designed, conducted and analysed reporter assay studies and flow cytometry studies of RIFIN mutants

designed, conducted and analysed reporter assay studies and flow cytometry studies of RIFIN mutants. a supported lipid bilayer system, which mimics NK cell activation by antibody- dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the NK cell immunological synapse and reduce cell activation, as measured by perforin mobilisation. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress NK cell function. To determine the molecular basis for RIFIN-mediated SPHINX31 LILRB1 activation, we assessed expression of different fragments of a LILRBl-binding RIFIN ectodomain from the 3D7 strain of (PF3D7_1254800)5. RIFIN ectodomains contain N-terminal semiconserved domains and C-terminal variable domains, followed by transmembrane helices6,7. While the full-length ectodomain did not express in a folded form, we produced the variable region (residues 165-274), previously shown to bind LILRB1, and the constant SPHINX31 region (residues 39-139) (Extended Data Figure 1)5. While the constant region showed no binding, the variable region bound LILRB1 with KD = 570 130nM. This is comparable to the binding affinities of LILRB1 for MHC class I molecules, which range from 2 to 7 M8. The monomeric variable domain was complexed with the complete extracellular domain of LILRB1, containing four immunoglobulin-like domains (Extended Data Figure 1b), allowing formation of crystals which diffracted to 3.0 ?. The structure was determined by molecular replacement, using existing structures of LILRB1 domains9,10 as search models (Figure 1, Extended Data Table 1). The LILRBl-binding region of the RIFIN adopts a primarily a-helical structure, consisting of three main helices. These are connected by extensive loops, which contain short helical segments (Figure 1a). Other proteins of the infected erythrocyte, such as DBL and CIDR domains of PfEMPl, are also built from small helical scaffolds11, but do not share the topology of the RIFIN variable domain. In the RIFIN, cysteine C223 is unpaired and exposed to solvent, possibly forming an additional disulphide bond with a cysteine in the constant region when in the full ectodomain. For all subsequent experiments, we therefore designed a shorter and more stable RIFIN, removing disordered regions and mutating this free cysteine to serine Rabbit Polyclonal to CPB2 (C223S). Surface plasmon resonance studies showed C223S to bind LILRB1 with KD = 700 5 nM (Extended Data Figure 2). The remaining two cysteines form a disulphide bond that stabilises the complex loop that links the second and third main helices and SPHINX31 forms most of the LILRB1 contact surface (Figure 1b,c). Open in a separate window Figure 1 The structure of the RIFIN:LILRB1 complex. a. The structure of RIFIN 1254800 variable region in a rainbow representation with SPHINX31 N- terminus blue and C-terminus red. b. The SPHINX31 structure of the RIFIN variable region (orange) bound to the LILRB1 ectodomain (blue). c. The interface between the RIFIN and LILRB1 with interacting residues and disulphide bonding cysteines of the RIFIN labelled in orange. The RIFIN binding site is contained within the two N-terminal, membrane distal immunoglobulin-like domains of LILRB1, as confirmed by surface plasmon resonance measurements (Extended Data Figure 2). Binding is mediated by loops at the interface between these two LILRB1 domains. The four LILRB1 domains form a linear zig-zag arrangement. Previous structures of LILRB1 domains 1 and 2, alone12 and in complex with.