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. The structures of two plasmepsins, PL-1 and HAP, including their complexes with inhibitors, provided information useful for design of anti-malarial drugs.Lectins and antibodies for the prevention of AIDSWe 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. A number of constructs of griffithsin containing tandem repeats of multiple monomers were prepared and shown to increase their antiviral activity. We have also solved atomic-resolution structure of another lectin, scytovirin, and conducted extensive studies aimed at elucidating its disulfide patterns. We have solved structures of Fab constructs of antibodies directed against HIV-1 gp41 complexed with different gp41 mimics. These structures are being interpreted in order to find why some closely related antibodies are neutralizing, while others are not.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 transcription factorsTranscriptional activation by C/EBP proteins relies on recruitment of the histone acetyl transferases (HATs) CBP/p300 to the promoter/enhancer region. Members of the C/EBP family bind to the Taz2 domain of p300/CBP and trigger p300/CBP phosphorylation. Two short sequence motifs, homology boxes A and B, which are conserved between C/EBP activators and comprise their minimal TADs, are necessary for p300/CBP binding. In order to gain insights into C/EBP:p300/CBP interactions we initiated x-ray crystallographic studies of the p300 Taz2 complexed to various peptide ligands. We determined the crystal structure of a segment of the human p300 protein (residues 1723-1836) corresponding to the extended zinc-binding Taz2 domain. 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 on Taz2. Based on the analysis of crystal contacts and molecular modeling, we proposed a hypothetical model of the binding of phosphorylated p53 to Taz2. Subsequently, the interactions between the core TAD of C/EBPepsilon and human p300 Taz2 were determined from the crystal structure (PDB ID:3T92) of a chimeric protein composed of p300 Taz2 (residues 1723-1818) and C/EBPepsilon (residues 37-61). The segment corresponding to the C/EBPepsilon TAD forms two helices connected by a short linker and interacts with the core structure of Taz2 from the symmetry-related molecule. The C/EBPepsilon binding site on Taz2 overlaps with the known binding site of STAT1, suggesting competition between the members of the two families. To confirm that we found the specific binding site, we showed that structure-guided mutations in Taz2 diminished tenfold the affinity of interaction with synthetic peptide encompassing C/EBP/epsilon minimal TAD. We postulate that other members of the C/EBP family interact with the Taz2 domain in the same manner. We also propose a possible structural framework for C/EBPbeta-dependent phosphorylation of the p300 C-terminus by protein kinase HIPK2 and suggest that phosphorylation of the p300/CBP C terminus may be executed by other proline-directed kinases which dock to sites N-terminal to homology box A of C/EBP proteins. This study thus provides structural insights important for understanding the molecular mechanisms underlying the role of C/EBP proteins in chromatin remodeling and transcriptional regulation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010348-13
Application #
8552677
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2012
Total Cost
$1,577,338
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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