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A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

ABSTRACT: The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492-499 and 573-580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.

SUBMITTER: Perry AJ

PROVIDER: S-EPMC3668739 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

Perry Andrew J AJ Wijeyewickrema Lakshmi C LC Wilmann Pascal G PG Gunzburg Menachem J MJ D'Andrea Laura L Irving James A JA Pang Siew Siew SS Duncan Renee C RC Wilce Jacqueline A JA Whisstock James C JC Pike Robert N RN

The Journal of biological chemistry 20130416 22

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the ...[more]

PMID: 23592783

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Project description:Effective clearance of apoptotic cells by phagocytes prevents the release of intracellular alarmins and manifestation of autoimmunity. This prompt efferocytosis is complemented by intracellular proteolytic degradation that occurs within the apoptotic cells and in the efferosome of the phagocytes. Although the role of extracellular proteases in apoptotic cells clearance is unknown, the strong association of congenital C1s deficiency with Systemic Lupus Erythematosus highlights the protective nature that this extracellular protease has against autoimmunity. The archetypical role of serine protease C1s as the catalytic arm of C1 complex (C1qC1r2C1s2) involve in the propagation of the classical complement pathway could not provide the biological basis for this association. However, a recent observation of the ability of C1 complex to cleave a spectrum of intracellular cryptic targets exposed during apoptosis provides a valuable insight to the underlying protective mechanism. High-mobility group box 1 (HMGB1), an intracellular alarmin that is capable of inducing the formation of antinuclear autoantibodies and causes lupus-like conditions in mice, is identified as a novel potential target by bioinformatics analysis. This is verified experimentally with C1s, both in its purified and physiological form as C1 complex, cleaving HMGB1 into defined fragments of 19 and 12?kDa. This cleavage diminishes HMGB1 ability to enhance lipopolysaccharide mediated pro-inflammatory cytokines production from monocytes, macrophages and dendritic cells. Further mass spectrometric analysis of the C1 complex treated apoptotic cellular proteins demonstrated additional C1s substrates and revealed the complementary role of C1s in apoptotic cells clearance through the proteolytic cleavage of intracellular alarmins and autoantigens. C1 complex may have evolved as, besides the bacteriolytic arm of antibodies in which it activates the complement cascade, a tissue renewal mechanism that reduces the immunogenicity of apoptotic tissue debris and decreases the likelihood of autoimmunity.

Project description:The C1s protease of the classical complement pathway propagates the initial activation of this pathway of the system by cleaving and thereby activating the C4 and C2 complement components. This facilitates the formation of the classical pathway C3 convertase (C4bC2a). C1s has a Lys residue located at position 628 (192 in chymotrypsin numbering) of the SP domain that has the potential to partially occlude the S2-S2' positions of the active site. The 192 residue of serine proteases generally plays an important role in interactions with substrates. We therefore investigated the role of Lys628 (192) in interactions with C4 by altering the Lys residue to either a Gln (found in many other serine proteases) or an Ala residue. The mutant enzymes had altered specificity profiles for a combinatorial peptide substrate library, suggesting that this residue does influence the active site specificity of the protease. Generally, the K628Q mutant had greater activity than wild type enzyme against peptide substrates, while the K628A residue had lowered activity, although this was not always the case. Against peptide substrates containing physiological substrate sequences, the K628Q mutant once again had generally higher activity, but the activity of the wild type and mutant enzymes against a C4 P4-P4' substrate were similar. Interestingly, alteration of the K628 residue in C1s had a marked effect on the cleavage of C4, reducing cleavage efficiency for both mutants about fivefold. This indicates that this residue plays a different role in cleaving protein versus peptide substrates and that the Lys residue found in the wild type enzyme plays an important role in interacting with the C4 substrate. Understanding the basis of the interaction between C1s and its physiological substrates is likely to lead to insights that can be used to design efficient inhibitors of the enzyme for use in treating diseases caused by inflammation as result of over-activity of the classical complement pathway.

Dataset Information

A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

Publications

A molecular switch governs the interaction between the human complement protease C1s and its substrate, complement C4.

Similar Datasets

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