Structural details of NKR-P1D : Clrb interaction elucidated by protein cross-linking and mass spectrometry

Pavel Hanč1, Josef Chmelík1, Karel Bezouška1,2 and Petr Novák1,2


1Institute of Microbiology, Academy of Sciences of the Czech Republic, Praha 4, Czech Republic
2Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, Praha 2, Czech Republic

Interaction between murine NKR-P1D and Clrb receptors was originally described as a novel type of „MHC class-I independent missing-self recognition“ and was shown to confer protection from killing by natural killer cells.[1] However, further studies suggested that it is not in fact NKR-P1D but rather NKR-P1B, an allelic form expressed in different strains of mice, which binds Clrb and that NKR-P1D binds with significantly lower affinity if at all.[2]

In order to address the issues arising from these conflicting results, we have recombinantly expressed the extracellular domains of both receptors in E. coli cells and refolded the proteins in vitro. The quality of refolding was confirmed both by determining the disulphide bonding pattern using FT-MS and measuring 1H/15N-HSQC spectra using 600MHz NMR spectrometer. Interaction between the proteins in solution was immobilized using protein cross-linking technique. Three cross-linking reagents, EDC, DSG and DSS with spacer arm lengths of 0A, 7.5A and 11.5A respectively were used. The reaction mixture was separated by means of SDS-PAGE and protein bands corresponding to dimers were digested in gel.  Using FT-MS we were able to find peptides from both proteins connected by the cross-linkers.

Using recently resolved structures of extracellular domains of NKR-P1A and Clrg receptors bearing 86% and 76% sequence identity with NKR-P1D and Clrb respectively, we were able to build homology models of both NKR-P1D and Clrb and a model of the interacting pair. Part of the data obtained from cross-linking experiments fitted nicely into the model of homodimers of both proteins interacting in a face-to-face fashion as would be expected; however significant portion of the observed cross-links could not be explained by this model.  In order to allow for these data we suggest that these receptors do not only interact in the face-to-face fashion but also in a chain-like or cluster-like fashion with each homodimer contacting two homodimers of the second protein at the same time.

To our knowledge this would be the first interaction of this kind observed for this type of receptors.

1. Iizuka, K. et. al., Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors. Nat. Immunol., 2003, 4, 801 – 807

2. Carlyle, J. et. al., Missing self-recognition of Ocil Clr-b by inhibitory NKR-P1 natural killer cell receptors. PNAS, 2004, 101, 3527 – 3532

This work was supported by the grant P207/10/1040 of the Grant Agency of Academy of Sciences of the Czech Republic.