Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction.

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.

A novel human tectonin protein with multivalent beta-propeller folds interacts with ficolin and binds bacterial LPS.

BACKGROUND: Although the human genome database has been completed a decade ago, approximately 50% of the proteome remains hypothetical as their functions are unknown. The elucidation of the functions of these hypothetical proteins can lead to additional protein pathways and revelation of new cascades. However, many of these inferences are limited to proteins with substantial sequence similarity. Of particular interest here is the Tectonin domain-containing family of proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have identified hTectonin, a hypothetical protein in the human genome database, as a distant ortholog of the limulus galactose binding protein (GBP). Phylogenetic analysis revealed strong evolutionary conservation of hTectonin homologues from parasite to human. By computational analysis, we showed that both the hTectonin and GBP form beta-propeller structures with multiple Tectonin domains, each containing beta-sheets of 4 strands per beta-sheet. hTectonin is present in the human leukocyte cDNA library and immune-related cell lines. It interacts with M-ficolin, a known human complement protein whose ancient homolog, carcinolectin (CL5), is the functional protein partner of GBP during infection. Yeast 2-hybrid assay showed that only the Tectonin domains of hTectonin recognize the fibrinogen-like domain of the M-ficolin. Surface plasmon resonance analysis showed real-time interaction between the Tectonin domains 6 & 11 and bacterial LPS, indicating that despite forming 2 beta-propellers with its different Tectonin domains, the hTectonin molecule could precisely employ domains 6 & 11 to recognise bacteria. CONCLUSIONS/SIGNIFICANCE: By virtue of a recent finding of another Tectonin protein, leukolectin, in the human leukocyte, and our structure-function analysis of the hypothetical hTectonin, we propose that Tectonin domains of proteins could play a vital role in innate immune defense, and that this function has been conserved over several hundred million years, from invertebrates to vertebrates. Furthermore, the approach we have used could be employed in unraveling the characteristics and functions of other hypothetical proteins in the human proteome.

The macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules.

Although the innate immune response is triggered by the formation of a stable assembly of pathogen-recognition receptors (PRRs) onto the pathogens, the driving force that enables this PRR-PRR interaction is unknown. Here, we show that serine proteases, which are activated during infection, participate in associating with the PRRs. Inhibition of serine proteases gravely impairs the PRR assembly. Using yeast two-hybrid and pull-down methods, we found that two serine proteases in the horseshoe crab Carcinoscorpius rotundicauda are able to bind to the following three core members of PRRs: galactose-binding protein, Carcinolectin-5 and C-reactive protein. These two serine proteases are (1) Factor C, which activates the coagulation pathway, and (2) C2/Bf, a protein from the complement pathway. By systematic molecular dissection, we show that these serine proteases interact with the core "pathogen-recognition complex" via their complement control protein modules.