Rune T. Kidmose

Structural basis for activation of the complement system by component C4 cleavage.

An essential aspect of innate immunity is recognition of molecular patterns on the surface of pathogens or altered self through the lectin and classical pathways, two of the three well-established activation pathways of the complement system. This recognition causes activation of the MASP-2 or the C1s serine proteases followed by cleavage of the protein C4. Here we present the crystal structures of the 203-kDa human C4 and the 245-kDa C4⋅MASP-2 substrate⋅enzyme complex. When C4 binds to MASP-2, substantial conformational changes in C4 are induced, and its scissile bond region becomes ordered and inserted into the protease catalytic site in a manner canonical to serine proteases. In MASP-2, an exosite located within the CCP domains recognizes the C4 C345C domain 60 Å from the scissile bond. Mutations in C4 and MASP-2 residues at the C345C–CCP interface inhibit the intermolecular interaction and C4 cleavage. The possible assembly of the huge in vivo enzyme–substrate complex consisting of glycan-bound mannan-binding lectin, MASP-2, and C4 is discussed. Our own and prior functional data suggest that C1s in the classical pathway of complement activated by, e.g., antigen–antibody complexes, also recognizes the C4 C345C domain through a CCP exosite. Our results provide a unified structural framework for understanding the early and essential step of C4 cleavage in the elimination of pathogens and altered self through two major pathways of complement activation.

Structure of the Qβ replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qβ is composed of the viral catalytic β-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the β-subunit and the two host proteins to 2.5-Å resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the β-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the β-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.

Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes

Significance A salient feature of the immune system is its ability to discriminate self from nonself. We define the molecular mechanism governing activation of an ancient and central component: the lectin pathway of complement. The basis is the association of two proteases in distinct complexes with at least five pattern recognition molecules. Clustering of these complexes on ligand surfaces allows cross-activation of the proteases, which subsequently activate downstream factors to initiate a proteolytic cascade. This is conceptually similar to signaling by cellular receptors and could be viewed as cellular signaling turned inside out. Different pattern recognition complexes “talk to each other” to coordinate immune activation, which may impart differential activation based on recognition of simple vs. complex ligand patterns. Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement.

The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase

Hydroxyurea (HU) is a well tolerated ribonucleotide reductase inhibitor effective in HIV, sickle cell disease, and blood cancer therapy. Despite a positive initial response, however, most treated cancers eventually progress due to development of HU resistance. Although RNR properties influence HU resistance in cell lines, the mechanisms underlying cancer HU resistance in vivo remain unclear. To address this issue, we screened for HU resistance in the plant Arabidopsis thaliana and identified seventeen unique catalase mutants, thereby establishing that HU toxicity depends on catalase in vivo. We further demonstrated that catalase is a direct HU target by showing that HU acts as a competitive inhibitor of catalase-mediated hydrogen peroxide decomposition. Considering also that catalase can accelerate HU decomposition in vitro and that co-treatment with another catalase inhibitor alleviates HU effects in vivo, our findings suggests that HU could act as a catalase-activated pro-drug. Clinically, we found high catalase activity in circulating cells from untreated chronic myeloid leukemia, offering a possible explanation for the efficacy of HU against this malignancy.

Structural Basis for the Function of Complement Component C4 within the Classical and Lectin Pathways of Complement.

Complement component C4 is a central protein in the classical and lectin pathways within the complement system. During activation of complement, its major fragment C4b becomes covalently attached to the surface of pathogens and altered self-tissue, where it acts as an opsonin marking the surface for removal. Moreover, C4b provides a platform for assembly of the proteolytically active convertases that mediate downstream complement activation by cleavage of C3 and C5. In this article, we present the crystal and solution structures of the 195-kDa C4b. Our results provide the molecular details of the rearrangement accompanying C4 cleavage and suggest intramolecular flexibility of C4b. The conformations of C4b and its paralogue C3b are shown to be remarkably conserved, suggesting that the convertases from the classical and alternative pathways are likely to share their overall architecture and mode of substrate recognition. We propose an overall molecular model for the classical pathway C5 convertase in complex with C5, suggesting that C3b increases the affinity for the substrate by inducing conformational changes in C4b rather than a direct interaction with C5. C4b-specific features revealed by our structural studies are probably involved in the assembly of the classical pathway C3/C5 convertases and C4b binding to regulators.

Structure of TSA2 reveals novel features of the active-site loop of peroxiredoxins.

Saccharomyces cerevisiae TSA2 belongs to the family of typical 2-Cys peroxiredoxins, a ubiquitously expressed family of redox-active enzymes that utilize a conserved peroxidatic cysteine to reduce peroxides. Typical 2-Cys peroxiredoxins have been shown to be involved in protection against oxidative stress and in hydrogen peroxide signalling. Furthermore, several 2-Cys peroxiredoxins, including S. cerevisiae TSA1 and TSA2, are able to switch to chaperone activity upon hyperoxidation of their peroxidatic cysteine. This makes the sensitivity to hyperoxidation of the peroxidatic cysteine a very important determinant for the cellular function of a peroxiredoxin under different cellular conditions. Typical 2-Cys peroxiredoxins exist as dimers, and in the course of the reaction the peroxidatic cysteine forms a disulfide with a resolving cysteine located in the C-terminus of its dimeric partner. This requires a local unfolding of the active site and the C-terminus. The balance between the fully folded and locally unfolded conformations is of key importance for the reactivity and sensitivity to hyperoxidation of the different peroxiredoxins. Here, the structure of a C48S mutant of TSA2 from S. cerevisiae that mimics the reduced state of the peroxidatic cysteine has been determined. The structure reveals a novel conformation for the strictly conserved Pro41, which is likely to affect the delicate balance between the fully folded and locally unfolded conformations of the active site, and therefore the reactivity and the sensitivity to hyperoxidation. Furthermore, the structure also explains the observed difference in the pKa values of the peroxidatic cysteines of S. cerevisiae TSA1 and TSA2 despite their very high sequence identity.

Interacting with the Human Insulin Receptor.

Insulin is an essential regulator of glucose homeostasis. In this issue of Structure, Croll et al. (2016) reports a significantly improved model of the Fab-complexed IR ectodomain refined against a dataset extending to 3.3 Å.

Structural Studies of the DCC-Ribosome Complex

During neuronal development axons are guided by a gradient of the signal molecule netrin, which attracts extending neurons by binding to the DCC (Deleted in Colorectal Cancer) Receptor. It has been shown that the intracellular domain of the DCC receptor interacts directly with the ribosome, and that this interaction is crucial for axon guidance(1). However structural insights into this interaction are still lacking. Our aim is therefore to determine the crystal structure of the DCC-ribosome complex. As membrane proteins can be challenging to crystalize, especially together with a huge macromolecular complex as the ribosome, a cloned fragment of the intracellular domain of the human DCC receptor will be used. Tcherkezian et al. showed that the interaction with the ribosome occurs through the ribosomal protein L5(1), which is conserved from S. cerevisiae to humans. We therefore decided to use the 80S S. cerevisiae ribosome because crystallization conditions are known and well-established (2). Furthermore, in the crystals of S. cerevisiae there is a large solvent channel passing by ribosomal protein L5(2), making it likely that we can soak fragments of the DCC receptor into crystals of S. cerevisiae ribosome. In order to screen for soluble fragments of the DCC receptor that can be used for soaking, a random PCR expression approach first described by Kawasaki and Inagaki(3), where random PCR fragments are inserted into a vector containing GFP was used. This method resulted in several soluble fragments covering the P1 domain that was shown to be responsible for ribosome binding(1). These fragments are now being purified, and will be tested for ribosome binding, and subsequently in soaking and co-crystallisation experiments. Progress will be presented.