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Updated: Jul 9, 2026
Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography
Published on: September 20, 2016
Expression, crystallization and preliminary X-ray diffraction analysis of human paired Ig-like type 2 receptor alpha
Shigekazu Tabata1, Kimiko Kuroki, Nobuo Maita
1Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Insights
Human paired immunoglobulin-like type 2 receptor alpha (PILRalpha) suppresses immune cell function. Researchers determined the crystal structure of the PILRalpha V-set domain, providing insights into its inhibitory role in immune responses.
Area of Science:
- Immunology
- Structural Biology
- Biochemistry
Background:
- Paired immunoglobulin-like type 2 receptor alpha (PILRalpha) is a type I membrane protein found on immune cells like monocytes and dendritic cells.
- PILRalpha contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that recruits SHP-2 phosphatase, inhibiting immune cell activation signals.
Purpose of the Study:
- To determine the crystal structure of the extracellular V-set domain of human PILRalpha.
- To provide structural insights into the inhibitory function of PILRalpha in immune regulation.
Main Methods:
- Overexpression of the human PILRalpha V-set domain (residues 13-131) in E. coli.
- Refolding of inclusion bodies and protein purification.
- X-ray crystallography at 1.3 A resolution using synchrotron radiation (SPring-8 BL41XU).
Main Results:
- The PILRalpha V-set domain was successfully crystallized using the sitting-drop vapor-diffusion method at 293 K.
- The crystal belonged to space group P2(1)2(1)2(1) with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 A.
- The crystal structure revealed one molecule per asymmetric unit.
Conclusions:
- The high-resolution crystal structure of the human PILRalpha V-set domain was determined.
- This structural information can aid in understanding PILRalpha's role in immune suppression and potentially in developing targeted therapeutics.
Abstract:
Human paired immunoglobulin-like (Ig-like) type 2 receptor alpha (PILRalpha) is a type I membrane protein that is mainly expressed in immune-related cells such as monocytes, granulocytes and dendritic cells. PILRalpha can suppress the functions of such immune cells because it has the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region, which recruits the phosphatase Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-2) to inhibit phosphorylations induced by activation signals. The extracellular region of human PILRalpha comprises one immunoglobulin superfamily V-set domain and a stalk region. The V-set domain (residues 13-131) of human PILRalpha was overexpressed in Escherichia coli as inclusion bodies, refolded by rapid dilution and purified. The PILRalpha protein was successfully crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.3 A resolution at SPring-8 BL41XU; they belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 A, and contain one molecule per asymmetric unit.
