Photon Factory Activity Report 2017 #35 (2018)

BL-5A/2016G532

X-ray structures of Clostridium perfringens sortase B and its mutant form
Shigehiro Kamitori1,*, Hiromi Yoshida1, and Eiji Tamai1,2Life Science Research Center and Faculty of Medicine, Kagawa University
1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan2
Department of Infectious Disease, College of Pharmaceutical Science Matsuyama University
4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
1

1 Introduction
The pathogenesis and infectivity of Gram-positivebacteria are mediated by many surface proteins that arecovalently attached to peptidoglycans of the cell wall.
The covalent attachment of these proteins is catalyzed bysortases (Srts), a family of cysteine transpeptidases,
which are classified into six classes, A – F, based on theiramino acid sequences and biological roles. Clostridium
perfringens, one of the pathogenic clostridial species, hasa class B sortase (CpSrtB) with 249 amino acid residues.
X-ray structures of CpSrtB and its inactive mutant form
with the replacement of the catalytic Cys232 with Ser(CpSrtB_C232S), were determined at 2.2 Å and 1.8 Åresolutions, respectively. [1]-barrel. The conserved catalytic residues His136, Ser232(Cys) and Arg240 are located at the loop between B4 andH4, the end of B7, and the beginning of B8, respectively.
A groove for substrate-binding is formed by three strands (B4, B7 and B8) and two helices (H3 and H6)creating the sidewalls of the groove.

2 Experiment
Crystals of CpSrtB were grown at 293 K in a droplet
mixed with 1 l of protein solution (18.4 mg/ml in 20mM Tris-HCl, pH 7.5) and 1 l of reservoir solution (200mM sodium nitrate, 20% (w/v) PEG3350, pH6.8) with 50
l of the reservoir solution, using the sitting drop vapor
diffusion method. Crystals of CpSrtB_C232S wereprepared by using protein solution (30.2 mg/ml in 20 mM
Tris-HCl, pH 7.5) and reservoir solution (200 mM
ammonium phosphate monobasic, 20% (w/v) PEG3350,pH4.6). X-ray diffraction data for CpSrtB andCpSrtB_C232S were collected at 100 K using an ADSCQUANTUM 315R area detector system on the PF BL-5A
beam line in the KEK (Tsukuba, Japan).
3 Results and Discussion
CpSrtB was crystallized in space group P21212, and thestructure was refined to R-factor of 0.227 at 2.2 Åresolution. Crystals of CpSrtB_C232S were obtained inspace group C2, giving a higher resolution of data, andthe structure was refined to R-factor of 0.184 at 1.8 Åresolution. The overall structure of CpSrtB_C232S isshown in Fig. 1 with a topological diagram of secondary
structure elements. CpSrtB_C232S adopts a typicalsortase-protein fold with eight β-strands and seven helices.
There is a distorted -barrel at the center of the protein.
The two -helices (H1 and H2) form the -bundle at theN-terminal site. The connected loop between H1 and H2in CpSrtB_C232S is invisible due to the highly
disordered structure. A long loop including a short 310helix (H3) is inserted between B2 and B3. The 310 helix(H4) and -helix (H5) between B4 and B5 are across the-barrel. The two -helices (H6 and H7) between B6 andB7 are arranged in an anti-parallel manner, contacting theFig. 1: The overall structures of CpSrtB_C232S and thetopological diagram of the secondary elements. Threecatalytic residues and hydrophobic residues involved in
the formation of the hydrophobic cluster with H1 and H2
are shown with a stick model.
Acknowledgement
We thank the PF staff for support of data collection.
Reference[1] E. Tamai et al., Biochem Biophys Res Commun. 493,
1267 (2017). doi: 10.1016/j.bbrc.2017.09.144.
* kamitori@med.kagawa-u.ac.jp