Molecular Interfaces of the Galactose-binding Protein Tectonin Domains in Host-Pathogen Interaction

Diana Hooi Ping Low,Vladimir Frecer,Agnès Le Saux,Ganesh Anand Srinivasan,Bow Ho,Jianzhu Chen,Jeak Ling Ding

doi:10.1074/jbc.m109.059774

Abstract

Beta-propeller proteins function in catalysis, protein-protein interaction, cell cycle regulation, and innate immunity. The galactose-binding protein (GBP) from the plasma of the horseshoe crab, Carcinoscorpius rotundicauda, is a beta-propeller protein that functions in antimicrobial defense. Studies have shown that upon binding to Gram-negative bacterial lipopolysaccharide (LPS), GBP interacts with C-reactive protein (CRP) to form a pathogen-recognition complex, which helps to eliminate invading microbes. However, the molecular basis of interactions between GBP and LPS and how it interplays with CRP remain largely unknown. By homology modeling, we showed that GBP contains six beta-propeller/Tectonin domains. Ligand docking indicated that Tectonin domains 6 to 1 likely contain the LPS binding sites. Protein-protein interaction studies demonstrated that Tectonin domain 4 interacts most strongly with CRP. Hydrogen-deuterium exchange mass spectrometry mapped distinct sites of GBP that interact with LPS and with CRP, consistent with in silico predictions. Furthermore, infection condition (lowered Ca(2+) level) increases GBP-CRP affinity by 1000-fold. Resupplementing the system with a physiological level of Ca(2+) did not reverse the protein-protein affinity to the basal state, suggesting that the infection-induced complex had undergone irreversible conformational change. We propose that GBP serves as a bridging molecule, participating in molecular interactions, GBP-LPS and GBP-CRP, to form a stable pathogen-recognition complex. The interaction interfaces in these two partners suggest that Tectonin domains can differentiate self/nonself, crucial to frontline defense against infection. In addition, GBP shares architectural and functional homologies to a human protein, hTectonin, suggesting its evolutionarily conservation for approximately 500 million years, from horseshoe crab to human.

Highlights

The ␤-propeller protein family members have diverse functions: enzyme catalysis, protein-protein interactions, and cell cycle regulation [1, 2]
Native galactose-binding protein (GBP) Has a Propensity to Oligomerize—GBP was purified from the plasma, through binding to the repeating units of ␣-1,6-linked D-galactose and 3,6-anhydro-L-galactose on the Sepharose CL-6B
We evaluated the avidity was eluted by Glucosamine (Gln) N-Acetylglucosamine (GlcNAc), we envisaged that GBP binds to the of GBP for LPS, lipid A, and GlcNAc by Surface Plasmon Resonance Analysis (SPR) measurements sugar moieties of bacterial LPS (Fig. 2A)

Summary

Introduction

The ␤-propeller protein family members have diverse functions: enzyme catalysis, protein-protein interactions, and cell cycle regulation [1, 2]. A subset of this family of proteins has pathogen-binding properties [1, 3,4,5,6,7,8], indicating a role in defense against microbial infection. Within the subset of pathogen binding ␤-propeller protein family, several members are classified as having Tectonin domains [4, 5, 7,8,9]. It was proposed to bind PAMPs while interacting with other pattern-recognition receptors (PRRs) to form a pathogen-recognition interactome [11, 12]. Because of its relative abundance in the plasma and its propensity to form an PRR-interactome, GBP is a useful model for studying the role of Tectonin domain-containing proteins in antimicrobial defense. Yeast Two-hybrid Assay—Cotransformations of the different bait and prey plasmids into Saccharomyces cerevisiae were performed to study protein-protein interactions

Methods

Results

Conclusion