Hilkka Lankinen

The Yersinia adhesin YadA collagen-binding domain structure is a novel left-handed parallel beta-roll.

The crystal structure of the recombinant collagen‐binding domain of Yersinia adhesin YadA from Yersinia enterocolitica serotype O:3 was solved at 1.55 Å resolution. The trimeric structure is composed of head and neck regions, and the collagen binding head region is a novel nine‐coiled left‐handed parallel β‐roll. Before the β‐roll, the polypeptide loops from one monomer to the rest, and after the β‐roll the neck region does the same, making the transition from the globular head region to the narrower stalk domain. This creates an intrinsically stable ‘lock nut’ structure. The trimeric form of YadA is required for collagen binding, and mutagenesis of its surface residues allowed identification of a putative collagen‐binding surface. Furthermore, a new structure–sequence motif for YadA β‐roll was used to identify putative YadA‐head‐like domains in a variety of human and plant pathogens. Such domains may therefore be a common bacterial strategy for avoiding host response.

Complement Inhibitor Factor H Binding to Lyme Disease Spirochetes Is Mediated by Inducible Expression of Multiple Plasmid-Encoded Outer Surface Protein E Paralogs

Borrelia burgdorferi spirochetes can circumvent the vertebrate host’s immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.

Structural determinants of growth factor binding and specificity by VEGF receptor 2

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 2∶2 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.

Effective Suppression of Vascular Network Formation by Combination of Antibodies Blocking VEGFR Ligand Binding and Receptor Dimerization

Antibodies that block vascular endothelial growth factor (VEGF) have become an integral part of antiangiogenic tumor therapy, and antibodies targeting other VEGFs and receptors (VEGFRs) are in clinical trials. Typically receptor-blocking antibodies are targeted to the VEGFR ligand-binding site. Here we describe a monoclonal antibody that inhibits VEGFR-3 homodimer and VEGFR-3/VEGFR-2 heterodimer formation, signal transduction, as well as ligand-induced migration and sprouting of microvascular endothelial cells. Importantly, we show that combined use of antibodies blocking ligand binding and receptor dimerization improves VEGFR inhibition and results in stronger inhibition of endothelial sprouting and vascular network formation in vivo. These results suggest that receptor dimerization inhibitors could be used to enhance antiangiogenic activity of antibodies blocking ligand binding in tumor therapy.

Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses

ABSTRACT Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and form protrusions, or “spikes.” The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the GN-GC spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.

Susceptibility of human cells to Puumala virus infection

Nephropathia epidemica involves several organs including kidney, lung, liver and brain. To investigate the susceptibility of putative target cells to the agent responsible, Puumala virus, we screened established human cell lines of lung (WI-38, A-427, CCD-11Lu), kidney (A-704), liver (Hep G2), pharynx (Detroit 562), submaxillary gland (A-253) and neural (SK-N-MC, SH-SY5Y) origin as well as primary human kidney glomerular cells, endothelial cells and peripheral blood monocytes/macrophages. Propagation of the Sotkamo strain of Puumala virus was also tested in the primary kidney, spleen and lung cells of bank voles (the natural host of the virus). All of the primary cells and most of the established cell lines expressed viral protein, synthesized viral RNA and secreted infectious virus, except the neural SK-N-MC and SH-SY5Y cells. None of the tested cell types except the primary bank vole kidney cells could propagate the virus as efficiently as the Vero E6 cells. The observed host cell range is wide and consistent with a multiorgan involvement of Puumala virus. No cytopathic effects were seen in any of the infected cell cultures.

Antibacterial activities of temporin A analogs

Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL‐NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin‐sensitive and ‐resistant Staphylococcus aureus as well as vancomycin‐resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N‐terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all‐D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non‐chiral interactions with membrane lipids.

Hantavirus nucleocapsid protein interacts with the Fas-mediated apoptosis enhancer Daxx.

Hantaviruses cause two severe diseases, haemorrhagic fever with renal syndrome in Eurasia and hantavirus pulmonary syndrome in the Americas. To understand more about the molecular mechanisms that lead to these diseases, the associations of Puumala virus nucleocapsid protein (PUUV-N) with cellular proteins were studied by yeast two-hybrid screening. Daxx, known as an apoptosis enhancer, was identified from a HeLa cDNA library and its interaction with PUUV-N was confirmed by GST pull-down assay, co-immunoprecipitation and co-localization studies. Furthermore, domains of interaction were mapped to the carboxyl-terminal region of 142 amino acids in Daxx and the carboxyl-terminal 57 residues in PUUV-N, respectively. In pepscan assays, the binding sites of Daxx to PUUV-N were mapped further to two lysine-rich regions, of which one overlaps the sequence of the predicted nuclear localization signal of Daxx. These data suggest a direct link between host cell machinery and a hantavirus structural component.

Lysine-Dependent Multipoint Binding of the Borrelia burgdorferi Virulence Factor Outer Surface Protein E to the C Terminus of Factor H

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18–20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala151-Lys166) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.

Tula hantavirus triggers pro-apoptotic signals of ER stress in Vero E6 cells

Abstract Tula virus is a member of the Hantavirus genus of the family Bunyaviridae. Viruses of this family have an unusual pattern of intracellular maturation at the ER–Golgi compartment. We recently found that Tula virus, similar to several other hantaviruses, is able to induce apoptosis in cultured cells [Li, X.D., Kukkonen, S., Vapalahti, O., Plyusnin, A., Lankinen, H., Vaheri, A., 2004. Tula hantavirus infection of Vero E6 cells induces apoptosis involving caspase 8 activation. J. Gen. Virol. 85, 3261–3268.]. However, the cellular mechanisms remain to be clarified. In this study, we demonstrate that the progressive replication of Tula virus in Vero E6 cells initiates several death programs that are intimately associated with ER stress: (1) early activation of ER-resident caspase-12; (2) phosphorylation of Jun NH2-terminal kinase (JNK) and its downstream target transcriptional factor, c-jun; (3) induction of the pro-apoptotic transcriptional factor, growth arrest- and DNA damage-inducible gene 153, or C/EBP homologous protein (Gadd153/chop); and (4) changes in the ER-membrane protein BAP31 implying cross-talk with the mitochondrial apoptosis pathway. Furthermore, we confirmed that a sustained ER stress was induced marked by an increased expression of an ER chaperone Grp78/BiP. Taken together, we have identified involvement of ER stress-mediated death program in Tula virus-infected Vero E6 cells which provides a new approach to understand the mechanisms in hantavirus-induced apoptosis.