• Crystal structures explain functional differences in the two actin depolymerization factors of the malaria parasite.

      Singh, Bishal K; Sattler, Julia M; Chatterjee, Moon; Huttu, Jani; Schüler, Herwig; Kursula, Inari (2011-08-12)
      Apicomplexan parasites, such as the malaria-causing Plasmodium, utilize an actin-based motor for motility and host cell invasion. The actin filaments of these parasites are unusually short, and actin polymerization is under strict control of a small set of regulatory proteins, which are poorly conserved with their mammalian orthologs. Actin depolymerization factors (ADFs) are among the most important actin regulators, affecting the rates of filament turnover in a multifaceted manner. Plasmodium has two ADFs that display low sequence homology with each other and with the higher eukaryotic family members. Here, we show that ADF2, like canonical ADF proteins but unlike ADF1, binds to both globular and filamentous actin, severing filaments and inducing nucleotide exchange on the actin monomer. The crystal structure of Plasmodium ADF1 shows major differences from the ADF consensus, explaining the lack of F-actin binding. Plasmodium ADF2 structurally resembles the canonical members of the ADF/cofilin family.
    • Myelin 2',3'-cyclic nucleotide 3'-phosphodiesterase: active-site ligand binding and molecular conformation.

      Myllykoski, Matti; Raasakka, Arne; Han, Huijong; Kursula, Petri; Department of Biochemistry and Biocenter Oulu, University of Oulu, Oulu, Finland. (2012)
      The 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a highly abundant membrane-associated enzyme in the myelin sheath of the vertebrate nervous system. CNPase is a member of the 2H phosphoesterase family and catalyzes the formation of 2'-nucleotide products from 2',3'-cyclic substrates; however, its physiological substrate and function remain unknown. It is likely that CNPase participates in RNA metabolism in the myelinating cell. We solved crystal structures of the phosphodiesterase domain of mouse CNPase, showing the binding mode of nucleotide ligands in the active site. The binding mode of the product 2'-AMP provides a detailed view of the reaction mechanism. Comparisons of CNPase crystal structures highlight flexible loops, which could play roles in substrate recognition; large differences in the active-site vicinity are observed when comparing more distant members of the 2H family. We also studied the full-length CNPase, showing its N-terminal domain is involved in RNA binding and dimerization. Our results provide a detailed picture of the CNPase active site during its catalytic cycle, and suggest a specific function for the previously uncharacterized N-terminal domain.
    • Preliminary crystallographic analysis of the N-terminal PDZ-like domain of periaxin, an abundant peripheral nerve protein linked to human neuropathies.

      Han, Huijong; Kursula, Petri (2013-07)
      Periaxin (PRX) is an abundant protein in peripheral nerves and contains a predicted PDZ-like domain at its N-terminus. The large isoform, L-PRX, is required for the maintenance of myelin in the peripheral nervous system and its defects cause neurological disease. Here, the human periaxin PDZ-like domain was crystallized and X-ray diffraction data were collected to 2.85 Å resolution using synchrotron radiation. The crystal belonged to the primitive hexagonal space group P3121 or P3221, with unit-cell parameters a = b = 80.6, c = 81.0 Å, γ = 120° and either two or three molecules in the asymmetric unit. The structure of PRX will shed light on its poorly characterized function in the nervous system.
    • Purification, crystallization and preliminary X-ray crystallographic analysis of MIL, a glycosylated jacalin-related lectin from mulberry (Morus indica) latex.

      Patel, Ashok K; Singh, Vijay K; Bergmann, Ulrich; Jagannadham, Medicherla V; Kursula, Petri (2011-05-01)
      A quantitatively major protein has been purified from the latex of Morus indica. The purified previously uncharacterized protein, M. indica lectin (MIL), was further shown to be a glycosylated tetramer and belongs to the family of jacalin-related lectins. Crystallization of MIL was also accomplished and the tetragonal crystals diffracted synchrotron X-rays to a resolution of 2.8 Å.
    • Structure of the dimeric autoinhibited conformation of DAPK2, a pro-apoptotic protein kinase.

      Patel, Ashok K; Yadav, Ravi P; Majava, Viivi; Kursula, Inari; Kursula, Petri (2011-06-10)
      The death-associated protein kinase (DAPK) family has been characterized as a group of pro-apoptotic serine/threonine kinases that share specific structural features in their catalytic kinase domain. Two of the DAPK family members, DAPK1 and DAPK2, are calmodulin-dependent protein kinases that are regulated by oligomerization, calmodulin binding, and autophosphorylation. In this study, we have determined the crystal and solution structures of murine DAPK2 in the presence of the autoinhibitory domain, with and without bound nucleotides in the active site. The crystal structure shows dimers of DAPK2 in a conformation that is not permissible for protein substrate binding. Two different conformations were seen in the active site upon the introduction of nucleotide ligands. The monomeric and dimeric forms of DAPK2 were further analyzed for solution structure, and the results indicate that the dimers of DAPK2 are indeed formed through the association of two apposed catalytic domains, as seen in the crystal structure. The structures can be further used to build a model for DAPK2 autophosphorylation and to compare with closely related kinases, of which especially DAPK1 is an actively studied drug target. Our structures also provide a model for both homodimerization and heterodimerization of the catalytic domain between members of the DAPK family. The fingerprint of the DAPK family, the basic loop, plays a central role in the dimerization of the kinase domain.