-filled channel reaching the chromophore area from the end cap of the beta-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu(68) and Ser(119), located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain.
Pletnev
Sergei
S
Synchrotron Radiation Research Section, Macromolecular Crystallography Laboratory, National Cancer Institute/SAIC-Frederick Inc., Frederick, Maryland 21702, USA.
Gurskaya
Nadya G
NG
Pletneva
Nadya V
NV
Lukyanov
Konstantin A
KA
Chudakov
Dmitri M
DM
Martynov
Vladimir I
VI
Popov
Vladimir O
VO
Kovalchuk
Mikhail V
MV
Wlodawer
Alexander
A
Dauter
Zbigniew
Z
Pletnev
Vladimir
V
D018360:Crystallography, X-Ray; D017484:Dermatitis, Phototoxic; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity*; D006801:Humans; D008027:Light; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D017319:Photosensitizing Agents / Q000737:chemistry* / Q000633:toxicity*; D011487:Protein Conformation
D017319:Photosensitizing Agents; C508771:killer red protein, Anthomedusae; D049452:Green Fluorescent Proteins