Photon Factory Activity Report 2017 #35 (2018)

BL-5A, BL-17A, AR-NW12A/2015G075

Crystallographic analysis of apo- and ligand-binding forms of the B-cellinhibitory co-receptor CD72
Nobutaka Numoto*
Medical Research Institute, Tokyo Medical and Dental University (TMDU),
Bunkyo-ku, Tokyo 113-8510, Japan
1 Introduction
B cells play a key role in the immune system by making
antibodies. CD72 is an inhibitory co-receptor that regulates
signaling through the B cell receptor (BCR). Activation ofCD72 prevent overstimulation of the B cells. Thus, CD72
is necessary to avoid autoimmune diseases and makeantibodies properly against exogenous antigens but notautoantigen. CD72 is a type II membrane protein forming
a homodimer, primarily expressed in B cells. The ligandbinding region of CD72 is located at the C-type lectin-likedomain (CTLD) in the C-terminal extracellular region.
However, due to the lack of structural information, themechanism of the ligand recognition of CD72 was unclear.
We have demonstrated that CD72 specifically binds toSm/RNP, an RNA-containing nuclear self-antigen, andinhibit B cell response, preventing production of antiSm/RNP antibody crucial for development ofsystemic lupus erythematosus (SLE) [1]. To
elucidate detailed model of the ligand binding site,and obtain structural bases to design novel ligandsthat regulate CD72 more efficiently, we have initiatedcrystallographic analysis of the CTLD of mouse CD72
(CD72-CTLD).
2 Experiment
RecombinantmouseCD72-CTLD
wasoverexpressed using BL21(DE3) cells and refolded using
His-Accept SF resin (Nacalai Tesque). Crystallization
conditions were screenedusingcommerciallyavailable screening kits (Hampton Research) withthe hanging-drop vapor diffusion method. X-ray
diffraction experiments were performed at beamelines
5A and 17A at PF, and NW12A at PF-AR, KEK. The
data were processed and scaled using program
HKL2000 [2] and were truncated using the CCP4
program suite [3]. The structure were solved by
the molecular replacement method using PHASER[4]. Several cycles of manual model rebuilding andrefinement were performed by using the program
COOT [5] and PHENIX [6], respectively.
3 Results and Discussion
The crystal structure of apo CD72-CTLD
was determined at 1.2 Å resolution [1]. The overallstructure (Fig. 1a) is very similar to that of the typical
CTLD fold but CD72-CTLD lacks the loop region
which forms the ligand-binding site in many otherCTLDs.
Analysis of the electrostatic potentials of CD72CTLD reveals that there is a highly positively charged
patch on the surface of the molecule (Fig. 1b).
Because themolecules with nucleic acids such as Sm/RNP possesses
negatively charged region derived from the sugarphosphate backbone, it is strongly suggested that thepositively charged patch of CD72-CTLD is related torecognition of the nucleic acids in Sm/RNP.
In addition, we calculated the homology model of CD72c,
which is an allele of CD72 in a model mouse of an
autoimmune disease. The model clearly shows that surface
charge distribution around the putative ligand binding
region is different from that of CD72, substitutions of someresidues in this area result in generation of a negativecharge patch. It would be disadvantageous for binding
Sm/RNP due to electrostatic repulsion with the negative
charge of RNA. In fact, binding affinity to Sm/RNP
is decreased in CD72c [1], which is considered tobe triggering autoimmune disease. Therefore, thecharge distribution on the molecular surface of CD72 isthought to play an important role in controlling therecognition and binding of Sm/RNP.
Further structural analysis of CD72-CTLD-lignad
complexes are in progress.

aPutative ligand
binding areabFig. 1: (a) Overall structures of CD72-CTLD. (b)Electrostatic surface representation around the putative
ligand-binding area.
References[1] C. Akatsu et al., J. Exp. Med., 213, 2691 (2016).
[2] Z. Otwinowski and W. Minor, Methods Enzymol. 276,307 (1997).
[3] M. D. Winn et al., Acta Cryst. D67, 235 (2011).
[4] A. J. McCoy et al., J. Appl. Cryst. 40, 658 (2007).
[5] P. Emsley et al., Acta Cryst. D66, 486 (2010).
[6] P. D. Adams et al., Acta Cryst. D66, 213 (2010).
* numoto.str@mri.tmd.ac.jp