Photon Factory Activity Report 2014 #32 (2015) B

NW12, NE3A, 5A/2013G618

Crystal structures of enzymes and transporters
involved in amino acid metabolism
Takeo TOMITA, Tetsuo KUBOTA, Ayako YOSHIDA1, Tetsu SHIMIZU1, Kento TAKAHASHI1,
Hiroyuki YAMAMOTO, Makoto NISHIYAMA*1
Biotechnology Research Center, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Introduction
Genome analysis of various organisms has revealed thatmembers of the Lrp (Leucine responsive regulatoryprotein) family of transcriptional regulators are widelydistributed among prokaryotes, both bacteria and archaea
and are responsible for various regulatory systems via
global transcriptional regulation. For example, the Lrp
from E. coli is a global regulator involved in modulating avariety of metabolic functions, including the catabolism
and anabolism of amino acids. Lrp consists of the Nterminal DNA binding domain and the C-terminal RAM(Regulation of Amino acid Metabolism) domain.
Although Thermus thermophilus does not possess
homologues of Lrp gene on its genome, it possesses agene encoding stand-alone RAM domain protein(TTC0493; SraA), which has similarity to C-terminal
domain of Lrp. Thus we hypothesized that SraA senses
amino acids as a signal and mediates global regulation ofmetabolism through interaction with other proteins. In
this study, we analysed the function and structure of SraA
to reveal the signal transduction system of amino acids inT. thermophilus. The crystal structure of SraA from T.
thermophiles HB8 has already been determined as aligand-free form. RAM domain of SraA can binds toamino acids, and we hypothesized that amino acids areligand of SraA and tried to determine the crystal structure
in complex with amino acids.
Materials and Methods
Purification of SraA
Strep-SraA was overexpressed in E. coli BL21-CodonPlus (DE3)-RIL as a host using 0.1 mM isopropyl-E-D-1thiogalactopyranoside (IPTG) for induction. From celllysate prepared by sonication, SraA was purified throughheat treatment, Streptactin affinity chromatography andSuperdex 200 gel filtration chromatography.
Elucidation of the oligomeric state of SraA
We performed gel filtration chromatography to elucidate
the oligomeric state of SraA, We mixed SraA (2.0mg/mL) with amino acid (5 mM) and applied to gel
filtration chromatography.
Crystallization of SraA
Screening of crystallization condition was initiallyperformed with Crystal screen I and II, Wizard classic I,
II, and III, and PEG/ION screening kits by hanging drop

vapor diffusion method. crystallization conditions werefurther optimized by changing pH and concentration ofprecipitant.
Data collection and processing.
The X-ray diffraction data of native proteins werecollected using the beamline, NW12, NE3 and 5A at PF.
The image sets were integrated and scaled usingHKL2000.
Results and Discussion
We examined the effect of amino acids on the oligomericstate of SraA with gel filtration chromatography. In theabsence of amino acids, SraA was present in an
equilibrium between dimer and decamer, whereas in thepresence of some amino acids, SraA decamer was muchinduced. This suggests that some amino acids binds SraA
and changes its conformation.
Furthermore, these results may suggest that SraA senses
various amino acids as signals to regulate a variety ofmetabolism by changing its conformation.
We tried to performed screening of crystallizationcondition of SraA with amino acids and obtained microor needle like- crystals from several conditions. Now weare trying to optimize the crystallization conditions.

* umanis@mail.ecc.u-tokyo.ac.jp