DNA binding properties of the purified Antennapedia homeodomain.

The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed. Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively. Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study. The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones. The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared.     

Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay.     

The des(1-6)antennapedia homeodomain: comparison of the NMR solution structure and the DNA-binding affinity with the intact Antennapedia homeodomain.

The nuclear magnetic resonance (NMR) solution structure of an N-terminally truncated mutant Antennapedia homeodomain, des(1-6)Antp(C39S), has been determined from 935 nuclear Overhauser effect upper distance constraints and 148 dihedral angle constraints by using the programs DIANA and OPAL. Twenty conformers representing the solution structure of des(1-6)Antp(C39S) have an average root-mean-square distance relative to the mean coordinates of 0.56 A for the backbone atoms of residues 8-59. Comparison with the intact Antp(C39S) homeodomain shows that the two proteins have identical molecular architectures. The removal of the N-terminal residues 1-6, which are flexibly disordered in the intact homeodomain, causes only strictly localized structure variations and does not noticeably affect the adjoining helix I from residues 10-21. The DNA-binding constant of des(1-6)Antp(C39S) is approximately 10-fold reduced relative to the intact Antp(C39S) homeodomain, which can now be attributed to the absence of the previously reported contacts of the N-terminal polypeptide segment of the intact Antp(C39S) homeodomain with the minor groove of the DNA duplex.     

DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos.

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.     

Theories of quasi-linkage and "affinity": some implications for population structure.

"Quasi-linkage" refers to nonrandom assortment of genes located on different chromosomes. Although this phenomenon has been widely observed in many organisms since the early part of this century, it is barely known. Interest in it was recently rekindled by the report of an association between quasi-linkage and the expression of genes belonging to a group of cancer viruses whose genomes are integrated in mouse chromosomes. This prompted an examination of the question whether "sustained meiotic affinity," which is one of the explanations proposed for quasi-linkage, can influence population structure in a manner unattainable by other known modes of heredity. It is shown for a two-locus two-allele system that equilibrium is attained with the gametic phase disequilibrium D greater than 0, leading to a permanent excess of the preferred genotypes. The possible relationship of these concepts to the inheritance of susceptibility to cancer and other diseases is discussed.     

Sequence-specific DNA binding by the vnd/NK-2 homeodomain of Drosophila

The ventral nervous system defective (vnd)/NK-2 homeodomain and some flanking amino acid residues were expressed in Escherichia coli, purified to homogeneity, and the protein was covalently coupled to Sepharose. Oligodeoxynucleotides that contained 16-bp random sequences were purified by vnd/NK-2 affinity column chromatography, cloned, and sequenced. The consensus nucleotide sequence of the vnd/NK-2 homeodomain binding site was shown to be T(T/C)AAGTG(G/C). The apparent equilibrium dissociation constant (K(D)) of the vnd/NK-2 homeodomain for the consensus sequence is 1.9 x 10(-10) M. In addition, results of competition between oligodeoxynucleotides for binding to the vnd/NK-2 homeodomain and determination of the apparent K(D) values of oligodeoxynucleotides that differ from the consensus sequence by only a single base pair demonstrate that the four central nucleotides, AAGT, in this sequence play a major role in determining the affinity of binding.     

DNA sequence recognition in the minor groove by crosslinked polyamides: The effect of N-terminal head group and linker length on binding affinity and specificity

Development of sequence-reading polyamides or "lexitropsins" with comparable DNA-binding affinities to cellular proteins raises the possibility of artificially regulated gene expression. Covalent linkage of polyamide ligands, with either a hairpin motif or crosslinking methylene bridge, has greatly improved binding affinity by ensuring their side-by-side register. Whereas hairpin polyamides have been investigated extensively, the optimized structure of crosslinked polyamides remains to be determined. This study examines a series of thiazole-imidazole-pyrrole (TIP) monomers and crosslinked dimers to evaluate the effects on selectivity and binding affinity of different N-terminal head groups attached to the leading thiazole ring and differing methylene linker lengths. Quantitative footprinting of a DNA sequence, containing potential match and mismatch sites for both maximum overlap and one-residue stagger binding modes, allowed measurement of binding constants at each putative site. Within an N-terminal amino TIP series, C7 and C8-linked compounds bound most strongly to these sites, whereas maximum binding affinity was observed for a C6 linker with a formyl head group. A C5 linker gave weak binding with either head group. A hydrogen or acetyl head group abrogated binding. Binding was confirmed by gel shift analyses. The highest specificity for the maximum overlap site over the one-residue stagger was observed with TIP-C7-amino. Selectivity of the leading thiazole was modulated by the head group, with N-terminal formyl TIP exhibiting up to 3-fold specificity for AGT over TGT, suggesting that N-formyl-thiazole may provide sequence discrimination of adenine over thymine. Moreover, the leading head group and methylene linker length significantly influences the binding characteristics of crosslinked polyamides.     

Sequence-specific DNA binding of individual cut repeats of the human CCAAT displacement/cut homeodomain protein.

CCAAT displacement protein (CDP), a nuclear protein of 180-190 kDa, contains a triplicated motif, the cut domain, similar (80-90% conserved) to three repeats of 60-65 amino acids first identified in Drosophila cut, a homeo-domain protein involved in cell-fate decisions in development. Cut repeats bind DNA and exhibit subtle differences in target-site recognition. DNA sequences specifically bound by cut repeats were isolated by PCR-mediated DNA target-site selection. Sequences selected for cut repeat 2 and 3 (CR2 and CR3) binding are A+T-rich and favor an ATA motif with similar, but not identical, flanking base preferences. CR2 and CR3 discriminate among similar target sequences. CR1, which is more divergent from CR2 and CR3, displays the most restricted pattern of DNA sequence recognition. Methylation interference analysis demonstrates different protein-DNA contacts for CR1 and CR3 binding to a target sequence. Thus, CDP/cut is a complex protein whose DNA-binding properties reflect the combinatorial interaction of four domains (three cut repeats and one homeodomain) with target DNA sequences.     

DNA binding specificity of the Arc and Mnt repressors is determined by a short region of N-terminal residues.

The Arc and Mnt repressors of phage P22 are related proteins that bind to different operator DNA sites. By creating a hybrid Arc-Mnt protein, we show that the binding specificity of Mnt can be switched to that of Arc by replacing six residues at the N terminus of Mnt with the corresponding nine residues from Arc.     

Human serum Paraoxonase/Arylesterase's retained hydrophobic N-terminal leader sequence associates with HDLs by binding phospholipids : apolipoprotein A-I stabilizes activity.

In serum, human paraoxonase/arylesterase (PON1) is found exclusively associated with high density lipoprotein (HDL) and contributes to its antiatherogenic properties by inhibiting low density lipoprotein (LDL) oxidation. Difficulties in purifying PON1 from apolipoprotein A-I (apoA-I) suggested that PON1's association with HDL may occur through a direct binding between these 2 proteins. An unusual property of PON1 is that the mature protein retains its hydrophobic N-terminal signal sequence. By expressing in vitro a mutant PON1 with a cleavable N-terminus, we demonstrate that PON1 associates with lipoproteins through its N-terminus by binding phospholipids directly rather than binding apoA-I. Nonetheless, apoA-I stabilized arylesterase activity more than did phospholipid alone, apoA-II, or apoE. Consequently, we studied the role of apoA-I in PON1 expression and HDL association in mice genetically deficient in apoA-I. Though present in HDL fractions at decreased levels, PON1 arylesterase activity was less stable than in control mice. Furthermore, PON1 could be competitively removed from HDL by phospholipids, suggesting that PON1's retained N-terminal peptide allows transfer of the enzyme between phospholipid surfaces. Thus, our data suggest that PON1 is stabilized by apoA-I, and its binding to HDL and physiological distribution are dependent on the direct binding of the retained hydrophobic N-terminus to phospholipids optimally presented in association with apoA-I.     

"DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites

We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.     

From the Cover: Defined spatial structure stabilizes a synthetic multispecies bacterial community

This paper shows that for microbial communities, “fences make good neighbors.” Communities of soil microorganisms perform critical functions: controlling climate, enhancing crop production, and remediation of environmental contamination. Microbial communities in the oral cavity and the gut are of high biomedical interest. Understanding and harnessing the function of these communities is difficult: artificial microbial communities in the laboratory become unstable because of “winner-takes-all” competition among species. We constructed a community of three different species of wild-type soil bacteria with syntrophic interactions using a microfluidic device to control spatial structure and chemical communication. We found that defined microscale spatial structure is both necessary and sufficient for the stable coexistence of interacting bacterial species in the synthetic community. A mathematical model describes how spatial structure can balance the competition and positive interactions within the community, even when the rates of production and consumption of nutrients by species are mismatched, by exploiting nonlinearities of these processes. These findings provide experimental and modeling evidence for a class of communities that require microscale spatial structure for stability, and these results predict that controlling spatial structure may enable harnessing the function of natural and synthetic multispecies communities in the laboratory.     

Hormone affinity and fibril formation of piscine transthyretin: the role of the N-terminal

Transthyretin (TTR) transports thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3) in the blood of vertebrates. TH-binding sites are highly conserved in vertebrate TTR, however, piscine TTR has a longer N-terminus which is thought to influence TH-binding affinity and may influence TTR stability. We produced recombinant wild type sea bream TTR (sbTTRWT) plus two mutants in which 6 (sbTTRM6) and 12 (sbTTRM12) N-terminal residues were removed. Ligand-binding studies revealed similar affinities for T3 (Kd=10.6+/-1.7nM) and T4 (Kd=9.8+/-0.97nM) binding to sbTTRWT. Affinity for THs was unaltered in sbTTRM12 but sbTTRM6 had poorer affinity for T4 (Kd=252.3+/-15.8nM) implying that some residues in the N-terminus can influence T4 binding. sbTTRM6 inhibited acid-mediated fibril formation in vitro as shown by fluorometric measurements using thioflavine T. In contrast, fibril formation by sbTTRM12 was significant, probably due to decreased stability of the tetramer. Such studies also suggested that sbTTRWT is more resistant to fibril formation than human TTR.