Native conformations of human complement components C3 and C4 show different dependencies on thioester formation

Abstract

The thioester bond in complement components C3 and C4 and the protease inhibitor alpha2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.

The Structure of Bovine Complement Component 3 Reveals the Basis for Thioester Function

“…During formation of the thioester, the distorted 3 10 helix (probably caused by the contacts between the MG8 domain and the thioester region) probably forms before the thioester bond, in agreement with mutation data suggesting that the thioester is not required directly for obtaining the native conformation. 29,30 Mutation of Pro1007 or Pro1020 (Pro1006 or 1019 in bovine C3) to glycine in human C3 leads to a thioester-deficient molecule cleavable by the C3 convertase. 30 Apparently, a highly specific conformation of the main chain around Cys1009 and Gln1012 is required for thioester formation.…”

Site‐Specific Cleavage—A Model System for the Identification of Lipid‐Modified Glutamate Residues in Proteins

“…The kinetics of product formation were investigated by HPLC analysis at different time points and quantified by correlation of the UV absorption with those of known concentrations of the chemically prepared intermediates. For this purpose, and to obtain supporting analytical data ( 1 H NMR, 13 C NMR spectra), the cleavage products were synthesized on a preparative scale.…”

“…1 H NMR spectra: Bruker AM 400 (400 MHz) and Bruker AM 300 (300 MHz) instruments, solvent as internal standard (CDCl 3 : d H = 7.24 ppm). 13 C NMR spectra: Bruker AM 400 (100 MHz) and Bruker AM 300 (75 MHz) instruments, solvent as internal standard (CDCl 3 : d C = 77.0 ppm). 13 C NMR spectra were recorded in broadband decoupled mode, and multiplicities were determined by use of a DEPT pulse sequence.…”

“…13 C NMR spectra: Bruker AM 400 (100 MHz) and Bruker AM 300 (75 MHz) instruments, solvent as internal standard (CDCl 3 : d C = 77.0 ppm). 13 C NMR spectra were recorded in broadband decoupled mode, and multiplicities were determined by use of a DEPT pulse sequence.…”

“…[7] Thioesters of glutamate side chains with internal cysteine residues occur in a2-macroglobulin, [8][9][10] in complements C3 and C4 [11][12][13] and in succinyl-CoA:3-keto acid transferase. [14] A remarkable feature of all these glutamic acid ester-or thioester-containing proteins is that they become labile with respect to fragmentation on elevation of the pH.…”