Table 1.
Crystallographic data collection and refinement statistics.
Table 2.
Steroid-binding data.
Figure 1.
Crystal structure of cleaved human CBG in complex with progesterone.
The central β-sheet A is shown in red, with the inserted RCL as part of it in blue. β-sheet C and β-sheet B are colored in green and yellow, respectively. On top of β-sheet B the bound steroid hormone progesterone is depicted in bright green. Electron density for residues 96 to 100 and residue 350 is missing. Chain breaks are shown with black dots.
Figure 2.
Alignment of reactive center loop sequences of CBG variants and of closely related AAT.
The RCL sequence shows a high degree of variance even within closely related proteins. The numbering of residues in the RCL of SERPINs is commonly in relation to the normal cleavage site in AAT between P1 and P1′. Already known cleavage sites are shown with grey boxes. The new cleavage site in human CBG observed in the structure is depicted in green. Introduced mutations in rat CBG-RCL1 through RCL3 are marked by orange boxes. The variant marked with an asterix bears in addition two mutations in the top of β-sheet A deleting a salt-bridge by converting it to amino acids present in human CBG (D323N, R174K). The wild-type protein sequences are annotated in the Uniprot knowledgebase with the following accession codes: rat CBG, P31211; human CBG, P08185; human AAT; P01009. The amino acids in the RCL are numbered following the convention of Schechter and Berger, 1967 [49].
Figure 3.
Insertion of the cleaved RCL into β-sheet A reveals a novel cleavage site.
The cleavage site utilized by neutrophil elastase is C-terminal of Val344 (yellow). The σA-weighted 2Fo-Fc electron density map contoured at 1 σ is displayed around residues 344 to 349, showing clearly the completion of β-sheet A. This elongation triggered by a different cleavage site leads to the formation of 6 additional hydrogen bonds. Residue Ser350 is not visible in the electron density maps. It is either part of this segment or of the new N-terminus.
Figure 4.
Stereo-views of the hormone-binding pocket.
(A) Coordination of progesterone in the binding site. Key residues for hormone binding are shown as stick representation. Water molecules surrounding the binding site are depicted as red dots. The σA-weighted 2Fo-Fc electron density map contoured at 1 σ is shown for the depicted amino acids, ligand and water molecules. The cation-π-stacking of R15 with W371, which has also been seen in the rat CBG structure [17], is well defined in the density. (B) Comparison of the binding pocket between cleaved human CBG in complex with progesterone and cleaved human CBG soaked with cortisol. The structures were aligned by the Cα chains of strand 4 and 5 of β-sheet B and helix H (residues 363–380 and 261–268, respectively). They show a very high degree of congruence, but differ slightly in the orientation of side chains which can form additional hydrogen bonds in the case of the ligand cortisol.
Figure 5.
The spectra confirm folded protein before and after cleavage of the RCL. Cleaved samples have been incubated with neutrophil elastase prior to buffer exchange. Native rat CBG-WT (green line) and cleaved rat CBG-RCL1 (blue broken line) show a similar curve shape with slight difference in signal strength, which is most probably due to errors in concentration measurements. In the comparison of native human CBG (yellow line) and cleaved human CBG (red broken line) a slight change in curve shape in the area around 208 nm is visible.
Figure 6.
Isothermal titration calorimetry measurements of steroid binding to rat and human CBG.
(A–F) Binding isotherms of titrations with hydrocortisone and corticosterone. (A) uncleaved rat CBG-RCL1 titrated with hydrocortisone (B) cleaved rat CBG-RCL1 titrated with hydrocortisone (C) uncleaved rat CBG-WT titrated with hydrocortisone (D) uncleaved human CBG-WT titrated with hydrocortisone (E) cleaved human CBG-WT titrated with hydrocortisone (F) uncleaved rat CBG-WT titrated with corticosterone. A reference titration with hydrocortisone in the absence of protein is shown as open dots in panel B.
Figure 7.
Thermal denaturation monitored by CD spectroscopy.
Measurement at a single wavelength while increasing temperature reveals the high thermal stability of cleaved human CBG. (A) Rat CBG shows no difference in thermal stability upon cleavage of the RCL. Native rat CBG-WT is shown in green, cleaved rat CBG-RCL1 in blue. The shape of the curves is largely similar. (B) Human CBG shows great differences in thermal stability between cleaved and native protein. The cleaved state of human CBG is highly thermostable (red), while the native state (yellow) shows a curve similar to rat CBG.