-
[show abstract]
[hide abstract]
ABSTRACT: Modular serine proteases are central to the complement cascade of the mammalian humoral immune system. These proteases form protein complexes through multi-domain interactions to achieve their proteolytic activity. We review the structural insights into complement initiation by auto-activation of the hetero-tetrameric proteases of the large danger-recognition protein complexes, amplification and labelling of particles by the formation and activity of C3 convertases, and regulation by convertase dissociation and degradation to prevent 'bystander' damage to healthy host cells and tissues. The data reveal that complex formation and large domain-domain rearrangements underlie the proteolytic reactions of the complement cascade, which enables the host to recognize and clear invading microbes and host debris from its blood and fluids surrounding tissues.
Current Opinion in Structural Biology 05/2012; 22(3):333-41. · 9.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.
Cell reports. 03/2012; 1(3):200-7.
-
[show abstract]
[hide abstract]
ABSTRACT: Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation-libration-screw (TLS) model. Modeling of 20 protein datasets at 1.1-3.1 Å resolution reduced cross-validated R(free) values by 0.3-4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a 'molten core' likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order-disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure-dynamics-function relationships.DOI:http://dx.doi.org/10.7554/eLife.00311.001.
eLife. 01/2012; 1:e00311.
-
Piet Gros
Nature Structural & Molecular Biology 02/2011; 18(4):401-2. · 12.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs.
Molecular Membrane Biology 02/2011; 28(3):158-70. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of the complement cascade induces inflammatory responses and marks cells for immune clearance. In the central complement-amplification step, a complex consisting of surface-bound C3b and factor B is cleaved by factor D to generate active convertases on targeted surfaces. We present crystal structures of the pro-convertase C3bB at 4 angstrom resolution and its complex with factor D at 3.5 angstrom resolution. Our data show how factor B binding to C3b forms an open "activation" state of C3bB. Factor D specifically binds the open conformation of factor B through a site distant from the catalytic center and is activated by the substrate, which displaces factor D's self-inhibitory loop. This concerted proteolytic mechanism, which is cofactor-dependent and substrate-induced, restricts complement amplification to C3b-tagged target cells.
Science 12/2010; 330(6012):1816-20. · 31.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The complement system is a crucial component of the innate immune response in humans. Recent studies in Staphylococcus aureus and Neisseria meningitidis have revealed how these bacteria escape complement-mediated killing. In addition, new structural data have provided detailed insights into the molecular mechanisms of host defence mediated by the complement system and how bacterial proteins interfere with this process. This information is fundamental to our understanding of bacterial pathogenesis and may facilitate the design of better vaccines.
Nature Reviews Microbiology 06/2010; 8(6):393-9. · 21.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Immune protection by the complement system critically depends on assembly of C3 convertases on the surface of pathogens and altered host cells. These short-lived protease complexes are formed through pro-convertases, which for the alternative pathway consist of the complement component C3b and the pro-enzyme factor B (FB). Here, we present the crystal structure at 2.2-A resolution, small-angle X-ray scattering and electron microscopy (EM) data of the pro-convertase formed by human FB and cobra venom factor (CVF), a potent homologue of C3b that generates more stable convertases. FB is loaded onto CVF through its pro-peptide Ba segment by specific contacts, which explain the specificity for the homologous C3b over the native C3 and inactive products iC3b and C3c. The protease segment Bb binds the carboxy terminus of CVF through the metal-ion dependent adhesion site of the Von Willebrand factor A-type domain. A possible dynamic equilibrium between a 'loading' and 'activation' state of the pro-convertase may explain the observed difference between the crystal structure of CVFB and the EM structure of C3bB. These insights into formation of convertases provide a basis for further development of complement therapeutics.
The EMBO Journal 08/2009; 28(16):2469-78. · 9.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Factor H (FH) is an abundant regulator of complement activation and protects host cells from self-attack by complement. Here we provide insight into the regulatory activity of FH by solving the crystal structure of the first four domains of FH in complex with its target, complement fragment C3b. FH interacted with multiple domains of C3b, covering a large, extended surface area. The structure indicated that FH destabilizes the C3 convertase by competition and electrostatic repulsion and that FH enables proteolytic degradation of C3b by providing a binding platform for protease factor I while stabilizing the overall domain arrangement of C3b. Our results offer general models for complement regulation and provide structural explanations for disease-related mutations in the genes encoding both FH and C3b.
Nature Immunology 08/2009; 10(7):728-33. · 26.01 Impact Factor
-
Suzan H M Rooijakkers,
Jin Wu,
Maartje Ruyken,
Robert van Domselaar,
Karel L Planken,
Apostolia Tzekou,
Daniel Ricklin,
John D Lambris,
Bert J C Janssen,
Jos A G van Strijp, Piet Gros
[show abstract]
[hide abstract]
ABSTRACT: Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.
Nature Immunology 08/2009; 10(7):721-7. · 26.01 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The first crystal structures of lipases that have been covalently modified through site-selective inhibition by different organometallic phosphonate-pincer-metal complexes are described. Two ECE-pincer-type d(8)-metal complexes, that is, platinum (1) or palladium (2) with phosphonate esters (ECE = [(EtO)-(O=)P(-O-C(6)H(4)-(NO(2))-4)(-C(3)H(6)-4-(C(6)H(2)-(CH(2)E)(2))](-); E = NMe(2) or SMe) were introduced prior to crystallization and have been shown to bind selectively to the Ser(120) residue in the active site of the lipase cutinase to give cut-1 (platinum) or cut-2 (palladium) hybrids. For all five presented crystal structures, the ECE-pincer-platinum or -palladium head group sticks out of the cutinase molecule and is exposed to the solvent. Depending on the nature of the ECE-pincer-metal head group, the ECE-pincer-platinum and -palladium guests occupy different pockets in the active site of cutinase, with concomitant different stereochemistries on the phosphorous atom for the cut-1 (S(P)) and cut-2 (R(P)) structures. When cut-1 was crystallized under halide-poor conditions, a novel metal-induced dimeric structure was formed between two cutinase-bound pincer-platinum head groups, which are interconnected through a single mu-Cl bridge. This halide-bridged metal dimer shows that coordination chemistry is possible with protein-modified pincer-metal complexes. Furthermore, we could use NCN-pincer-platinum complex 1 as site-selective tool for the phasing of raw protein diffraction data, which shows the potential use of pincer-platinum complex 1 as a heavy-atom derivative in protein crystallography.
Chemistry 03/2009; 15(17):4270-80. · 5.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3'-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca(2+)-dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded beta-barrel, reveals that the active site is located between the barrel wall and an alpha-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca(2+) forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.
Proceedings of the National Academy of Sciences 02/2009; 106(6):1960-4. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cobra venom factor (CVF) is a structural and functional analog of complement C3 isolated from cobra venom. Both CVF and C3b can bind factor B and subsequently form the bimolecular C3/C5 convertases CVF,Bb or C3b,Bb, respectively. The two homologous enzymes exhibit several differences of which the difference in physico-chemical stability is most important, allowing continuous activation of C3 and C5 by CVF,Bb, leading to serum complement depletion. Here we describe the detailed functional properties of two hybrid proteins in which the 113 or 315 C-terminal residues of C3 were replaced with corresponding CVF sequences. Both hybrid proteins formed stable convertases that exhibited C3-cleaving activity, although at different rates. Neither convertase cleaved C5. Both convertases showed partial resistance to inactivation by factors H and I, allowing them to deplete complement in human serum. These data demonstrate that functionally important structural differences between CVF and C3 are located in the very C-terminal region of both homologous proteins, and that small substitutions in human C3 with homologous CVF sequence result in C3 derivatives with CVF-like functions. Such hybrid proteins are important tools to study the structure/function relationships in both C3 and CVF, and these "humanized CVF" proteins may become reagents for therapeutic complement depletion.
Developmental & Comparative Immunology 09/2008; 33(1):105-16. · 3.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Complement in mammalian plasma recognizes pathogenic, immunogenic and apoptotic cell surfaces, promotes inflammatory responses and marks particles for cell lysis, phagocytosis and B-cell stimulation. At the heart of the complement system are two large proteins, complement component C3 and protease factor B. These two proteins are pivotal for amplification of the complement response and for labelling of the target particles, steps that are required for effective clearance of the target. Here we review the molecular mechanisms of complement activation, in which proteolysis and complex formation result in large conformational changes that underlie the key offensive step of complement executed by C3 and factor B. Insights into the mechanisms of complement amplification are crucial for understanding host defence and pathogen immune evasion, and for the development of complement-immune therapies.
Nature Reviews Immunology 02/2008; 8(1):48-58. · 32.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compstatin in complex with C3c, a major proteolytic fragment of C3. The structure reveals that the compstatin-binding site is formed by the macroglobulin (MG) domains 4 and 5. This binding site is part of the structurally stable MG-ring formed by domains MG 1-6 and is far away from any other known binding site on C3. Compstatin does not alter the conformation of C3c, whereas compstatin itself undergoes a large conformational change upon binding. We propose a model in which compstatin sterically hinders the access of the substrate C3 to the convertase complexes, thus blocking complement activation and amplification. These insights are instrumental for further development of compstatin as a potential therapeutic.
Journal of Biological Chemistry 11/2007; 282(40):29241-7. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Slit2 and Roundabout 1 (Robo1) provide a key ligand-receptor interaction for the navigation of commissural neurons during the development of the central nervous system. Slit2 is a large multidomain protein containing an unusual domain organization of four tandem leucine-rich repeat (LRR) domains at its N-terminus. These domains are well known to mediate protein-protein interactions; indeed, the Robo1-binding region has been mapped to the concave face of the second LRR domain. It has also been shown that the fourth LRR domain may mediate Slit dimerization and that both the first and second domains can bind heparin. Thus, while roles have been ascribed for three of the LRR domains, there is still no known role for the third domain. Each of the four LRR domains from human Slit2 have now been successfully expressed in milligram quantities using expression in mammalian cells. Here, the crystallization of the second and third LRR domains and the structure of the third LRR domain are presented. This is the first structure of an LRR domain from human Slit2, which has an extra repeat compared with the Drosophila homologue. It is proposed that a highly conserved patch of surface residues on the concave face may mediate any protein-protein interactions involving this LRR domain, a result that will be useful in guiding further studies on Slit2.
Acta Crystallographica Section D Biological Crystallography 10/2007; 63(Pt 9):961-8. · 12.62 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8alpha at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane beta hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8alpha is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion.
Science 10/2007; 317(5844):1552-4. · 31.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 A resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.
The Journal of Immunology 10/2007; 179(5):2989-98. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1-2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.
Proceedings of the National Academy of Sciences 10/2007; 104(38):14923-8. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of Roundabout 1 (Robo1) by Slit proteins results in axon repulsion from the midline. Robo1 is a large transmembrane receptor expressed on the axon growth cone and the minimal Robo1-binding region required for Slit activation has been mapped to the N-terminal Ig1-2 domains. The cDNA encoding the first two Ig domains of Robo1 has been cloned and the protein has been expressed in HEK293 EBNA-1 mammalian cells. Here, the purification and crystallization conditions of this Robo1 construct are reported. The crystals are orthorhombic, space group P2(1)2(1)2, with unit-cell parameters a = 38.8, b = 69.4, c = 103.3 A and one molecule in the asymmetric unit. X-ray diffraction data have been collected to 2.8 A resolution on beamline ID29 at the ESRF.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2007; 63(Pt 8):689-91. · 0.51 Impact Factor
