Tadashi Tomizawa

Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left–right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1–4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin α1/α6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS.

NMR solution structures of actin depolymerizing factor homology domains

Actin is one of the most conserved proteins in nature. Its assembly and disassembly are regulated by many proteins, including the family of actin‐depolymerizing factor homology (ADF‐H) domains. ADF‐H domains can be divided into five classes: ADF/cofilin, glia maturation factor (GMF), coactosin, twinfilin, and Abp1/drebrin. The best‐characterized class is ADF/cofilin. The other four classes have drawn much less attention and very few structures have been reported. This study presents the solution NMR structure of the ADF‐H domain of human HIP‐55‐drebrin‐like protein, the first published structure of a drebrin‐like domain (mammalian), and the first published structure of GMF β (mouse). We also determined the structures of mouse GMF γ, the mouse coactosin‐like domain and the C‐terminal ADF‐H domain of mouse twinfilin 1. Although the overall fold of the five domains is similar, some significant differences provide valuable insights into filamentous actin (F‐actin) and globular actin (G‐actin) binding, including the identification of binding residues on the long central helix. This long helix is stabilized by three or four residues. Notably, the F‐actin binding sites of mouse GMF β and GMF γ contain two additional β‐strands not seen in other ADF‐H structures. The G‐actin binding site of the ADF‐H domain of human HIP‐55‐drebrin‐like protein is absent and distorted in mouse GMF β and GMF γ.

Solution structure of an atypical WW domain in a novel β-clam-like dimeric form

The WW domain is known as one of the smallest protein modules with a triple‐stranded β‐sheet fold. Here, we present the solution structure of the second WW domain from the mouse salvador homolog 1 protein. This WW domain forms a homodimer with a β‐clam‐like motif, as evidenced by size exclusion chromatography, analytical ultracentrifugation and NMR spectroscopy. While typical WW domains are believed to function as monomeric modules that recognize proline‐rich sequences, by using conserved aromatic and hydrophobic residues that are solvent‐exposed on the surface of the β‐sheet, this WW domain buries these residues in the dimer interface.

Structure of the C-terminal phosphotyrosine interaction domain of Fe65L1 complexed with the cytoplasmic tail of amyloid precursor protein reveals a novel peptide binding mode

Fe65L1, a member of the Fe65 family, is an adaptor protein that interacts with the cytoplasmic domain of Alzheimer amyloid precursor protein (APP) through its C-terminal phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domain. In the present study, the solution structures of the C-terminal PID domain of mouse Fe65L1, alone and in complex with a 32-mer peptide (DAAVTPEERHLSKMQQNGYENPTYKFFEQMQN) derived from the cytoplasmic domain of APP, were determined using NMR spectroscopy. The C-terminal PID domain of Fe65L1 alone exhibits a canonical PID/PTB fold, whereas the complex structure reveals a novel mode of peptide binding. In the complex structure, the NPTY motif forms a type-I β-turn, and the residues immediately N-terminal to the NPTY motif form an antiparallel β-sheet with the β5 strand of the PID domain, the binding mode typically observed in the PID/PTB·peptide complex. On the other hand, the N-terminal region of the peptide forms a 2.5-turn α-helix and interacts extensively with the C-terminal α-helix and the peripheral regions of the PID domain, representing a novel mode of peptide binding that has not been reported previously for the PID/PTB·peptide complex. The indispensability of the N-terminal region of the peptide for the high affinity of the PID-peptide interaction is consistent with NMR titration and isothermal calorimetry data. The extensive binding features of the PID domain of Fe65L1 with the cytoplasmic domain of APP provide a framework for further understanding of the function, trafficking, and processing of APP modulated by adapter proteins.

Letter to the Editor: NMR assignment of the hypothetical ENTH-VHS domain At3g16270 from Arabidopsis thaliana

Blanca Lopez-Mendeza, David Pantoja-Ucedaa, Tadashi Tomizawaa, Seizo Koshibaa, Takanori Kigawaa, Mikako Shirouzua,b, Takaho Teradaa,b, Makoto Inouea, Takashi Yabukia, Masaaki Aokia, Eiko Sekia, Takayoshi Matsudaa, Hiroshi Hirotaa, Mayumi Yoshidaa, Akiko Tanakaa, Takashi Osanaia, Motoaki Sekia, Kazuo Shinozakia, Shigeyuki Yokoyamaa,b,c & Peter Gunterta,∗ aRIKEN Genomic Sciences Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan; bRIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki, Sayo, Hyogo 679-5148, Japan; cDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan

ZF21 Protein, a Regulator of the Disassembly of Focal Adhesions and Cancer Metastasis, Contains a Novel Noncanonical Pleckstrin Homology Domain

Directional migration of adherent cells on an extracellular matrix requires repeated formation and disassembly of focal adhesions (FAs). Directional migration of adherent cellsWe have identified ZF21 as a regulator of disassembly of FAs and cell migration, and increased expression of the gene has been linked to metastatic colon cancer. ZF21 is a member of a protein family characterized by the presence of the FYVE domain, which is conserved among Fab1p, YOPB, Vps27p, and EEA1 proteins, and has been shown to mediate the binding of such proteins to phosphoinositides in the lipid layers of cell membranes. ZF21 binds multiple factors that promote disassembly of FAs such as FAK, β-tubulin, m-calpain, and SHP-2. ZF21 does not contain any other known protein motifs other than the FYVE domain, but a region of the protein C-terminal to the FYVE domain is sufficient to mediate binding to β-tubulin. In this study, we demonstrate that the C-terminal region is important for the ability of ZF21 to induce disassembly of FAs and cell migration, and to promote an early step of experimental metastasis to the lung in mice. In light of the importance of the C-terminal region, we analyzed its ternary structure using NMR spectroscopy. We demonstrate that this region exhibits a structure similar to that of a canonical pleckstrin homology domain, but that it lacks a positively charged interface to bind phosphatidylinositol phosphate. Thus, ZF21 contains a novel noncanonical PH-like domain that is a possible target to develop a therapeutic strategy to treat metastatic cancer.

Solution structure of the C-terminal DUF1000 domain of the human thioredoxin-like 1 protein.

Solution structure of the C-terminal DUF1000 domain of the human thioredoxin-like 1 protein Alexander K. Goroncy, Seizo Koshiba, Naoya Tochio, Tadashi Tomizawa, Makoto Inoue, Akiko Tanaka, Sumio Sugano, Takanori Kigawa, and Shigeyuki Yokoyama* 1 RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan 2Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan 3Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology,

The NMR solution structures of the five constituent cold-shock domains (CSD) of the human UNR (upstream of N-ras) protein.

Upon cold shock, the amounts of most proteins dramatically decrease from normal levels, but those of cold shock proteins (CSPs) and proteins containing cold-shock domains (CSDs) greatly increase. Although their biological function is still not completely clear, cold-shock proteins might control translation via RNA chaperoning. Many cold-shock proteins contain the motifs (Y/F)GFI and (V/F)(V/F)H, which are known as ribonucleoprotein (RNP)-1 and RNP-2 motifs implicated in RNA/DNA binding. We determined the solution NMR structures of all five constituent CSDs of the human UNR (upstream of N-ras) protein. The spatial arrangements of the sidechains in the RNP-1 and RNP-2 motifs are mostly conserved; however, the conformations of the following residues in the first CSD are different: F43 and H45 (the first phenylalanine residue and the histidine residue in the putative binding site RNP-2) and Y30 (the first residue in the putative binding site RNP-1). F43 and H45 affect each other, and H45 is further influenced by C46. The altered binding site of the first CSD, and its putatively enhanced intrinsic stability, may provide an explanation for the observation that the first CSD has 20-fold higher RNA-binding activity than the fifth CSD. It also lends support to the hypothesis that the UNR protein arose by repeated duplication of a protein that originally contained just one CSD, and that the proto-UNR protein acquired cysteine C46 by mutation during evolution.

Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a β‐clam‐like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355–Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 × 102 M−1. We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin‐relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C‐terminal extension (Leu355–Glu401), as the formation of an extra α‐helix was predicted. An NMR structural determination confirmed the formation of an α‐helix in the extended C‐terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp‐to‐Ser mutation gained apparent stability in a protein concentration‐dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C‐terminal α‐helix stabilized the global fold by facilitating contacts between the N‐terminal linker region and the main body of the WW domain.

Structural basis for the recognition of nucleophosmin-anaplastic lymphoma kinase oncoprotein by the phosphotyrosine binding domain of Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target-2

The nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion oncoprotein, formed by the t(2;5) chromosomal translocation in anaplastic large-cell lymphomas, has constitutive tyrosine kinase activity and interacts with a number of signaling molecules. One of the interacting partners of NPM-ALK is the adaptor protein, Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT), and mutations that deprive NPM-ALK of all three of the SNT-binding sites significantly reduced the transforming activity. In this study, the interactions of the three binding sites in NPM-ALK with the phosphotyrosine binding (PTB) domain of SNT-2 were analyzed. First, by isothermal titration calorimetry, we found that the phosphorylation-independent binding site in NPM-ALK interacts with the SNT-2 PTB domain more tightly than the phosphorylation-dependent binding sites. Second, the solution structure of the SNT-2 PTB domain in complex with the nonphosphorylated NPM-ALK peptide was determined by nuclear magnetic resonance spectroscopy. The NPM-ALK peptide interacts with the hydrophobic surface of the PTB domain and intermolecularly extends the PTB β-sheet. This interaction mode is much broader and more extensive than those of the phosphorylation-dependent binding sites. Our results indicate that the higher binding activity of the phosphorylation-independent binding site is caused by additional hydrophobic interactions.