title: Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction.
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.
authors.affiliation: Computational and Systems Biology, Singapore-Massachusetts Institute of Technology Alliance, Singapore 117576; Departments of Biological Sciences, Singapore 117543.
authors.forename: Diana Hooi Ping
authors.initials: DHP
authors.lastname: Low
authors.affiliation: Laboratory of Molecular Biostructural and Nanomaterial Modeling, AREA Science Park, Trieste 34149, Italy; Cancer Research Institute, Slovak Academy of Sciences, 83391 Bratislava, Slovakia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Frecer
authors.affiliation: Departments of Biological Sciences, Singapore 117543.
authors.forename: Agnès
authors.initials: A
authors.lastname: Le Saux
authors.affiliation: Departments of Biological Sciences, Singapore 117543.
authors.forename: Ganesh Anand
authors.initials: GA
authors.lastname: Srinivasan
authors.affiliation: Microbiology, National University of Singapore, Singapore 117543.
authors.forename: Bow
authors.initials: B
authors.lastname: Ho
authors.affiliation: Computational and Systems Biology, Singapore-Massachusetts Institute of Technology Alliance, Singapore 117576; Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142.
authors.forename: Jianzhu
authors.initials: J
authors.lastname: Chen
authors.affiliation: Computational and Systems Biology, Singapore-Massachusetts Institute of Technology Alliance, Singapore 117576; Departments of Biological Sciences, Singapore 117543. Electronic address: dbsdjl@nus.edu.sg.
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005075:Biological Evolution; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D017124:Conserved Sequence; D006737:Horseshoe Crabs; D054884:Host-Pathogen Interactions; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
chemical_list: D002135:Calcium-Binding Proteins; D008024:Ligands; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; C111170:tectonin I