Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography.
We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.
Sakamoto
Kensaku
K
RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Murayama
Kazutaka
K
Oki
Kenji
K
Iraha
Fumie
F
Kato-Murayama
Miyuki
M
Takahashi
Masahiro
M
Ohtake
Kazumasa
K
Kobayashi
Takatsugu
T
Kuramitsu
Seiki
S
Shirouzu
Mikako
M
Yokoyama
Shigeyuki
S
D000123:Acetyltransferases / Q000737:chemistry / Q000378:metabolism; D001191:Arylamine N-Acetyltransferase / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D017016:Methanococcaceae / Q000201:enzymology / Q000378:metabolism; D007470:Monoiodotyrosine / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology
C523964:3-iodotyrosine; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D000123:Acetyltransferases; C521051:RimL protein, E coli; D001191:Arylamine N-Acetyltransferase; D007470:Monoiodotyrosine