Regulators of complement activity mediate inhibitory mechanisms through a common C3b‐binding mode

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A Molecular Insight into Complement Evasion by the Staphylococcal Complement Inhibitor Protein Family

Staphylococcus aureus possesses an impressive arsenal of complement evasion proteins that help the bacterium escape attack of the immune system. The staphylococcal complement inhibitor (SCIN) protein exhibits a particularly high potency and was previously shown to block complement by acting at the level of the C3 convertases. However, many details about the exact binding and inhibitory mechanism remained unclear. In this study, we demonstrate that SCIN directly binds with nanomolar affinity to a functionally important area of C3b that lies near the C terminus of its -chain. Direct competition of SCIN with factor B for C3b slightly decreased the formation of surface-bound convertase. However, the main inhibitory effect can be attributed to an entrapment of the assembled convertase in an inactive state. Whereas native C3 is still able to bind to the blocked convertase, no generation and deposition of C3b could be detected in the presence of SCIN. Furthermore, SCIN strongly competes with the binding of factor H to C3b and influences its regulatory activities: the SCIN-stabilized convertase was essentially insensitive to decay acceleration by factor H and the factor I- and H-mediated conversion of surface-bound C3b to iC3b was significantly reduced. By targeting a key area on C3b, SCIN is able to block several essential functions within the alternative pathway, which explains the high potency of the inhibitor. Our findings provide an important insight into complement evasion strategies by S. aureus and may act as a base for further functional studies. The Journal of Immunology, 2009, 183: 2565–2574. T he bacterium Staphylococcus aureus is a widely dissem-inated and persistent human pathogen that has a long-standing and increasingly negative impact on human health. It is a primary etiologic agent of numerous disorders tha

Mechanisms of regulation of complement activity

The complement system has been characterized as a “double-edged sword, since on one hand it acts to protect the host from infection, but on the other hand it can cause harm when not effectively regulated. More and more inflammatory and autoimmune diseases are being added to the list of those in which complement is found to be involved such as age-related macular degeneration, anti-phopsholipid syndrome, etc., indicating the need for development of complement inhibitors. Among the complement activation pathways, the alternative is especially important since it includes a positive-feedback loop that amplifies the activation initiated by all pathways and results in the activation of multiple C3 molecules to C3a and C3b. It is believed that inhibition of the alternative pathway is an effective means of treatment of certain conditions. In order to develop these inhibitors it is necessary to gain a better understanding of the activation and regulation mechanisms of the C3 convertase of the alternative pathway, C3bBb. For that purpose, here I studied the structures of the convertase and pro-convertase (C3bB, as well as its complex with FD), and analysed the interactions among their constituents using biophysical and biochemical assays. The results indicate the structural features of these proteins that are responsible for their high binding specificity, as well as the basis of the convertase activation and regulation mechanisms. This knowledge opens the way for the rational design of alternative pathway inhibitors for therapeutic use. In parallel, I studied two existing complement inhibitors: the bacterial “Staphylococcal Complement INhibitor” (SCIN) and the synthetic peptide compstatin. The first molecule is one of many regulators produced by Staphylococcus aureus, in order to escape the complement attack. The structural basis of its unique inhibition mechanism (which is the entrapment of the the C3bBb convertase into an inactive state) is described here. Although SCIN itself is immunogenic and probably not appropriate as theurapeutic, it could function as a template for the design of new inhibitors. Compstatin is a promising peptidic inhibitor currently in clinical trials for the treatment of age-related macular degeneration. Its increased use as a therapeutic in different disease models entails the search for new analogues with improved activity and stability. Here I present new analogues where the disulfide bond that keeps the molecule in a cyclized form has been replaced by a thioether bond which is stable against reduction (linearization). These analogues might prove useful in conditions in which the reductive capacity of the blood is increased, such as sepsis and hemolysis.Το σύστημα του συμπληρώματος έχει τα τελευταία χρόνια χαρακτηριστεί ως ένα «δίκοπο μαχαίρι», το οποίο από τη μια πλευρά προστατεύει τον οργανισμό από μολύνσεις ενώ από την άλλη μπορεί να προκαλέσει βλάβη σε αυτόν όταν αφεθεί ανεξέλεγκτο. Όλο και περισσότερες φλεγμονώδεις και αυτοάνοσες παθήσεις προστίθενται στη λίστα αυτών στις οποίες έχει βρεθεί ότι εμπλέκεται η υπερβολική ενεργοποίηση του συμπληρώματος (π.χ. ηλικιακή εκφύλιση της ωχράς κηλίδας, σύνδρομο αντιφωσφολιπιδίων κ.ά.), δείχνοντας την ανάγκη για την ανάπτυξη θεραπευτικών αναστολέων. Από τις οδούς ενεργοποίησης του συμπληρώματος ιδιαίτερη σημασία έχει η εναλλακτική, αφού περιλαμβάνει έναν κύκλο θετικής ανάδρασης που πολλαπλασιάζει την απόκριση όλων των οδών και ενεργοποιεί πολλαπλά μόρια C3 σε C3b και C3a. Η αναστολή της οδού αυτής πιστεύεται ότι θα ήταν πολύ αποτελεσματική για την αντιμετώπιση συγκεκριμένων ασθενειών. Για την ανάπτυξη αναστολέων για αυτή την οδό είναι απαραίτητη μια βαθύτερη γνώση του μηχανισμού ενεργοποίησης και ρύθμισης της κονβερτάσης του C3, C3bBb. Για το σκοπό αυτό εδώ μελετήθηκαν οι δομές της κονβερτάσης και της προκονβερτάσης (C3bB - καθώς και σε συνδυασμό με το FD) και οι αλληλεπιδράσεις μεταξύ των συστατικών του ενζύμου αυτού σε βιοφυσικό και βιοχημικό επίπεδο. Τα αποτελέσματα ανέδειξαν τα δομικά στοιχεία που προσδίδουν την υψηλή ειδικότητα της πρόσδεσης των πρωτεϊνών αυτών και τη βάση του μηχανισμού ενεργοποίησης και ρύθμισης της κονβερτάσης. Αυτές οι γνώσεις ανοίγουν το δρόμο για το σχεδιασμό νέων αναστολέων της εναλλακτικής οδού για θεραπευτική χρήση. Παράλληλα μελετήθηκαν δύο υπάρχοντες αναστολείς του συμπληρώματος, ο βακτηριακής προέλευσης «αναστολέας του συμπληρώματος του σταφυλόκοκκου» (SCIN) και η τεχνητή compstatin. Το πρώτο μόριο είναι ένα από πολλούς ρυθμιστές του συμπληρώματος που παράγει ο χρυσίζοντας σταφυλόκκος για να διαφύγει από αυτό. Εδώ βρέθηκε η δομική βάση του μοναδικού μηχανισμού δράσης του που συνίσταται στην παγίδευση του C3bBb σε μια ανενεργό μορφή. Το μόριο αυτό είναι ανοσογενές και πιθανώς ακατάλληλο για φάρμακο, αλλά θα μπορούσε να χρησιμεύσει ως εκμαγείο για το σχεδιασμό νέων αναστολέων. Η compstatin είναι ένας πολλά υποσχόμενος πεπτιδικός αναστολέας που βρίσκεται σε κλινικές δοκιμές για τη θεραπεία της ηλικιακής εκφύλισης της ωχράς κηλίδας. Η αυξανόμενη χρήση της σε όλο και περισσότερα μοντέλα ασθενειών επιβάλλει τη βελτίωση της δραστικότητας και της σταθερότητάς της. Εδώ παρουσιάζονται νέα ανάλογα της compstatin, όπου ο δισουλφιδικός δεσμός που συγκρατεί το μόριο σε κυκλική μορφή έχει αντικατασταθεί από έναν θειοαιθερικό δεσμό ο οποίος είναι ανθεκτικός στην αναγωγή (διάσπαση). Τα ανάλογα αυτά πιθανόν να φανούν χρήσιμα σε καταστάσεις όπου το αίμα εμφανίζει αυξημένη «αναγωγική δυνατότητα», όπως η σήψη και η αιμόλυση

Crystallization of human complement component C3b in the presence of a staphylococcal complement-inhibitor protein (SCIN)

The crystallization and results of multiwavelength anomalous diffraction studies of a recombinant C3-inhibitory fragment of Efb from S. aureus are reported

Philippines d Cardiovascular Biology Research Program

a b s t r a c t Therapeutic modulation of the complement system has become increasingly important in line with the growing recognition of the role of complement in numerous diseases. Compstatin, a peptidic inhibitor that acts at the central level of the complement cascade, is currently in clinical evaluation but routes to improve its efficacy have not yet been fully explored. Here, we report improvements in both the inhibitory potency and pharmacokinetic parameters of compstatin that broaden its clinical applications. Selective modification of the compstatin N-terminus with non-proteinogenic amino acids resulted in the first analogue with subnanomolar binding affinity (K D = 0.5 nM) and other similarly potent derivatives with improved solubility in clinically relevant solvents. Detailed structure-activity relationship studies based on biophysical and computational methods revealed key structural determinants for the observed improvements. Importantly, pharmacokinetic evaluation in non-human primates revealed target-driven elimination kinetics with plasma half-life values exceeding expectations for peptidic drugs (close to 12 h). This successful optimization strategy is expected to pave the way for systemic administration of compstatin in a range of clinical conditions

Allosteric inhibition of complement function by a staphylococcal immune evasion protein

The complement system is a major target of immune evasion by Staphylococcus aureus. Although many evasion proteins have been described, little is known about their molecular mechanisms of action. Here we demonstrate that the extracellular fibrinogen-binding protein (Efb) from S. aureus acts as an allosteric inhibitor by inducing conformational changes in complement fragment C3b that propagate across several domains and influence functional regions far distant from the Efb binding site. Most notably, the inhibitor impaired the interaction of C3b with complement factor B and, consequently, formation of the active C3 convertase. As this enzyme complex is critical for both activation and amplification of the complement response, its allosteric inhibition likely represents a fundamental contribution to the overall immune evasion strategy of S. aureus