The Use of Mass Spectrometry and Molecular Modeling to Design Structural Model of Mouse NKR-P1 Proteins

Daniel Rozbesky^{1, 2}; Petr Man^{1, 2}; Zdenek Kukacka^{1, 2}; Zofie Sovova^{3, 4}; Rudiger Ettrich^{3, 4}; Julien Marcoux⁵; Carol V. Robinson⁵; Petr Novak^{1, 2}

¹Institute of Microbiology, Prague, Czech Republic;
²Faculty of Sciences, Charles University, Prague, Czech Republic;
³Institute of Nanobiology and Structural Biology, Nove Hrady, Czech Republic;
⁴Faculty of Sciences, University of South Bohemia, Nove Hrady, Czech Republic;
⁵Department of Chemistry, University of Oxford, Oxford, United Kingdom

Introduction
Determination of protein conformation has traditionally been realized by X-ray crystallography and NMR spectroscopy. Although these techniques provide high resolution atomic data, they have some limitations. Both NMR and X-ray require large amounts of pure analyte and are time-consuming techniques. Mass spectrometry combined with chemical cross-linking offers alternative approach to identify the protein fold. This method is fast and uses small amounts of material. Our aim was to gain insight into low-resolution structure of NKR-P1C receptors. NKR-P1C is an activating immune receptor expressed on the surface of mouse natural killer cells. Using distance constraints derived from chemical cross-linking and disulfide arrangement in combination with computational methods, protein conformation was designed. The validation of structural model was addressed using ion-mobility mass spectrometry.

Methods
In order to design structural model of NKR-P1C, protein was cross-linked using homobifunctional cross-linking reagents disuccinimidyl suberate (DSS) and disuccinimidyl glutarate (DSG). After cross-linking reaction, SDS-PAGE of cross-linking reaction mixture was performed. Also, disulfide bound arrangement was determined after non-reducing SDS-PAGE.  The band of cross-linked or non-reduced protein was excised and subjected to in gel digestion by Asp-N and trypsin. The peptide mixtures from the enzymatic digest were analyzed by LC/ESI-FT-ICR MS. Cross-links and disulfide-linked products were identified using Links software. These distance constraints were used for molecular modeling. Homology modeling followed by a short steepest descent minimization was performed using the MODELLER 9v7 package. To verify the fold of NKR-P1C, native mass spectrometry with ion mobility measurements were performed.

Preliminary Data
Restraint-based computational modeling was used to generate a model that represents the experimentally determined constraints with a minimum of violations. Molecular dynamics was used to refine the model and to describe the most populated protein conformers in solution. In addition to the positional constraints obtained from the disulfide mapping and from cross-linking experiments the model needs to preserve the overall C-type lectin-like fold in these simulations, as the protein core is strongly conserved, and the template and modeled structure share a sequence identity of 88%. However, as crystal structures are rigid contrary to protein dissolved in solution we allowed the side chains in the core to be more flexible and adapt to the given experimentally determined constraints. Specific attention was paid to the extended loop region proposed to be involved in protein-ligand interactions and ligand specificity. The only crystal structure published to date for the entire NKR-P1 family, mouse NKR-P1A, shows this extended loop pointing away from the protein core, in a conformation in which the loop would be fully exposed to the solvent. Such a conformation could be clearly excluded from the cross-links of the protein in solution. This was further supported by IM-MS measurements corresponding to the compact form of the molecule based on the experimentally derived collisional cross section. Therefore, in the most populated conformation in solution, NKR-P1C most likely adopts the conformation similar to the solution structure of NKR-P1A. Our model enables us to describe this conformation on an atomic scale.

This work has been financially supported by the Grant Agency of the Czech Republic (GACR P207/10/1040), the Ministry of Education, Youth and Sports of the Czech Republic (Centre for Microbiology CZ.1.07/2.3.00/20.0055), and by the Institutional Research Project of the Institute of Microbiology (RVO61388971).