Mass spectrometry for the study of protein-protein interactions.

The identification of subpicomolar amounts of protein by mass spectrometry (MS) coupled with two-dimensional methods to separate complex protein mixtures is fueling the field of proteomics, and making feasible the notion of cataloging and comparing all of the expressed proteins in a biological sample. Functional proteomics is a complementary effort aimed at the characterization of functional features of proteins, such as their interactions with other proteins. Proteins comprise modular domains, many of which are noncatalytic modules that direct protein-protein interactions. Capturing proteins of interest and their interacting proteins by using high-affinity antibodies presents a simple method to prepare relatively simple protein mixtures easily resolved in one-dimensional formats. Individual or mixtures of proteins identified as stained bands in polyacrylamide gels are subjected to in situ digestion with the protease trypsin, and the extracted peptide fragments are analyzed by MS. The quality, quantity, and complexity of the tryptic digest, the species origin of the proteins, and the quality of the corresponding databases of genomic and protein information greatly influence the subsequent MS analysis in terms of degree of difficulty and methodological approach required to make an unambiguous protein identification. In this article we report the isolation of associated proteins from a complex cell-derived lysate by using an epitope-directed antibody. The protein pICLn engineered to carry an epitope tag was purified from cultured human embryonic kidney cells, and found to associate with a variety of proteins including the spliceosomal proteins smE and smG. By application of this general approach, the systematic identification of protein complexes and assignment of protein function are possible.

CIA, a novel estrogen receptor coactivator with a bifunctional nuclear receptor interacting determinant.

Coregulators for nuclear receptors (NR) are factors that either enhance or repress their transcriptional activity. Both coactivators and corepressors have been shown to use similar but functionally distinct NR interacting determinants containing the core motifs LxxLL and PhixxPhiPhi, respectively. These interactions occur through a hydrophobic cleft located on the surface of the ligand-binding domain (LBD) of the NR and are regulated by ligand-dependent activation function 2 (AF-2). In an effort to identify novel coregulators that function independently of AF-2, we used the LBD of the orphan receptor RVR (which lacks AF-2) as bait in a yeast two-hybrid screen. This strategy led to the cloning of a nuclear protein referred to as CIA (coactivator independent of AF-2 function) that possesses both repressor and activator functions. Strikingly, we observed that CIA not only interacts with RVR and Rev-ErbAalpha in a ligand-independent manner but can also form complexes with estrogen receptor alpha (ERalpha) and ERbeta in vitro and enhances ERalpha transcriptional activity in the presence of estradiol (E(2)). CIA-ERalpha interactions were found to be independent of AF-2 and enhanced by the antiestrogens EM-652 and ICI 182,780 but not by 4-hydroxytamoxifen and raloxifene. We further demonstrate that CIA-ERalpha interactions require the presence within CIA of a novel bifunctional NR recognition determinant containing overlapping LxxLL and PhixxPhiPhi motifs. The identification and functional characterization of CIA suggest that hormone binding can create a functional coactivator interaction interface in the absence of AF-2.

Mutations in an Abf1p binding site in the promoter of yeast RPO26 shift the transcription start sites and reduce the level of RPO26 mRNA.

A binding site for the transcription factor Abf1p was identified as an important promoter element of the gene that encodes Rpo26, a subunit common to all three yeast nuclear RNA polymerases (RNAP). Mutations in the Abf1p binding site were identified among a pool of rpo26 mutant alleles that confer synthetic lethality in combination with a temperature-sensitive mutation (rpo21-4) in the gene that encodes the largest subunit of RNAPII (Rpo21p). In the presence of the wild-type allele of RPO21 these rpo26 promoter mutations confer a cold-sensitive growth defect. Electrophoretic mobility-shift assays using purified Abf1p demonstrated that Abf1p binds to the RPO26 promoter and that the promoter mutations abolish this binding in vitro. Quantitation of the amount of RPO26 mRNA showed that mutations in the Abf1p binding site reduce the expression of RPO26 by approximately 60%. Mutations that affect Abf1p binding also result in a shift of the RPO26 transcriptional start sites to positions further upstream than normal. These results suggest that binding of the Abf1p transcription factor to the RPO26 promoter is important not only in establishing the level of transcription for this gene, but also in positioning the initiation sites of transcription.

Functional analysis of the ABF1-binding sites within the Ya regions of the MATa and HMRa loci of Saccharomyces cerevisiae.

Cell type in the yeast Saccharomyces cerevisiae is determined by information present at the MAT locus. Cells can switch mating types when cell-type information located at a silent locus, HML or HMR, is transposed to the MAT locus. The HML and HMR loci are kept silent through the action of a number of proteins, one of which is the DNA-binding protein, ABF1. We have identified a binding site for ABF1 within the Ya region of MATa and HMRa. In order to examine the function of this ABF1-binding site, we have constructed strains that lack the site in the MATa or HMRa loci. Consistent with the idea that ABF1 plays a redundant role in silencing, it was found that a triple deletion of the ABF1-binding sites at HMRE, Ya and I did not permit the expression of HMRa. We have also shown that chromosomal deletion of the binding site at MATYa had no effect on the level of cutting by the HO endonuclease nor on the amount of mating-type switching observed. Similarly, chromosomal deletion of all three ABF1-binding sites at HMRa had no effect on the directionality of mating-type switching.

Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor.

The ROR alpha isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct amino-terminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR alpha 1 isoform and have now identified the minimal sequence both in its amino- and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.

The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions.

ROR alpha 1 and ROR alpha 2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR alpha is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.

Contacts of the ABF1 protein of Saccharomyces cerevisiae with a DNA binding site at MATa.

The ABF1 protein of Saccharomyces cerevisiae is a multifunctional DNA-binding protein that is required for cell viability. The ABF1 protein has previously been shown to bind to a number of yeast sequences having a consensus of: 5'-A/G TC A/G C/T C/T NNNNACG-3'. A heretofore undiscovered ABF1-binding site was found in the MATa region. We have used missing contact analysis of this ABF1-binding site to show that removal of the conserved bases, as well as of some bases which are not conserved, reduces binding. We have probed contacts of ABF1 with the DNA-binding site using dimethyl sulfate and potassium permanganate and find that the protein makes extensive contacts with both the major and minor grooves. Ethylation interference studies indicate that numerous phosphate contacts are also important for ABF1 binding. Interference studies indicate that the ABF1 protein is also in close proximity to the DNA that is 5' and 3' of the conserved bases of the binding site. The extensive DNA contacts exhibited by ABF1 may play a role in the protein-induced bending of the DNA target (McBroom, L. D. B., and Sadowski, P. D. (1994) J. Biol. Chem. 169, 16461-16468).

DNA bending by Saccharomyces cerevisiae ABF1 and its proteolytic fragments.

The ABF1 protein of the yeast Saccharomyces cerevisiae has been found to bend the DNA containing the target site for DNA binding. A bend angle of about 120 degrees was measured and the bend center was 7 base pairs to the 5' end of the ABF1 consensus-containing sequence. Phasing analysis showed that intact ABF1 bends the DNA towards the minor groove. We have subjected ABF1 to partial proteolysis and have found that proteolytic fragments were able to bind to the DNA-binding site and induce partial bends in the DNA. Interestingly, the locations of the bend centers, the bend angles, and the plane of the bends induced by the proteolytic peptides of ABF1 were different from those of the intact protein. We present a model for the mechanism of bending of DNA by ABF1.