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Crystal structure of the usher:chaperone:adhesin subunit complex – insights into pilus assembly mechanism

2011; Wiley; Volume: 67; Issue: a1 Linguagem: Inglês

10.1107/s0108767311099533

1600-5724

Gilles Phan, Han Remaut, William J. Allen, Sebastian Geibel, Andrey A. Lebedev, Nadine S. Henderson, David G. Thanassi, Gabriel Waksman,

Enzyme Structure and Function

Activation of the complement cascade involves multiple proteolytic reactions mediated by large macromolecular complexes.Upon initiation, the three complement pathways converge into the critical step of the conversion of C3 to its activated form C3b by means of short-lived enzymatic complexes called C3 convertases.These convertases amplify C3b production near target surfaces, resulting in opsonization of target cells, activation of the complement terminal pathway and stimulation of the adaptive immune response.Generation and activation of complement convertases are multi-step processes that require localization of serine protease-containing proenzymes on large protein subunits.The interplay between formation of large multi-domain enzymes, extensive conformational changes and specific proteolytic cleavages highlights the complexity of the complement system.We investigated the process of convertase formation in the alternative pathway of complement.In this pathway, the C3 convertase complex is generated when the proenzyme factor B (FB) interacts with surface-bound C3b to form the pro-convertase C3bB, which is then specifically cleaved by the soluble, self-inactivated serine protease factor D (FD), yielding the active yet unstable C3 convertase C3bBb.We present here the crystal structures of the pro-convertase complex C3bB, formed by C3b (12 domains, 160 kDa) with factor B (5 domains, 90 kDa) at 4-Å resolution and the transient complex formed by C3bB with an inactive mutant of factor D (1 domain, 27 kDa) at 3.5-Å resolution.In agreement with previously published electron microscopy data, our structures highlight the equilibrium between an initial (closed/loading) state and a subsequent (open/activation) state of factor B. The structures reveal unexpected conformational changes that create the "open" state which exposes the scissile loop of FB for proteolytic cleavage.In the C3bBD * complex, we show how the open state of FB provides a docking platform for factor D distant from its catalytic site, which has been caught in an activated conformation.Additional biochemical and biophysical analyses confirm the observed structural features and reveal a highly concerted and specific activation mechanism based on cofactor-dependent and substrateinduced proteolysis, which provides an important "double-safety" catch to restrict complement amplification to C3b-tagged target cells.By adding new frames to the "structural movie" of complement activation, our data also provide new valuable information for potential structurebased drug design of complement inhibitors.