Photon Factory Activity Report 2019 #37 (2020)

BL-1A, AR-NW12A/2016G638

High resolution crystal structure analyses of the complexes of CD28 family moleculesand signaling proteins
Nobutaka NUMOTO1,*
1
Medical Research Institute, Tokyo Medical and Dental University (TMDU),
1-5-45 Yushima Bunkyo-ku, Tokyo, 113-8510, Japan
1 Introduction
T cell regulation plays an important role in the humanimmune system and requires two independent signals. One
is an antigen-specific signal via the T cell receptor (TCR)
upon binding to the major histocompatibility complex(MHC), and the other is an antigen non-specific signal via
the CD28 family molecule upon binding to the B7
molecule group. The intracellular region of CD28 consistsof about 40 residues and transmits a signal into the cell byrecruiting other molecules. The tyrosine residue in theamino acid sequence of YMNM in the intracellular regionof CD28 is phosphorylated by the Tyr kinase, and thephospholylated Tyr (pY) containing pYMNM sequence isrecognized by the signaling molecules such as Growth
factor receptor-bound protein 2 (Grb2). The SH2 domainof Grb2 specifically binds to the consensus sequence ofpYXNX, which matches the pYMNM sequence in theCD28. Grb2 exhibits a monomer - dimer equilibrium thatis thought to regulate normal and abnormal functions. We
have previously reported the crystal structure of Grb2 SH2domain in complex with the CD28 phosphopeptide,
SDpYMNMTP, and elucidated that the recognition
mechanism of pY and the Asn residue in the consensus
sequence.
In this study, we have determined the crystal structuresof the SH2 domain of Grb2 (Grb2-SH2) without thephosphopeptide at 1.15 Å resolution and a domainswapped dimer of Grb2-SH2 at 2.0 Å resolution [1].
Obtained structures reveal that Trp121 is the key residue atthe hinge region of C-terminal helix swapping, and thedihedral angle in the main-chain of Trp121 is energetically
preferred in the domain swapped dimer structure than thatin the monomer structure.
2 Experiment
Grb2-SH2 was overexpressed in Escherichia coli andpurified as described previously. Crystallization wasperformed by the hanging-drop vapor-diffusion method at20ºC. Prior to data collection, the crystals were soaked incryo-protectant solutions containing 20% (v/v) glycerol
along with their respective reservoir buffers and flashfrozen using nitrogen gas stream at 95 K.
X-ray diffraction experiments were performed at thebeamlines BL-1A and NW12A at PF and PF-AR, KEK. Alldata were processed and scaled using XDS [2]. The initial
phases were determined by the molecular replacement
method using the program PHASER [3]. Several cycles ofmanual model rebuilding by using COOT [4] andrefinement by using PHENIX [5] were performed.
3 Results and Discussion
Crystals of the monomeric Grb2-SH2 were obtained inthe buffer containing 0.1 M sodium chloride, 0.1 M
HEPES (pH 7.5), 1.6 M ammonium sulfate. The structure
was determined at 1.15 Å resolution, which is the highest
resolution analysis among over 30 structures of the peptide
bound/unbound Grb2-SH2.
Crystals of the domain-swapped dimer of Grb2-SH2
were unexpectedly obtained from the monomer fraction of
the purified protein, using the reservoir solution containing
0.1 M MES (pH 6.5), 12% (w/v) polyethylene glycol
20,000. The structure was determined at 2.00 Å resolution.
Trp121 was the hinge residue of the extended, domain
swapped form compared to the monomeric globularstructure (Fig. 1). The dihedral angles in the main-chain of
Trp121 in the monomer structure, φ = –133º and ψ = –82º,are energetically disadvantageous, while those in the dimerstructure, φ = –153º and ψ = 147º, are energeticallypreferred. The peptide plane flipping at this residue seemedto cause the large conformational change for the Cterminal extended form.

W121

Fig. 1: Structures of monomeric (green) anddomain-swapped (yellow, one protomer) form of
Grb2-SH2.
References[1] Y. Hosoe et al., Biophys. Physicobiol. 16, 80 (2019).
[2] W. Kabsh, Acta Cryst. D66, 125 (2010).
[3] A. J. McCoy et al., J. Appl. Crystallogr. 40, 658 (2007).
[4] P. Emsley et al., Acta Cryst. D66, 486 (2010).
[5] P. D. Adams et al., Acta Cryst. D66, 213 (2010).
* numoto.str@mri.tmd.ac.jp