C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

Abstract

C1 inhibitor, a member of the serpin family, is a major downregulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded ␤-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpinproteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.C1 inhibitor belongs to the group of serpin-type proteinase inhibitors in blood plasma. Serpins act as pseudosubstrates of serine and cysteine proteinases (although there are noninhibitory serpins as well) (1). A conformational change is triggered in the serpin upon peptide bond cleavage, which distorts the active site of proteinase and traps it in an inactive, covalently linked serpin-enzyme complex (2, 3). Serpins are vital downregulator components of proteolytic signal amplification cascades. Human C1 inhibitor (C1-inh) 4 is the only inhibitor that acts on early components of the classical pathway (C1r and C1s) and on that of the lectin pathway (MASP-1 and MASP-2) of the complement system (4). Complement mediates host defense against pathogens and altered host cells, but its uncontrolled activation could be harmful. Other physiologically crucial targets include plasma kallikrein and activated factor XII (fXIIa) of the contact activation and activated factor XI (fXIa) of the intrinsic coagulation systems (4). Apart from proteinase inhibition, it was recently discovered that C1-inh binds the central component C3b of complement (5), endotoxins from bacteria (6) and E-, P-selectin adhesion proteins on endothelial cells (7). C1-inh is a single-chain glycoprotein that has atypical two-domain architecture (8) with the C-terminal serpin and the unique N-terminal domains (9). C1-inh is extensively modified post-translationally, bearing six N-linked carbohydrates. Sequencing analysis revealed 14 potential O-glycosylation sites (9), seven of which had been verified by carbohydrate analysis (10). Most of the sugars are present in the N-terminal domain and do not affect proteinase inhibition (11, 12), but affinity to endotoxins and selectins depends on the N-glycans.The importance of C1-inh is underlined by its def...

Functional Characterization of the Recombinant Human C1 Inhibitor Serpin Domain: Insights into Heparin Binding

“…The expression yields ranged from 3-10 mg/l and, therefore, were comparable to that reported previously for the production of full-length C1 inhibitor using the same expression system (16). In keeping with the report by Wolff et al (16) and in contrast with the highly heterogeneous material produced in P. pastoris (12,19), the C1 inhibitor serpin domain expressed in the current study was homogeneous in terms of size, as shown by SDS-PAGE and MALDI mass spectrometry analyses, indicating relative homogeneity of the N-linked oligosaccharides. In this regard, the mass spectrometry analyses performed on the recombinant proteins are consistent with the occurrence of short, oligomannose carbohydrates comprising two N-acetylglucosamine residues and three to four mannose residues (calculated mass = 893-1055), in keeping with previous analyses (16).…”

“…This value is slightly higher but comparable to those measured for the interaction of heparin with C1 inhibitor and C1s. Therefore, this result seems to be consistent with a "sandwich" mechanism rather than with a "bridging" mechanism (19); in the latter case, the oligomer size necessary to achieve half-maximal potentiation would be expected to be much higher. Similar potentiation curves were obtained using C1inhD97 instead of full-length C1 inhibitor (data not shown).…”

“…However, in contrast to native C1 inhibitor, this value was shifted to 47˚C in the presence of heparin, indicating that interaction with the isolated serpin domain of C1 inhibitor significantly decreased its thermal stability. Interestingly, a minor negative peak was also observed at 66˚C, possibly reflecting the presence of low amounts of a latent form of the serpin domain (19).…”

“…Active C1 inhibitor was also expressed at high yield in Pichia pastoris and purified (17). Various constructs corresponding to the serpin domain of C1 inhibitor were also produced in COS-1 cells (10), Escherichia coli (18), and recently in P. pastoris (19). Using the latter recombinant protein, the crystal structure of the C1 inhibitor serpin domain has been solved, revealing a latent conformation with the uncleaved reactive center loop inserted into a seven-stranded antiparallel b-sheet (19).…”

Serum MASP-1 in complex with MBL activates endothelial cells

“…C1-inh is a serpin (serine protease inhibitor) that makes a covalent complex with the protease molecule, distorting its structure and preventing cleavage of further substrate molecules (Beinrohr et al, 2007). We wanted to unambiguously exclude the possibility that the C1-inh mediated inhibition of the cell activating ability of MASP-1 is due to steric reasons; i.e.…”