The innate immune system recognizes invading microbes by means of pattern recognition receptors (PRRs) that bind unique products of microbial metabolism (pathogen-associated molecular patterns, or PAMPs). Examples of PAMPs recognized by PRRs such as peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain-containing proteins (NODs), and the extracellular matrix protein mindin include LPS and peptidoglycan (PGN). Although much is known about the function of PRRs in host defense, far less is known about how they recognize PAMPs. Accordingly, we determined the crystal structure of human PGRP-Ia in complex with a muramyl tripeptide (MTP) representing the core of Lys-type PGNs. The peptide stem of MTP is buried at the deep end of a long binding groove, with N-acetylmuramic acid in the middle of the groove, whose shallow end could accommodate a linked N-acetylglucosamine. Most interactions are with the peptide, rather than glycan, suggesting that PGRPs mainly recognize the variable peptide stems of PGNs. This work provides a starting point for detailed structure-function analysis of PRR-PAMP interactions. In addition to PGRPs, we will focus on two other key PRRs: NODs, which mediate intracellular detection of PGNs, and mindin, which interacts with LPS and is essential for initiation of immune responses. The specific objectives of the proposed studies are: 1. Determination of the structures of selected human and Drosophila PGRPs in complex with both Lys- and Dap-type PGN analogs in order to explain the ability of PGRPs to discriminate between Gram-positive and Gram-negative bacteria. Structures will be correlated with the affinity of PGRP-PGN interactions measured by ITC or SPR. 2. Determination of the structure of Drosophila PGRP-LC, a cell surface receptor required for Imd pathway activation, PGRP-LC exists as three splice isoforms (LCa, LCx, LCy) that form homo- or heterodimers with distinct PGN specificities. To understand ligand recognition by this receptor, we will examine dimerization of PGRP-LC isoforms in solution, followed by structure determination of homo- and heterodimers. 3. Determination of the basis for PGN recognition by NODI and NOD2 in order to define the relationship between their PGN binding mode and that of PGRPs. We will engineer recombinant forms of NOD1 and NOD2 and determine their structures bound to PGN ligands. 4. Identification of LPS determinants recognized by mindin. We have expressed recombinant mindin for use in ITC and SPR binding experiments to synthetic LPS oligosaccharides and lipid A derivatives, and for crystallization of specific mindin-ligand complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI065612-05
Application #
7599682
Study Section
Cellular and Molecular Immunology - B (CMI)
Program Officer
Palker, Thomas J
Project Start
2005-07-01
Project End
2010-04-24
Budget Start
2009-04-01
Budget End
2010-04-24
Support Year
5
Fiscal Year
2009
Total Cost
$276,259
Indirect Cost
Name
University of MD Biotechnology Institute
Department
Type
Organized Research Units
DUNS #
603819210
City
Baltimore
State
MD
Country
United States
Zip Code
21202
Li, Yili; Cao, Chunzhang; Jia, Wei et al. (2009) Structure of the F-spondin domain of mindin, an integrin ligand and pattern recognition molecule. EMBO J 28:286-97
Tasumi, Satoshi; Velikovsky, C Alejandro; Xu, Gang et al. (2009) High-affinity lamprey VLRA and VLRB monoclonal antibodies. Proc Natl Acad Sci U S A 106:12891-6
Velikovsky, C Alejandro; Deng, Lu; Tasumi, Satoshi et al. (2009) Structure of a lamprey variable lymphocyte receptor in complex with a protein antigen. Nat Struct Mol Biol 16:725-30
Cho, Sangwoo; Wang, Qian; Swaminathan, Chittoor P et al. (2007) Structural insights into the bactericidal mechanism of human peptidoglycan recognition proteins. Proc Natl Acad Sci U S A 104:8761-6
Swaminathan, Chittoor P; Brown, Patrick H; Roychowdhury, Abhijit et al. (2006) Dual strategies for peptidoglycan discrimination by peptidoglycan recognition proteins (PGRPs). Proc Natl Acad Sci U S A 103:684-9
Kumar, Sanjay; Roychowdhury, Abhijit; Ember, Brian et al. (2005) Selective recognition of synthetic lysine and meso-diaminopimelic acid-type peptidoglycan fragments by human peptidoglycan recognition proteins I{alpha} and S. J Biol Chem 280:37005-12