Structural insights into the dual strategy of recognition by peptidoglycan recognition protein, PGRP-S: structure of the ternary complex of PGRP-S with lipopolysaccharide and stearic acid

Abstract

Peptidoglycan recognition proteins (PGRPs) are part of the innate immune system. The 19 kDa Short PGRP (PGRP-S) is one of the four mammalian PGRPs. The concentration of PGRP-S in camel (CPGRP-S) has been shown to increase considerably during mastitis. The structure of CPGRP-S consists of four protein molecules designated as A, B, C and D forming stable intermolecular contacts, A-B and C-D. The A-B and C-D interfaces are located on the opposite sides of the same monomer leading to the the formation of a linear chain with alternating A-B and C-D contacts. Two ligand binding sites, one at C-D contact and another at A-B contact have been observed. CPGRP-S binds to the components of bacterial cell wall molecules such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) from both gram-positive and gram-negative bacteria. It also binds to fatty acids including mycolic acid of the Mycobacterium tuberculosis (Mtb). Previous structural studies of binary complexes of CPGRP-S with LPS and stearic acid (SA) have shown that LPS binds to CPGRP-S at C-D contact (Site-1) while SA binds to it at the A-B contact (Site-2). The binding studies using surface plasmon resonance showed that LPS and SA bound to CPGRP-S in the presence of each other. The structure determination of the ternary complex showed that LPS and SA bound to CPGRP-S at Site-1 and Site-2 respectively. LPS formed 13 hydrogen bonds and 159 van der Waals contacts (distances ≤4.2 Å) while SA formed 56 van der Waals contacts. The ELISA test showed that increased levels of productions of pro-inflammatory cytokines TNF-α and IFN-γ due to LPS and SA decreased considerably upon the addition of CPGRP-S.

Figure 1. Initial difference Fourier map (Fo−Fc) contoured at 2.0 σ for (A) SA and (B) LPS.

Figure 2. Sensogram for the binding of (A) LPS and (C) SA.

(B) and (D) regions corresponding to injection stage.

Figure 3. Inhibition of LPS+SA induced pro-inflammatory cytokines, TNF-α and IFN-γ when CPGRP-S was added to the medium along with LPS and SA.

Figure 4. Structure of the ternary complex of CPGRP-S with LPS and LTA.

The binding sites are shown in different colours. SA and LPS are shown as space fitting models in blue and green colours respectively.

Figure 5. View of the structure of the ternary complex of CPGRP-S showing four crystallographically independent molecules in the asymmetric unit which is indicated by dashed lines.

CPGRP-S molecules assemble as a linear polymer forming A–B and C–D contacts alternatingly. The bound molecules of SA at Site-2 and LPS at Site-1 are also shown as space filling models.

Figure 6. The binding of SA and LPS to CPGRP-S, (A) A section of A–B contact showing a bound SA molecule in the cleft.

The binding is essentially stabilized by van der Waals contacts. (B) A section of C–D contact showing a bound LPS molecule in the cleft. The binding is stabilized by several hydrogen bonds and a network of van der Waals contacts.