ABSTRACT
Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific Tâ cell responses.
Subject(s)
Azo Compounds/chemistry , HLA-A Antigens/chemistry , Peptides/chemistry , Amino Acid Sequence , Azo Compounds/immunology , Dithionite/chemistry , Epitopes/chemistry , Epitopes/immunology , HLA-A Antigens/immunology , Humans , Ligands , Models, Molecular , Peptides/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/immunologyABSTRACT
FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ) domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases. We have used NMR spectroscopy to determine the solution structure of this domain, revealing that the domain shares close structural similarity with SET domains. Titration with S-adenosyl-L-homocysteine, the cofactor product synonymous with SET domain methyltransferase activity, indicated that the FOG PR domain is not, however, likely to function as a methyltransferase in the same fashion. We also sought to define the function of this domain using both pulldown experiments and gel shift assays. However, neither pulldowns from mammalian nuclear extracts nor yeast two-hybrid assays reproducibly revealed binding partners, and we were unable to detect nucleic-acid-binding activity in this domain using our high-diversity Pentaprobe oligonucleotides. Overall, our data demonstrate that FOG1 is a member of the PRDM (PR domain containing proteins, with zinc fingers) family of transcriptional regulators. The function of many PR domains, however, remains somewhat enigmatic for the time being.
Subject(s)
Nuclear Proteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Animals , Cell Line, Tumor , Conserved Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Mice , Models, Molecular , Molecular Sequence Data , Nuclear Proteins/genetics , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , S-Adenosylhomocysteine/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Transcription Factors/geneticsABSTRACT
Chromatin-modifying complexes such as the NuRD complex are recruited to particular genomic sites by gene-specific nuclear factors. Overall, however, little is known about the molecular basis for these interactions. Here, we present the 1.9 Å resolution crystal structure of the NuRD subunit RbAp48 bound to the 15 N-terminal amino acids of the GATA-1 cofactor FOG-1. The FOG-1 peptide contacts a negatively charged binding pocket on top of the RbAp48 ß-propeller that is distinct from the binding surface used by RpAp48 to contact histone H4. We further show that RbAp48 interacts with the NuRD subunit MTA-1 via a surface that is distinct from its FOG-binding pocket, providing a first glimpse into the way in which NuRD assembly facilitates interactions with cofactors. Our RbAp48·FOG-1 structure provides insight into the molecular determinants of FOG-1-dependent association with the NuRD complex and into the links between transcription regulation and nucleosome remodeling.
Subject(s)
Histone Deacetylases , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Nuclear Proteins , Repressor Proteins , Retinoblastoma-Binding Protein 4 , Transcription Factors , Transcription, Genetic/physiology , Amino Acid Sequence , Animals , Binding Sites/physiology , Cells, Cultured , Conserved Sequence , Crystallography, X-Ray , Histone Deacetylases/chemistry , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histones/chemistry , Histones/genetics , Histones/metabolism , Mi-2 Nucleosome Remodeling and Deacetylase Complex/chemistry , Mi-2 Nucleosome Remodeling and Deacetylase Complex/genetics , Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Interaction Domains and Motifs/physiology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism , Retinoblastoma-Binding Protein 4/chemistry , Retinoblastoma-Binding Protein 4/genetics , Retinoblastoma-Binding Protein 4/metabolism , Spodoptera , Trans-Activators , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
MED-1 is a member of a group of divergent GATA-type zinc finger proteins recently identified in several species of Caenorhabditis. The med genes are transcriptional regulators that are involved in the specification of the mesoderm and endoderm precursor cells in nematodes. Unlike other GATA-type zinc fingers that recognize the consensus sequence (A/C/T)GATA(A/G), the MED-1 zinc finger (MED1zf) binds the larger and atypical site GTATACT(T/C)(3). We have examined the basis for this unusual DNA specificity using a range of biochemical and biophysical approaches. Most strikingly, we show that although the core of the MED1zf structure is similar to that of GATA-1, the basic tail C-terminal to the zinc finger unexpectedly adopts an alpha-helical structure upon binding DNA. This additional helix appears to contact the major groove of the DNA, making contacts that explain the extended DNA consensus sequence observed for MED1zf. Our data expand the versatility of DNA recognition by GATA-type zinc fingers and perhaps shed new light on the DNA-binding properties of mammalian GATA factors.
