DOI: 10.6084/m9.figshare.25515391.v1
title: <b>Crystal structure of</b><b> </b><b><i>Trichinella spiralis</i></b><b> </b><b>calreticulin and the structural basis of its complement evasion mechanism involving C1q</b>

description: Helminths produce calreticulin(CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth <i>Trichinella spiralis</i> produces calreticulin (TsCRT) which binds C1q to inhibit activation of the complement classical pathway. We used X-ray crystallography to resolve the structure of truncated TsCRT (TsCRT<sup>Δ</sup>), the first structure of helminth-derived CRT. TsCRT<sup>Δ</sup> was observed to share the same binding region on C1q with IgG based on the structure and molecular docking and Hydrogen Deuterium Exchange Mass Spectrometry(HDX-MS). Here we displayed the raw data of HDX-MS, which demonstrate the sites involved in the interaction both on C1q and TsCRT<sup>Δ</sup>.MethodsThrough monitoring of the proton-exchange capacity of the amide skeleton, HDX-MS enables mapping of interaction sites (possible peptides involved in interaction) between proteins(James et al. 2022). ​When the protein-protein complex is placed in a buffer with or without deuterium water for a certain duration, the rate of deuterium exchange at the sites involved in the interaction changes in the complex relative to the monomeric protein. High-performance liquid chromatography was used to separate the peptides obtained by enzyme digestion. Finally, based on the mass difference between hydrogen and deuterium, MS was used to monitor the change in hydrogen-deuterium exchange by measuring the peptide center-of-mass shift. ​ The specific procedure was as follows:For the HDX-MS experiment, 135 µl of 6 mg/ml TsCRT<sup>Δ</sup> / 2 mg/ml C1q alone and in the presence of the corresponding ligand were prepared. The buffer, whose pH was 8.0, contained 50 mM Hepes, 150 mM NaCl, and 2 mM CaCl<sub>2</sub>. To initiate deuterium labeling, 5 μL of each 180 μM protein solution was diluted with 45 μL of labeling buffer (20 mM Tris, 500 mM (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 99% D<sub>2</sub>O, pH 8.5) at 25 ℃ for 30 s, 60 s, 90 s, and 300 s, and 50 μL of ice-cold quenching buffer (4 M guanidine hydrochloride, 200 mM citric acid, and 500 mM TECP in water solution at pH 1.8, 100% H<sub>2</sub>O) was added to quench the labeling. The reaction tube was then put on ice. Next, 5 μL of 1 μM pepsin solution was added for digestion. At 2 min, the centrifugated sample was placed into the auto-sampler of the Ultimate 3000 HPLC (Thermo, USA) for injection. Then, 50 μL of sample was loaded onto and separated by a ACQUITY UPLC 1.7 μm BEH C18 2.1*50 mm column (Waters, UK). Mobile phase A consisted of 1% v/v formic acid, and mobile phase B consisted of 100% acetonitrile and 1% formic acid. The polypeptide was separated by gradient elution at a flow rate of 115 µl/min for 20 min. Mass spectrometry analysis was performed on a Q Exactive Orbitrap mass spectrometer (Thermo, USA). The deuterium exchange levels were determined by subtracting the centroid mass of undeuterated peptide from the centroid mass of deuterated peptide using HDExaminer (Version PD1.4, Thermo-Fisher Scientific, USA).Contents:<br>There are 18 raw data files, 17 excel files and two fasta files. The raw data files can be open with Xcalibur software.