Structural basis of heparin binding to camel peptidoglycan recognition protein-S

Abstract

Short peptidoglycan recognition protein (PGRP-S) is a member of the innate immunity system in mammals. PGRP-S from Camelus dromedarius (CPGRP-S) is found to be highly potent against bacterial infections. It is capable of binding to a wide range of pathogen-associated molecular patterns (PAMPs) including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). The heparin-like polysaccharides have also been observed in some bacteria such as the capsule of K5 Escherichia coli thus making them relevant for determining the nature of their interactions with CPGRP-S. The binding studies of CPGRP-S with heparin disaccharide in solution using surface plasmon resonance gave a value 3.3×10(-7) M for the dissociation constant (Kd). The structure of the heparin bound CPGRP-S determined at 2.8Å resolution revealed the presence of a bound heparin molecule in the binding pocket of CPGRP-S. It was found anchored tightly to the protein with the help of several ionic and hydrogen bonded interactions. Three sulphate groups of heparin S1, S2 and S3 have been found to interact with residues, Arg-31, Lys-90, Thr- 97, Asn-99 Asn-140, Gln-150 and Arg-170 of CPGRP-S. The binding site includes two subsites, S-I and S-II with cleft-like structures. Heparin disaccharide is bound in subsite S-I. Previously determined structures of the complexes of CPGRP-S with LPS, LTA and PGN also showed that their glycan moieties were also held in subsite S-I indicating that heparin disaccharide also represents an important element for the recognition by CPGRP-S.

Keywords: LPS; LTA; PAMPs; PGN; PGRP-S; crystal structure; heparin.

Figure 1

(A) Stereoview of the initial (Fo-Fc) electron density map at 2σ cut off. The neighbouring residues from molecules A, B, C and D are also indicated. (B) Stereoview of the omit map calculated during final stages of the refinement of by omitting heparin disaccharide and surrounding residues of the binding subsite S-I at 2.5σ cut off.

Figure 2

(A) SPR sensograms showing the association and dissociation curves for the binding of heparin disaccharide. CPGRP-S was immobilized on CM- 5 chip and three concentrations (1.4 mM, 2.5 mM and 5.0 mM) of analyte heparin disaccharide were used in the mobile phase. The corresponding curves are indicated by a, b and c respectively. (B) The fitted curves are shown by dotted lines.

Figure 3

(A) Ribbon diagram for four crystallographically independent molecules A, B, C and D representing A-B and C-D units of neighbouring CPGRP-S linear polymers. Heparin disaccharide is bound to protein molecule C at subsite SI. The ligand binding cleft in the tetramer is indicated by segments which are highlighted in red. The molecules of tartrate and glycerol are also shown (in ball and stick representation). (B) Heparin molecule with atom numbering is shown.

Figure 4

The columns of linear polymers of CPGRP-S with alternating contacts A-B and C-D. Heparin disaccharide molecules are bound to molecule C at C-D contact site.

Figure 5

The interactions involving protein atoms (CPGRP-S) with sulphate moieties of heparin disaccharide and protein residues. The sulphate moieties S1 (PDB name: S26) forms an ionic interaction with Arg-A170, S2 (PDB name: S16) forms a salt bridge with Lys-C90 while S3 (PDB name: S20) forms three hydrogen bonds with Ala-C94, Asn-C99 and Glu- D150.

Figure 6

Showing two pockets SI and SII on the ligand binding site of CPGRP-S. Heparin disaccharide binds at subsite I.

Figure 7

(A) The bindings of LPS (PDB: 3RT4) and LTA (PDB: 3O4K) to CPGRP-S. The glycan moieties are fitted in the glycan binding pocket while their hydrocarbon chains are adjusted along the molecular surface. (B) The binding of PGN (PDB: 3NW3) to CPGRP-S. The glycan moiety is fixed at glycan binding pocket. The peptide chain is adjusted along the surface of the protein molecule.

Figure 8

(A) Muramyl dipeptide bound to HPGRP-IαC with peptide moiety at subsite, S-I and glycan moiety at subsite, S-II. (B) Superimposition of subsite S-I from CPGRP-S and HPGRP-S showing an obstruction by His99 in HPGRP-S. The corresponding residue in CPGRP-S is Pro-96. Furthermore, the presence of Glu-24 and Lys-90 in the binding site in CPGRP-S are helpful in the recognition of PAMPs. The corresponding residues in HPGRP-S are Gln-27 and Thr-93.