Single-chain repressors containing engineered DNA-binding domains of the phage 434 repressor recognize symmetric or asymmetric DNA operators

Single-chain (sc) DNA-binding proteins containing covalently dimerized N-terminal domains of the bacteriophage 434 repressor cI have been constructed. The DNA-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the N and C-terminal domains of the intact repressor. Compared to the isolated N-terminal DNA-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a slightly stronger repression in vivo. The recognition of the symmetric O(R)1 operator sequence by this sc homodimer was indistinguishable from that of the naturally dimerized repressor in terms of binding affinity, DNase I protection pattern and in vivo repressor function. Using the new, sc framework, mutant proteins with altered DNA-binding specificity have also been constructed. Substitution of the DNA-contacting amino acid residues of the recognition helix in one of the domains with the corresponding residues of the Salmonella phage P22 repressor c2 resulted in a sc heterodimer of altered specificity. This new heterodimeric molecule recognized an asymmetric, artificial 434-P22 chimeric operator with high affinity. Similar substitutions in both 434 domains have led to a new sc homodimer which showed high affinity binding to a natural, symmetric P22 operator. These findings, supported by both in vitro and in vivo experiments, show that the sc architecture allows for the introduction of independent changes in the binding domains and suggest that this new protein framework could be used to generate new specificities in protein-DNA interaction.     

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.     

Specific residues in the Pbx homeodomain differentially modulate the DNA-binding activity of Hox and Engrailed proteins

Two classes of homeodomain proteins, Hox and Engrailed, have been shown to act in concert with the atypical homeodomain proteins Pbx and extradenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Engrailed gene products. Within the N-terminal region of the Pbx homeodomain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with Engrailed-2 (En-2). Furthermore, there are similarities between heterodimeric interactions involving the yeast mating type proteins MATa1 and MATalpha2 and those that allow the formation of Pbx/Hox and Pbx/En-2 heterodimers. Specifically, residues located in the a1 homeodomain that were previously shown to form a hydrophobic pocket allowing the alpha2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/En-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involving both Hox and En-2. Replacement of the three residues located in the turn between helix 1 and helix 2 of the Pbx homeodomain with those of the atypical homeodomain proteins controlling cell fate in the basidiomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactions that control cell fate in Saccharomyces cerevisiae and in the basidiomycetes may well be conserved in part in multicellular organisms.     

A conserved C-terminal domain in PBX increases DNA binding by the PBX homeodomain and is not a primary site of contact for the YPWM motif of HOXA1

HOX proteins are dependent upon cofactors of the PBX family for specificity of DNA binding. Two regions that have been implicated in HOX/PBX cooperative interactions are the YPWM motif, found N-terminal to the HOX homeodomain, and the GKFQ domain (also known as the Hox cooperativity motif) immediately C-terminal to the PBX homeodomain. Using derivatives of the E2A-PBX oncoprotein, we find that the GKFQ domain is not essential for cooperative interaction with HOXA1 but contributes to the stability of the complex. By contrast, the YPWM motif is strictly required for cooperative interactions in vitro and in vivo, even with mutants of E2A-PBX lacking the GKFQ domain. Using truncated PBX proteins, we show that the YPWM motif contacts the PBX homeodomain. The presence of the GKFQ domain increases monomer binding by the PBX homeodomain 5-fold, and the stability of the HOXA1.E2A-PBX complex 2-fold. These data suggest that the GKFQ domain acts mainly to increase DNA binding by PBX, rather than providing a primary contact site for the YPWM motif of HOXA1. We have identified 2 residues, Glu-301 and Tyr-305, required for GKFQ function and suggest that this is dependent on alpha-helical character.     

AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins

Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets.     

An atypical homeodomain in SATB1 promotes specific recognition of the key structural element in a matrix attachment region

SATB1 is a cell type-specific nuclear matrix attachment region (MAR) DNA-binding protein, predominantly expressed in thymocytes. We identified an atypical homeodomain and two Cut-like repeats in SATB1, in addition to the known MAR-binding domain. The isolated MAR-binding domain recognizes a certain DNA sequence context within MARs that is highly potentiated for base unpairing. Unlike the MAR-binding domain, the homeodomain when isolated binds poorly and with low specificity to DNA. However, the combined action of the MAR-binding domain and the homeodomain allows SATB1 to specifically recognize the core unwinding element within the base-unpairing region. The core unwinding element is critical for MAR structure, since point mutations within this core abolish the unwinding propensity of the MAR. The contribution of the homeodomain is abolished by alanine substitutions of arginine 3 and arginine 5 in the N-terminal arm of the homeodomain. Site-directed mutagenesis of the core unwinding element in the 3' MAR of the immunoglobulin heavy chain gene enhancer revealed the sequence 5'-(C/A)TAATA-3' to be essential for the increase in affinity mediated by the homeodomain. SATB1 may regulate T-cell development and function at the level of higher order chromatin structure through the critical DNA structural elements within MARs.     

Pathogenesis of experimentally induced rabies in domestic ferrets

Isl-1 is a member of a family of Homeodomains containing proteins that possess an N-terminal pair of zinc binding LIM domains. The Isl-1 gene in rat codes for a protein that binds to the insulin gene enhancer and is also involved in regulation of amylin and proglucagon genes. A DNA sequence coding for 66 amino acid residues containing the C-terminal homeodomain fragment of Isl-1 was expressed as a soluble protein in Escherichia coli. Here, we describe a procedure which allows the rapid native purification of recombinant homeodomain protein fused to an N-terminal tag of six histidines. The purified homeodomain showed DNA-binding activity to its cognate DNA sequence. An enhanced binding activity is observed in the presence of a reducing agent in electrophoretic mobility shift assays. The DNA binding was further characterized by circular dichroism spectroscopy. Addition of DNA to the homeodomain did not change the overall secondary structure content, but the thermal and chemical denaturing profiles were altered. A stabilization of the secondary structure was observed upon DNA binding. The free energy of unfolding at 23 degrees C was 7 kJ mol(-1) in absence of DNA and 29 kJ mol(-1) in the presence of DNA.     

Recognition of DNA by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs

Single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized DNA-binding domains (DBD) which are connected with a peptide linker in a head-to-tail arrangement. The prototype RR69 contains two wild-type DBDs, while RR*69 contains a wild-type and an engineered DBD. In this latter domain, the DNA- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related P22 repressor. We have used binding site selection, targeted mutagenesis and binding affinity studies to define the optimum DNA recognition sequence for these single-chain proteins. It is shown that RR69 recognizes DNA sequences containing the consensus boxes of the 434 operators in a palindromic arrangement, and that RR*69 optimally binds to non-palindromic sequences containing a 434 operator box and a TTAA box of which the latter is present in most P22 operators. The spacing of these boxes, as in the 434 operators, is 6 bp. The DNA-binding of both single-chain repressors, similar to that of the 434 repressor, is influenced indirectly by the sequence of the non-contacted, spacer region. Thus, high affinity binding is dependent on both direct and indirect recognition. Nonetheless, the single-chain framework can accommodate certain substitutions to obtain altered DNA-binding specificity and RR*69 represents an example for the combination of altered direct and unchanged indirect readout mechanisms.     

Mutational analysis of the engrailed homeodomain recognition helix by phage display

The homeodomain (HD) is a ubiquitous protein fold that confers DNA binding function on a superfamily of eukaryotic gene regulatory proteins. Here, the DNA binding of recognition helix variants of the HD from the engrailed gene of Drosophila melanogaster was investigated by phage display. Nineteen different combinations of pairwise mutations at positions 50 and 54 were screened against a panel of four DNA sequences consisting of the engrailed consensus, a non-specific DNA control based on the lambda repressor operator OR1 and two model sequence targets con-taining imperfect versions of the 5'-TAAT-3' consensus. The resulting mutant proteins could be divided into four groups that varied with respect to their affinity for DNA and specificity for the engrailed consensus. The altered specificity phenotypes of several mutant proteins were confirmed by DNA mobility shift analysis. Lys50/Ala54 was the only mutant protein that exhibited preferential binding to a sequence other than the engrailed consensus. Arginine was also demonstrated to be a functional replacement for Ala54. The functional combinations at 50 and 54 identified by these experiments recapitulate the distribution of naturally occurring HD sequences and illustrate how the engrailed HD can be used as a framework to explore covariation among DNA binding residues.     

Okadaic acid-induced lens epithelial cell apoptosis requires inhibition of phosphatase-1 and is associated with induction of gene expression including p53 and bax

We report the 2.2 A resolution structure of the Drosophila engrailed homeodomain bound to its optimal DNA site. The original 2.8 A resolution structure of this complex provided the first detailed three-dimensional view of how homeodomains recognize DNA, and has served as the basis for biochemical studies, structural studies and molecular modeling. Our refined structure confirms the principal conclusions of the original structure, but provides important new details about the recognition interface. Biochemical and NMR studies of other homeodomains had led to the notion that Gln50 was an especially important determinant of specificity. However, our refined structure shows that this side-chain makes no direct hydrogen bonds to the DNA. The structure does reveal an extensive network of ordered water molecules which mediate contacts to several bases and phosphates (including contacts from Gln50), and our model provides a basis for detailed comparison with the structure of an engrailed Q50K altered-specificity variant. Comparing our structure with the crystal structure of the free protein confirms that the N and C termini of the homeodomain become ordered upon DNA-binding. However, we also find that several key DNA contact residues in the recognition helix have the same conformation in the free and bound protein, and that several water molecules also are "preorganized" to contact the DNA. Our structure helps provide a more complete basis for the detailed analysis of homeodomain-DNA interactions.