title: Pathophysiological condition changes the conformation of a flexible FBG-related protein, switching it from pathogen-recognition to host-interaction.
abstract: Although homeostatic disturbance of the blood pH and calcium in the vicinity of tissue injury/malignancy/local infection seems subtle, it can cause substantial pathophysiological consequences, a phenomenon which has remained largely unexplored. The fibrinogen-related proteins (FREPs) containing fibrinogen-like domain (FBG) represent a conserved protein family with a common calcium-binding region, implying the presence of elements responsive to physiological perturbation. Here, we studied the molecular interaction between a representative FREP, the M-ficolin, and an acute phase blood protein, the C-reactive protein (CRP), both of which are known to trigger and control seminal pathways in infection and injury. Using hydrogen-deuterium exchange mass spectrometry, we showed that the C-terminal region of M-ficolin FBG underwent dramatic conformational change upon pH and calcium perturbations. Biochemical and biophysical assays showed that under defined pathophysiological condition (pH 6.5, 2.0 mM calcium), the FBG:CRP interaction occurred more strongly compared to that under physiological condition (pH 7.4, 2.5 mM calcium). We identified the binding interface between CRP and FBG, locating it to the pH- and calcium-sensitive C-terminal region of FBG. By site-directed mutagenesis, we determined H284 in the N-acetylglucosamine (GlcNAc)-binding pocket of the FBG, to be the critical CRP-binding residue. This conformational switch involving H284, explains how the pathophysiologically-driven FBG:CRP interaction diverts the M-ficolin away from GlcNAc/pathogen-recognition to host protein-protein interaction, thus enabling the host to regain homeostatic control. Our elucidation of the binding interface at the flexible FBG domain provides insights into the bioactive centre of the M-ficolin, and possibly other FREPs, which might aid future development of immunomodulators.
authors.affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Lifeng
authors.initials: L
authors.lastname: Yang
authors.forename: Ganesh Srinivasan
authors.initials: GS
authors.lastname: Anand
authors.forename: Bow
authors.initials: B
authors.lastname: Ho
authors.forename: Jeak Ling
authors.initials: JL
authors.lastname: Ding
mesh_terms: D002097:C-Reactive Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D000097604:Ficolins
chemical_list: D002097:C-Reactive Protein; D037102:Lectins; D000097604:Ficolins