In the past several years, our work has concentrated in several distinct areas. Crystallographic studies of proteases Crystallographic studies of proteases have been an important area of research of this Section since its establishment. We have been particularly active in the investigation of structure-function relationship in aspartic proteases, including clinically important retroviral enzymes. Our studies of HIV protease, although no longer a major target of active research, are still ongoing and concentrate on the investigation of drug-resistant variants and their complexes with inhibitors. We have investigated retroviral proteases from several other sources such as FIV, RSV, HTLV-1, and, most recently, XMRV. A number of inhibitor complexes of HTLV-1 PR have been analyzed, with the aim of assisting in the development of drugs against HTLV-caused leukemia. XMRV protease was found to share properties of both dimeric retroviral aspartic protease, as well as monomeric pepsin-like enzymes. Complexes of XMRV PR with several inhibitors, including an AIDS drug, have been solved and analyzed. Cockroach allergen Bla g 2 was shown to be an inactive aspartic protease and we solved the structures of two complexes with different specific antibodies. We have also been investigating a bacterial ATP-dependent protease Lon, finding that is proteolytic domain has a unique fold and thus establishes a new family of proteases with a Ser-Lys catalytic dyad. The structures of two plasmepsins, PL-1 and HAP, including their complexes with inhibitors, provided information useful for design of anti-malarial drugs. Lectins with antiviral activity We have been involved in studies of several lectins with antiviral activities, some of them currently being developed in pre-clinical trials as potential drugs preventing HIV infection. We have solved the structure of griffithsin, as free protein and complexed with a number of mono- and disaccharides, explaining the structural basis for its tight binding to branched mannose-rich carbohydrates. We have reengineered griffithsin into a monomeric form and solved its structure with a complex oligosaccharide, elucidating the basis of its antiviral properties. We have also solved atomic-resolution structure of another lectin, scytovirin, and conducted extensive studies aimed at elucidating its disulfide patterns. Cytokines and cytokine receptors Our Section has been investigating the crystal structures of several cytokines and has made progress in preparing their receptor complexes. We have purified and crystallized complexes of IL-10 with its specific receptor and are studying complexes of several other cytokines related to IL-10, such as IL-19, IL-20, and IL-22. We have solved the structure of interferon lambda-1 complexed with its receptor, finding considerable differences in the receptor-ligand interactions between different family members. Crystallographic studies of the Taz2 domain of p300 and its interactions with p53 and C/EBP transcription factors. CBP and its paralogue p300 are histone acetyl transferases that regulate gene expression by interacting with multiple transcription factors via specialized domains. We determined the crystal structure of a segment of human p300 protein (residues 1723-1836) corresponding to the extended zinc-binding Taz2 domain. The crystal structure was solved utilizing an anomalous diffraction signal of the bound Zn ions. The structure comprises an atypical helical bundle stabilized by three Zn ions and closely resembles the solution structures determined previously for shorter peptides. Residues 1813-1834 from this construct form a helical extension of the C-terminal helix and make extensive crystal contact interactions with the peptide binding site of Taz2, providing additional insights into the mechanism of recognition of diverse transactivation domains (TADs) by Taz2. Based on the analysis of these contacts and molecular modeling we proposed a hypothetical model of the binding of phosphorylated p53 to Taz2. Currently, we use the crystal contact interactions to investigate Taz2 binding to C/EBP proteins. A chimera protein, Taz2(1723-1818)C/EBPbeta(37-61), where 25 C-terminal residues from the initial Taz2 construct were replaced by amino acids corresponding to the minimal TAD from the C/EBPbeta transcription factor was produced by the Protein Purification Core of MCL. The protein crystallized in the P6(5) group and X-Ray diffraction data extending to 1.5 resolution were collected at synchrotron SER-CAT beamline at Argonne. The segment of the human C/EBPbeta protein (residues 37-60) adopts helix-turn-helix conformation and interacts with the core structure of Taz2 from the symmetry related molecule. This putative C/EBPbeta binding site overlaps with the known binding site of STAT1 on Taz2.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010348-12
Application #
8348987
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2011
Total Cost
$1,593,257
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Ottaiano, Tatiana F; Andrade, Sheila S; de Oliveira, Cleide et al. (2017) Plasma kallikrein enhances platelet aggregation response by subthreshold doses of ADP. Biochimie 135:72-81
Wlodawer, Alexander (2017) Stereochemistry and Validation of Macromolecular Structures. Methods Mol Biol 1607:595-610
Wlodawer, Alexander; Dauter, Zbigniew (2017) `Atomic resolution': a badly abused term in structural biology. Acta Crystallogr D Struct Biol 73:379-380
Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek (2016) Crystal Structure of a Complex of the Intracellular Domain of Interferon ? Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1. J Mol Biol 428:4651-4668
Woodfolk, Judith A; Glesner, Jill; Wright, Paul W et al. (2016) Antigenic Determinants of the Bilobal Cockroach Allergen Bla g 2. J Biol Chem 291:2288-301
Dauter, Zbigniew; Wlodawer, Alexander (2016) Progress in protein crystallography. Protein Pept Lett 23:201-10
Raczynska, Joanna E; Wlodawer, Alexander; Jaskolski, Mariusz (2016) Prior knowledge or freedom of interpretation? A critical look at a recently published classification of ""novel"" Zn binding sites. Proteins 84:770-6
Lubkowski, Jacek; Durbin, Sarah V; C Silva, Mariana C et al. (2016) Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth. FEBS J :
Leal, Ana Rita; Cruz, Rui; Bur, Daniel et al. (2016) Enzymatic properties, evidence for in vivo expression, and intracellular localization of shewasin D, the pepsin homolog from Shewanella denitrificans. Sci Rep 6:23869
Wlodawer, Alexander (2016) Serendipity, politics, and crystal structures, or how I became a protein crystallographer. Postepy Biochem 62:239-241

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