Fig. 5: Folding determinants shape the evolutionary pattern of CRP.

Conservation scores of CRP were calculated with Consurf⁴⁰ using 121 available amino acid sequences of different species. a Conservation scores were mapped onto the crystal structure of the human CRP subunit (PDB 1B09)⁵⁶. Side chains of residues critical to PC-, C1q-, and FcγR-binding are shown (binding-decisive) (left), and their conservation scores are compared with scores of other CRP residues (right). b Strands of the hydrophobic core that folds spontaneously³⁹ are indicated (left). The effects of mutations on CRP folding were evaluated (right). c Side chains of residues critical to calcium binding and disulfide bonding (folding-decisive) were shown with mapped conservation scores. Binding-decisive residues were significantly more variable than folding-decisive residues and those not directly involved in binding and folding (others). Mutating residues within the hydrophobic core did not abrogate its spontaneous formation (Step 1; 10 mutants examined, Supplementary Table 1), whereas mutating residues involved in disulfide bonding or calcium binding abrogated the subsequent nonspontaneous folding (Step 2; 8 mutants examined, Supplementary Table 1). Each data point represents the relative folding of a different mutant (n = 3 independent experiments). d The effects of mutations at folding determinants on CRP secretion by prokaryotic E. coli (44 mutants examined; Supplementary Table 2) and eukaryotic COS-7 cells (16 mutants examined; Supplementary Table 2). Though these mutations could not be secreted by COS-7 cells, they were still able to be efficiently secreted by E. coli cells. Each data point represents the relative secretion of a different mutant (n = 3 independent experiments). e Most part of CBS is buried within the native structure of human CRP. The crystal structure of CRP subunit (1B09)⁵⁶ is shown in both Ribbon (left) and Surface modes (right). CBS is colored in purple with the side chain of Cys36 also indicated. f The sequence logo of CBS generated with WebLogo 3⁶⁶. Sequences of mouse, rat, and human CBS are also shown. The three pattern-decisive residues are highly conserved, whereas the rest are rather variable allowing fine adjustment of CBS activities. g IDPs (disorder contents of soluble protein or that of the extracellular region of membrane proteins >50%) in human (98), rodents (mouse and rat; 33), other multicellular organisms (drosophila, nematode, and arabidopsis; 22), and yeast (34) were retrieved from DisProt database⁴⁷ (Supplementary Table 3). Ratios of secretory IDPs with a signal peptide are much lower even when corrected for the ratio of secretory proteins versus the entire proteome (36%). h Ratios of disordered sequences in extracellular/luminal (non-cytoplasmic; median ratio = 0) versus cytoplasmic portions of all membrane IDPs (median ratio = 1) from all species regardless of their overall disordered contents or cellular localization (Supplementary Table 4; n = 130; left). The sequence lengths of the extracellular/luminal and cytoplasmic portions of these membrane IDPs are also shown (right). Disordered sequences are significantly enriched in cytoplasmic over extracellular/luminal portions of membrane proteins, whereas the overall lengths of the two portions are comparable. In box plots, centerline represents the median; box limits represent upper and lower quartiles; whiskers represent 1.5× interquartile range; points represent outliers. *p < 0.05; ***p < 0.001, Kolmogorov–Smironv tests, two-sided.