Interactions of sulfur oxidation repressor with its promoters involve different binding geometries

The divergently transcribed sulfur oxidation (sox) operon of a sulfur chemolithotrophs, Pseudaminobacter salicylatoxidans KCT001, comprising sox TRS-VW-XYZABCD, is regulated by a repressor (SoxR). SoxR binds to two disparate operators, sv (present in between soxS and soxV) and wx (present in between soxW and soxX). Here we report details of the interaction between SoxR and these two operator regions of the sox operon, using methylation interference and hydroxyl radical footprinting. We propose that the sv operator is symmetric and compact, while the wx operator is asymmetric and extended. We report an interesting difference between the SoxR-sv interaction and the SoxR-wx interaction through a competition assay involving groove-specific ligands. SoxR binds in the major groove of the sv operator, but binds in the minor groove of the wx operator. The structural flexibility of the SoxR helps it to act differentially in its interactions with these two operators. Mutational analysis shows that SoxR uses different amino acid residues when binding to the sv operator versus the wx operator. Taken together, the results indicate that interaction between SoxR and the two operator sites involves different binding geometries. This makes SoxR the only known example of a ArsR-family protein that binds differentially to different operators.     

Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons

Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human. The central insight utilized is that functional contact residues would be conserved among pairs of orthologous receptors, but considerably less conserved among paralogous pairs. Using judiciously selected subsets of 218 ortholog pairs and 518 paralog pairs, we have identified 22 sequence positions that are both highly conserved among the putative orthologs and variable among paralogs. These residues are disposed on transmembrane helices 2 to 7, and on the second extracellular loop of the receptor. Strikingly, although the prediction makes no assumption about the location of the binding site, these amino acid positions are clustered around a pocket in a structural homology model of ORs, mostly facing the inner lumen. We propose that the identified positions constitute the odorant binding site. This conclusion is supported by the observation that all but one of the predicted binding site residues correspond to ligand-contact positions in other rhodopsin-like GPCRs.     

Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli

mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.     

Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit

BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of approximately 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an alpha-helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal alpha-helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional (1)H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.     

Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis

Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.