Abstract
C-reactive protein (CRP) is an acute phase reactant secreted by hepatocytes as a pentamer. The structure formation of pentameric CRP has been demonstrated to proceed in a stepwise manner in live cells. Here, we further dissect the sequence determinants that underlie the key steps in cellular folding and assembly of CRP. The initial folding of CRP subunits depends on a leading sequence with a conserved dipeptide that licenses the formation of the hydrophobic core. This drives the bonding of the intra-subunit disulfide requiring a favorable niche largely conferred by a single residue within the C-terminal helix. A conserved salt bridge then mediates the assembly of folded subunits into pentamer. The pentameric assembly harbors a pronounced plasticity in inter-subunit interactions, which may form the basis for a reversible activation of CRP in inflammation. These results provide insights into how sequence constraints are evolved to dictate structure and function of CRP.
Highlights
C-reactive protein (CRP) is a major human acute phase reactant mainly produced by hepatocytes as a pentamer
The Relatively Conserved P29/L30 Dipeptide in N-Terminal Is Critical for the Formation of the Hydrophobic Core a.a. 1–31 has been shown to be required for the spontaneous folding of the N-terminal hydrophobic core, as evidence by the fact that CRP mutant lacking this sequence showed marginal cytoplasmic stability and greatly impaired capacity to form intrasubunit disulfide bond [7]
Sequential truncations here revealed that the absence of a.a. 1–11 or 1–22 did not impair the cytoplasmic stability of non-secretory CRP in E. coli cells
Summary
C-reactive protein (CRP) is a major human acute phase reactant mainly produced by hepatocytes as a pentamer. The N-terminal half of the two-layered β sheet, i.e., strands C to I termed the hydrophobic core (a.a. 32–111), and the a.a. 168–176 helix first fold spontaneously, leading to the formation of the intra-subunit disulfide bond [7]. This event drives the following non-spontaneous stage of subunit folding, which involves a global conformation remodeling that requires the aid of cellular factors [7]. Folded subunits assemble into the native cyclic pentamer [7] It is unclear what sequence features underlie the stepwise pathway of folding and assembly of CRP in live cells. Our results further reveal the presence of a pronounced plasticity in pentameric assembly of CRP that underlies its conditional activation
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