10 research outputs found
Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition
c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIHāthe FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control
Structural Interactions Dictate the Kinetics of Macrophage Migration Inhibitory Factor Inhibition by Different Cancer-Preventive Isothiocyanates
Regulation of cellular processes by dietary nutrients
is known
to affect the likelihood of cancer development. One class of cancer-preventive
nutrients, isothiocyanates (ITCs), derived from the consumption of
cruciferous vegetables, is known to have various effects on cellular
biochemistry. One target of ITCs is macrophage migration inhibitory
factor (MIF), a widely expressed protein with known inflammatory,
pro-tumorigenic, pro-angiogenic, and anti-apoptotic properties. MIF
is covalently inhibited by a variety of ITCs, which in part may explain
how they exert their cancer-preventive effects. We report the crystallographic
structures of human MIF bound to phenethylisothiocyanate and to l-sulforaphane (dietary isothiocyanates derived from watercress
and broccoli, respectively) and correlate structural features of these
two isothiocyanates with their second-order rate constants for MIF
inactivation. We also characterize changes in the MIF structure using
nuclear magnetic resonance heteronuclear single-quantum coherence
spectra of these complexes and observe many changes at the subunit
interface. While a number of chemical shifts do not change, many of
those that change do not have features similar in magnitude or direction
for the two isothiocyanates. The difference in the binding modes of
these two ITCs provides a means of using structureāactivity
relationships to reveal insights into MIF biological interactions.
The results of this study provide a framework for the development
of therapeutics that target MIF
Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.
Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 Ć
crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites
RCSB Protein Data bank: Tools for visualizing and understanding biological macromolecules in 3D
Now in its 52nd year of continuous operations, the Protein Data Bank (PDB) is the premiere open-access global archive housing three-dimensional (3D) biomolecular structure data. It is jointly managed by the Worldwide Protein Data Bank (wwPDB) partnership. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) is funded by the National Science Foundation, National Institutes of Health, and US Department of Energy and serves as the US data center for the wwPDB. RCSB PDB is also responsible for the security of PDB data in its role as wwPDB-designated Archive Keeper. Every year, RCSB PDB serves tens of thousands of depositors of 3D macromolecular structure data (coming from macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro-electron diffraction). The RCSB PDB research-focused web portal (RCSB.org) makes PDB data available at no charge and without usage restrictions to many millions of PDB data consumers around the world. The RCSB PDB training, outreach, and education web portal (PDB101.RCSB.org) serves nearly 700āK educators, students, and members of the public worldwide. This invited Tools Issue contribution describes how RCSB PDB (i) is organized; (ii) works with wwPDB partners to process new depositions; (iii) serves as the wwPDB-designated Archive Keeper; (iv) enables exploration and 3D visualization of PDB data via RCSB.org; and (v) supports training, outreach, and education via PDB101.RCSB.org. New tools and features at RCSB.org are presented using examples drawn from high-resolution structural studies of proteins relevant to treatment of human cancers by targeting immune checkpoints