Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3.
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Isaac L, Isenman DE
Structural requirements for thioester bond formation in human complement component C3. Reassessment of the role of thioester bond integrity on the conformation of C3.
J Biol Chem. 1992 May 15;267(14):10062-9.
- PubMed ID
- 1577777 [ View in PubMed]
- Abstract
A unique thioester bond, formed between the side chains of neighboring C and Q residues, is present in complement components C3 and C4 and the protease inhibitor alpha 2-macroglobulin. This structure is essential for mediating covalent attachment to target acceptors and also for maintaining these proteins in their native conformation. An examination of the residues in the immediate vicinity of the C and Q reveals a very high degree of sequence similarity among the three proteins which crosses species barriers. The following is the sequence flanking the thioester residues in C3, the highly conserved amino acids being underlined and the the thioester-forming residues being indicated by italics: 1005V-T-P-S-G-C-G-E-Q-N-M-I-G-M-T-P-T1021. Through a site-directed mutagenesis and cDNA expression approach, we have examined the importance of the conserved amino acids in the formation, stability, and function of the thioester bond in C3. The behavior of the mutants fell into three categories. The potential loss in peptide backbone flexibility by the replacement of G1009 by A or S was permissive to thioester formation and function as was replacement of M1015 by the still fairly bulky residue F. In contrast, replacement of M1015 by A resulted in an alpha-chain which was highly unstable toward proteolytic degradation. The third category, which included mutant molecules P1007G, P1020G, E1012Q, and Q1013N, displayed an unusual phenotype in which both the autolytic fragmentation and the hemolytic activity characteristics of thioester-intact molecules were absent. However, like their wildtype counterpart, these molecules retained the ability to be cleaved by C3 convertase (C4b2a), a conformation-dependent property that is normally lost in the conversion of native C3 to thioester-hydrolyzed C3(H2O). Since an identical functional profile was obtained when the thioester was deliberately prevented from forming in the mutant C1010A, we conclude that if a stable thioester fails to form during biosynthesis, at least parts of the mature protein can adopt a more native-like conformation than is the case when the thioester is first formed and then hydrolyzed in the mature protein. In view of these new findings, the interpretation of the previously observed correlation between the loss of thioester integrity and the adoption of a C3b-like conformation must be reassessed.