Photon Factory Activity Report 2022 #40 (2023)

BL-5A/2022G538

X-ray structure of Clostridium perfringens pili protein B N-terminal domain
Shigehiro KAMITORI1,*, Mitsugu YAMADA2, Hiroshi SEKIYA3 and Eiji TAMAI3
Research Facility Center for Science & Technology and Faculty of Medicine, Kagawa University,
1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
2
Japan Aerospace Exploration Agency (JAXA), 2-1-1 Sengen, Tsukuba, Ibaraki 305-8505, Japan3
Department of Infectious Disease, College of Pharmaceutical Sciences, Matsuyama University, 4-2
Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan
11 Introduction
Sortase-mediated pili are flexible rod proteins composedof major and minor/tip pilins, playing important roles in
the initial adhesion of bacterial cells to host tissues. The
pilus shaft is formed by covalent polymerization of major
pilins, and minor/tip pilin is covalently attached to the tipof the shaft involved in adhesion to the host cell. The
Gram-positive bacterium Clostridium perfringens has
major pilin, and minor/tip pilin (CppB) with the collagenbinding motif. Bioinformatic analysis predicted that CppB
has five domains (D1 - D5) of an Ig-like fold, and collagenbinding assays of CppB domain variants showed that Nterminal two consecutives domains of CppB (CppBD1D2) has a high collagen-binding activity. X-raystructure of CppB-D1D2 were determined, and only 64%
of partial structure of D1 could be built in the resultant
electron due to a disordered structure. In order to obtain thecomplete structure of D1, X-ray structure determination ofN-terminal domain of CppB (CppB-D1) was performed.
2 Experiment
Crystals of CppB-D1 (Glu33 - Tyr164) were grown at
20°C in a droplet mixed with 1 L of protein solution (110mg/mL) and 1 L of reservoir solution against 50 L of thereservoir solution (50 mM bis-tris pH 6.5, 50 mM
ammonium sulfate, 30% v/v pentaerythritol ethoxylate(15/4 EO/OH)), using the sitting drop vapor diffusion
method. Crystal was flash-cooled in liquid nitrogen at 100
K using 25% v/v glycerol in reservoir solutions as
cryoprotectants. X-ray diffraction data were collected on
PF-BL5A in KEK (Tsukuba, Japan). Using the partialmodel of D1 (64%) and a model derived from the program
AlphaFold2, the structure of CppB-D1 was solved bymolecular replacement with the program MOLREP, andstructure was refined using the programs Refmac5 at 1.55
Å resolution (PDB ID: 8GSY) [1].
3 Results and Discussion
The overall structure is shown in Fig. 1A. CppB-D1 iscomprised of two antiparallel -sheets and a helix. The sheet S1 consisting of B5, B8, B3, and B1 faces the -sheet
S2 consisting of B2, B10, B9, B4, B7, and B6, in which ashort -strand B2 is irregularly parallel to the neighboring-strand B10. Most of the loops between -strands lie
across the two -sheets, except B6-B7 and B9-B10 loops.
A short helix, H1, locates on the B3-B4 loop. To generate
a complete structure of CppB-D1D2, the structure ofCppB-D1 is introduced into the structure of CppB-D1D2(Fig. 1B). The structure adopts an L-shaped structure
considered to be in open form by a structural comparison
with Staphylococcus aureus collagen-binding adhesin incomplex with the synthetic collagen peptide (PDB: 2F6A).

AB

Fig. 1: Structures of CppB-D1 and CppB-D1D2. (A)Overall structure of CppB-D1 is illustrated with blue strands, green loops, and red -helix. (B) Overall structureof CppB-D1D2 is illustrated. The disordered regions in D1
are shown in light gray. The amino acids involved in theinteractions between domains are shown by a stick model.
Acknowledgement
We thank the PF staff for support of data collection.
References[1] E. Tamai et al., FEBS Letters 597, 1317 (2023).
* kamitori.shigehiro@kagawa-u.ac.jp