Photon Factory Activity Report 2022 #40 (2023)

AR-NE3A/2020G681, 2022G031

X-ray crystallographic analysis of a scavenger receptor cysteine-rich domain(SRCRD) from Salivary scavenger and agglutinin (SALSA)
Changyu Zhang1, Peng LU1* and Koji NAGATA1,2
1
Department of Applied Biological Chemistry,2
Agricultural Bioinformatics Research Unit,
Graduate School of Agricultural and Life Sciences, The
University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
1 Introduction
Salivary scavenger and agglutinin (SALSA), a 300to 400-kDa glycoprotein also as known as deleted inmalignant brain tumors 1 (DMBT1) and salivaryagglutinin (SAG), is a member of the scavenger receptor
family, which was thought to be involved in the oral
clearance of microorganisms due to its bacterial
agglutination properties. It contains 14 SRCRDs
separated by SRCR-interspersed domains (SIDs). Dueto its ability to bind and agglutinate Streptococcus
mutans, salivary agglutinin has been considered to play
an important role in preventing dental caries.
As SALSA recognized a various of biological ligands,
one of its main physiological activities is to mediate theadherence of Streptococcus mutans to teeth surface bybinding to cell surface protein antigen c (PAc). The AVP
region located at the morphological end of PAc protein
was demonstrated to contain the binding affinity for
SALSA. The ligand recognition by the SRCRD usuallyneeds divalent metal ions, such as Ca2+. Calcium
induces secondary structural changes, increase thethermal stability and binding affinity to a Fe3+ binding
protein, lactoferrin, which are essential for preventing
the adherence of oral Streptococci. Most describedligand-binding interaction of SRCRDs was dependent
on the presence of calcium ions.

Crystals of SRCRD were obtained using a reservoir
composition of 0.2 M Lithium citrate tribasic
tetrahydrate and 20% w/v Polyethylene glycol 3,350
from PEG/ion kit. The crystals were picked up with
Mounted CryoLoop and cryoprotected in reservoir
solution containing 20% glycerol. Then the crystals
were flash frozen in liquid nitrogen and transferred tounipuck. X-ray diffraction experiments for SRCRD
crystals were performed on beamline AR-NE3A at
Photon Factory. The data were indexed, integrated, andscaled using the XDS program. Initial structural modelsof SRCRD were obtained by molecular replacementwith Morlep using the structure of GP340 SRCRD 8(PDB 6SA5) as the search model. Further modelbuilding and refinement were performed withREFMAC5, Coot, and Phenix.

2 Experiment
The DNA encoding SRCRD11 (1371-1489) from
Deleted in malignant brain tumors 1 (DMBT1) protein
was cloned into pET-32a expression vector. The verified
recombinant plasmids were transformed intoEscherichia coli strain SHuffle T7 cells. SRCRDprotein was refolded using glutathione as a redox systemand purified by ion exchange chromatography. The
purified protein was then concentrated to ~10 mg/ml forcrystallization.
Initial crystallization screening was performed
Wizard Ⅰ & Ⅱ (Rigaku Reagents, Inc.), Crystal Screen
HT™ (Hampton Research), and PEG Ion Screen™
(Hampton Research). The sitting-drop vapor diffusion
method was adopted at 20℃ in 96-well VIOLAMO
Protein Crystallization Plates (As One), and 0.5 µL
protein solution and 0.5 µL reservoir solution weremixed in one drop to grow the crystals.

Fig.1 Crystal structure of SRCRD with Fo – Fc map
coutoured at 1 σ.
3 Results and Discussion
The diffraction data from the SRCRD crystals werecollected to ~1.8 Å (Fig. 1). The crystal structure ofSRCRD from SALSA reveals a typical globular SRCRfold. The fold contains one α-helix at the N-terminus andfour β-strand at both N- and C-termini. The SRCRD
contains eight cysteines that form four pairs of disulfide
bonds, giving the SRCRD a complete and stablestructure, which is evidence of the success of therefolding process. According to the surface chargedistribution map of SRCRD, there is a region of negative
charge on the surface of SRCRD, which is considered to
be a binding site for calcium ions.

Acknowledgement
We would like to thank the beamline staff members
Photon Factory AR-NE3A for their help with X-ray
data collection and processing.
* porterlu@g.ecc.u-tokyo.ac.jp