A humanized antibody that regulates the alternative pathway convertase: potential for therapy of renal disease associated with nephritic factors.

Dysregulation of the complement alternative pathway can cause disease in various organs that may be life-threatening. Severe alternative pathway dysregulation can be triggered by autoantibodies to the C3 convertase, termed nephritic factors, which cause pathological stabilization of the convertase enzyme and confer resistance to innate control mechanisms; unregulated complement consumption followed by deposition of C3 fragments in tissues ensues. The mAb, 3E7, and its humanized derivative, H17, have been shown previously to specifically bind activated C3 and prevent binding of both the activating protein, factor B, and the inhibitor, factor H, which are opposite effects that complicate its potential for therapy. Using ligand binding assays, functional assays, and electron microscopy, we show that these Abs bind C3b via a site that overlaps the binding site on C3 for the Ba domain within factor B, thereby blocking an interaction essential for convertase formation. Both Abs also bind the preformed convertase, C3bBb, and provide powerful inhibition of complement activation by preventing cleavage of C3. Critically, the Abs also bound and inhibited C3 cleavage by the nephritic factor-stabilized convertase. We suggest that by preventing enzyme formation and/or cleavage of C3 to its active downstream fragments, H17 may be an effective therapy for conditions caused by severe dysregulation of the C3 convertase and, in particular, those that involve nephritic factors, such as dense deposit disease.

Structure of Yin Yang 1 oligomers that cooperate with RuvBL1-RuvBL2 ATPases.

Yin Yang 1 (YY1) is a transcription factor regulating proliferation and differentiation and is involved in cancer development. Oligomers of recombinant YY1 have been observed before, but their structure and DNA binding properties are not well understood. Here we find that YY1 assembles several homo-oligomeric species built from the association of a bell-shaped dimer, a process we characterized by electron microscopy. Moreover, we find that YY1 self-association also occurs in vivo using bimolecular fluorescence complementation. Unexpectedly, these oligomers recognize several DNA substrates without the consensus sequence for YY1 in vitro, and DNA binding is enhanced in the presence of RuvBL1-RuvBL2, two essential AAA+ ATPases. YY1 oligomers bind RuvBL1-RuvBL2 hetero-oligomeric complexes, but YY1 interacts preferentially with RuvBL1. Collectively, these findings suggest that YY1-RuvBL1-RuvBL2 complexes could contribute to functions beyond transcription, and we show that YY1 and the ATPase activity of RuvBL2 are required for RAD51 foci formation during homologous recombination.

The structures of cytosolic and plastid-located glutamine synthetases from Medicago truncatula reveal a common and dynamic architecture.

The first step of nitrogen assimilation in higher plants, the energy-driven incorporation of ammonia into glutamate, is catalyzed by glutamine synthetase. This central process yields the readily metabolizable glutamine, which in turn is at the basis of all subsequent biosynthesis of nitrogenous compounds. The essential role performed by glutamine synthetase makes it a prime target for herbicidal compounds, but also a suitable intervention point for the improvement of crop yields. Although the majority of crop plants are dicotyledonous, little is known about the structural organization of glutamine synthetase in these organisms and about the functional differences between the different isoforms. Here, the structural characterization of two glutamine synthetase isoforms from the model legume Medicago truncatula is reported: the crystallographic structure of cytoplasmic GSII-1a and an electron cryomicroscopy reconstruction of plastid-located GSII-2a. Together, these structural models unveil a decameric organization of dicotyledonous glutamine synthetase, with two pentameric rings weakly connected by inter-ring loops. Moreover, rearrangement of these dynamic loops changes the relative orientation of the rings, suggesting a zipper-like mechanism for their assembly into a decameric enzyme. Finally, the atomic structure of M. truncatula GSII-1a provides important insights into the structural determinants of herbicide resistance in this family of enzymes, opening new avenues for the development of herbicide-resistant plants.

3D structure of the C3bB complex provides insights into the activation and regulation of the complement alternative pathway convertase.

Generation of the alternative pathway C3-convertase, the central amplification enzyme of the complement cascade, initiates by the binding of factor B (fB) to C3b to form the proconvertase, C3bB. C3bB is subsequently cleaved by factor D (fD) at a single site in fB, producing Ba and Bb fragments. Ba dissociates from the complex, while Bb remains bound to C3b, forming the active alternative pathway convertase, C3bBb. Using single-particle electron microscopy we have determined the 3-dimensional structures of the C3bB and the C3bBb complexes at approximately 27A resolution. The C3bB structure shows that fB undergoes a dramatic conformational change upon binding to C3b. However, the C3b-bound fB structure was easily interpreted after independently fitting the atomic structures of the isolated Bb and Ba fragments. Interestingly, the divalent cation-binding site in the von Willebrand type A domain in Bb faces the C345C domain of C3b, whereas the serine-protease domain of Bb points outwards. The structure also shows that the Ba fragment interacts with C3b separately from Bb at the level of the alpha'NT and CUB domains. Within this conformation, the long and flexible linker between Bb and Ba is likely exposed and accessible for cleavage by fD to form the active convertase, C3bBb. The architecture of the C3bB and C3bBb complexes reveals that C3b could promote cleavage and activation of fB by actively displacing the Ba domain from the von Willebrand type A domain in free fB. These structures provide a structural basis to understand fundamental aspects of the activation and regulation of the alternative pathway C3-convertase.

Coexistence of closed and open conformations of complement factor B in the alternative pathway C3bB(Mg2+) proconvertase.

Complement factor B (fB) circulates in plasma as a proenzyme that, upon binding to C3b in the presence of Mg(2+), is cleaved by factor D to produce Ba and Bb fragments. Activated Bb remains bound to C3b organizing the alternative pathway C3 convertase (C3bBb). Recently, we have visualized the stable C3bB(Ni(2+)) proconvertase using electron microscopy, revealing a large conformational change of the C3b-bound fB likely exposing the fD-cleavage site. In contrast, the crystal structure of the proconvertase formed by human fB and the cobra venom factor reveals fB in the closed conformation of the proenzyme. In this study, we have used single-particle electron microscopy and image processing to examine the C3bB(Mg(2+)) proconvertase. We describe two C3bB(Mg(2+)) conformations, one resembling cobra venom factor, likely representing the loading state of fB to C3b, and another identical with C3bB(Ni(2+)). These data illustrate the coexistence of C3b-bound fB in closed and open conformations that either exist in equilibrium or represent structural transitions during the assembly of the C3bB proconvertase.

Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes.

Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 A. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared with the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different than previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins.

Three-dimensional model for the isolated recombinant influenza virus polymerase heterotrimer.

The genome of influenza A virus is organized into eight ribonucleoprotein complexes (RNPs), each containing one RNA polymerase complex. This RNA polymerase has also been found non-associated to RNPs and is possibly involved in distinct functions in the infection cycle. We have expressed the virus RNA polymerase complex by co-tranfection of the PB1, PB2 and PA genes in mammalian cells and the heterotrimer was purified by the TAP tag procedure. Its 3D structure was determined by electron microscopy and single-particle image processing. The model obtained resembles the structure previously reported for the polymerase complex associated to viral RNPs but appears to be in a more open conformation. Detailed model comparison indicated that specific areas of the complex show important conformational changes as compared to the structure for the RNP-associated polymerase, particularly in regions known to interact with the adjacent NP monomers in the RNP. Also, the PB2 subunit seems to undergo a substantial displacement as a result of the association of the polymerase to RNPs. The structural model presented suggests that a core conformation of the polymerase in solution exists but the interaction with other partners, such as proteins or RNA, will trigger distinct conformational changes to activate new functional properties.

Analysis of the interaction of influenza virus polymerase complex with human cell factors.

The influenza virus polymerase is formed by the PB1, PB2 and PA subunits and is required for virus transcription and replication in the nucleus of infected cells. Here we present the characterisation of the complexes formed intracellularly by the influenza polymerase in human cells. The virus polymerase was expressed by cotransfection of the polymerase subunits cDNAs, one of which fused to the tandem-affinity purification (TAP) tag. The intracellular complexes were purified by the TAP approach, which involves IgG-Sepharose and calmodulin-agarose chromatography, under very mild conditions. The purified complexes contained the heterotrimeric polymerase and a series of associated proteins that were not apparent in purifications of untagged polymerase used as a control. Several influenza polymerase-associated proteins were identified by MALDI-MS and their presence in purified polymerase-containing complexes were verified by Western blot. Their relevance for influenza infection was established by colocalisation with virus ribonucleoproteins in human infected cells. Most of the associated human factors were nuclear proteins involved in cellular RNA synthesis, modification and nucleo-cytoplasmic export, but some were cytosolic proteins involved in translation and transport. The interactions recognised in this proteomic approach suggest that the influenza polymerase might be involved in steps of the infection cycle other than RNA replication and transcription.

The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription.

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I-Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

Crystal Structure of Hcp from Acinetobacter baumannii: A Component of the Type VI Secretion System.

The type VI secretion system (T6SS) is a bacterial macromolecular machine widely distributed in Gram-negative bacteria, which transports effector proteins into eukaryotic host cells or other bacteria. Membrane complexes and a central tubular structure, which resembles the tail of contractile bacteriophages, compose the T6SS. One of the proteins forming this tube is the hemolysin co-regulated protein (Hcp), which acts as virulence factor, as transporter of effectors and as a chaperone. In this study, we present the structure of Hcp from Acinetobacter baumannii, together with functional and oligomerization studies. The structure of this protein exhibits a tight ß barrel formed by two ß sheets and flanked at one side by a short α-helix. Six Hcp molecules associate to form a donut-shaped hexamer, as observed in both the crystal structure and solution. These results emphasize the importance of this oligomerization state in this family of proteins, despite the low similarity of sequence among them. The structure presented in this study is the first one for a protein forming part of a functional T6SS from A. baumannii. These results will help us to understand the mechanism and function of this secretion system in this opportunistic nosocomial pathogen.

Amyloidogenesis of bacterial prionoid RepA-WH1 recapitulates dimer to monomer transitions of RepA in DNA replication initiation.

Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross ß sheets. However, structures in which a whole protein domain acts as building block of an amyloid fiber are scarce, in spite of their relevance to understand amyloidogenesis. Here, we use electron microscopy (EM) and atomic force microscopy (AFM) to analyze the structure of amyloid filaments assembled by RepA-WH1, a winged-helix domain from a DNA replication initiator in bacterial plasmids. RepA-WH1 functions as a cytotoxic bacterial prionoid that recapitulates features of mammalian amyloid proteinopathies. RepA are dimers that monomerize at the origin to initiate replication, and we find that RepA-WH1 reproduces this transition to form amyloids. RepA-WH1 assembles double helical filaments by lateral association of a single-stranded precursor built by monomers. Double filaments then associate in mature fibers. The intracellular and cytotoxic RepA-WH1 aggregates might reproduce the hierarchical assembly of human amyloidogenic proteins.

The C-terminal SH3 domain contributes to the intramolecular inhibition of Vav family proteins.

Vav proteins are phosphorylation-dependent guanine nucleotide exchange factors (GEFs) that catalyze the activation of members of the Rho family of guanosine triphosphatases (GTPases). The current regulatory model holds that the nonphosphorylated, catalytically inactive state of these GEFs is maintained by intramolecular interactions among the amino-terminal domains and the central catalytic core, which block the binding of Vav proteins to GTPases. We showed that this autoinhibition is mechanistically more complex, also involving the bivalent association of the carboxyl-terminal Src homology 3 (SH3) region of Vav with its catalytic and pleckstrin homology (PH) domains. Such interactions occurred through proline-rich region-independent mechanisms. Full release from this double-locked state required synergistic weakening effects from multiple phosphorylated tyrosine residues, thus providing an optimized system to generate gradients of Vav GEF activity depending on upstream signaling inputs. This mechanism is shared by mammalian and Drosophila melanogaster Vav proteins, suggesting that it may be a common regulatory feature for this protein family.

Human C3 mutation reveals a mechanism of dense deposit disease pathogenesis and provides insights into complement activation and regulation.

Dense deposit disease (DDD) is a severe renal disease characterized by accumulation of electron-dense material in the mesangium and glomerular basement membrane. Previously, DDD has been associated with deficiency of factor H (fH), a plasma regulator of the alternative pathway (AP) of complement activation, and studies in animal models have linked pathogenesis to the massive complement factor 3 (C3) activation caused by this deficiency. Here, we identified a unique DDD pedigree that associates disease with a mutation in the C3 gene. Mutant C(3923ΔDG), which lacks 2 amino acids, could not be cleaved to C3b by the AP C3-convertase and was therefore the predominant circulating C3 protein in the patients. However, upon activation to C3b by proteases, or to C3(H2O) by spontaneous thioester hydrolysis, C(3923ΔDG) generated an active AP C3-convertase that was regulated normally by decay accelerating factor (DAF) but was resistant to decay by fH. Moreover, activated C(3b923ΔDG) and C3(H2O)(923ΔDG) were resistant to proteolysis by factor I (fI) in the presence of fH, but were efficiently inactivated in the presence of membrane cofactor protein (MCP). These characteristics cause a fluid phase-restricted AP dysregulation in the patients that continuously activated and consumed C3 produced by the normal C3 allele. These findings expose structural requirements in C3 that are critical for recognition of the substrate C3 by the AP C3-convertase and for the regulatory activities of fH, DAF, and MCP, all of which have implications for therapeutic developments.

3D structure of the influenza virus polymerase complex: localization of subunit domains.

The 3D structure of the influenza virus polymerase complex was determined by electron microscopy and image processing of recombinant ribonucleoproteins (RNPs). The RNPs were generated by in vivo amplification using cDNAs of the three polymerase subunits, the nucleoprotein, and a model virus-associated RNA containing 248 nt. The polymerase structure obtained is very compact, with no apparent boundaries among subunits. The position of specific regions of the PB1, PB2, and PA subunits was determined by 3D reconstruction of either RNP-mAb complexes or tagged RNPs. This structural model is available for the polymerase of a negative-stranded RNA virus and provides a general delineation of the complex and its interaction with the template-associated nucleoprotein monomers in the RNP.