Biomedical subjects

Jonathan M Wojciak

Publications and source records attributed to Jonathan M Wojciak.

4 recordsLinked to original sources

PubMed2005

CBP/p300 TAZ1 domain forms a structured scaffold for ligand binding.

The transcriptional coactivator protein CBP and its paralog p300 each contain two homologous zinc-containing TAZ domains, which constitute the interaction sites for a number of transcription factors. Previous reports of the three-dimensional structures of TAZ1 in complex with binding partners and of the isolated CBP TAZ2 domain show a distinctive topology composed of four amphipathic helices, organized by three zinc-binding clusters with HCCC-type coordination. The isolated CBP TAZ2 domain forms a stable three-dimensional structure in solution, but a recent report [Dial, R., Sun, Z., and Freedman, S. J. (2003) Biochemistry 42, 9937] suggested that the isolated p300 TAZ1 domain lacks a well-defined structure and behaves like a molten globule, even in the presence of Zn(2+), and that the formation of a stable three-dimensional structure requires binding of a protein partner. In marked contrast to this result, we find that both the CBP and p300 TAZ domains in the presence of stoichiometric concentrations of Zn(2+) adopt a well-defined structure in solution in the absence of binding partners. We have determined the three-dimensional structure of the isolated CBP TAZ1 domain by NMR methods and show that it has the same structure in the presence and absence of binding partners. This is an important finding: whether the free TAZ1 domain forms a folded structure or behaves as a molten globule will have a significant bearing on the mechanism of protein-protein recognition. Although TAZ1 and TAZ2 share many structural similarities, there is a major structural difference: the fourth helix is oriented in opposite directions in the TAZ1 and TAZ2 domains. The structure of the free TAZ1 domain suggests that this difference is an inherent feature that determines binding specificity and facilitates discrimination between different subsets of transcription factors by the two TAZ domains.

Acyltransferases↗

PubMed2003

Identification of the lambda integrase surface that interacts with Xis reveals a residue that is also critical for Int dimer formation.

Lambda integrase (Int) is a heterobivalent DNA-binding protein that together with the accessory DNA-bending proteins IHF, Fis, and Xis, forms the higher-order protein-DNA complexes that execute integrative and excisive recombination at specific loci on the chromosomes of phage lambda and its Escherichia coli host. The large carboxyl-terminal domain of Int is responsible for binding to core-type DNA sites and catalysis of DNA cleavage and ligation reactions. The small amino-terminal domain (residues 1-70), which specifies binding to arm-type DNA sites distant from the regions of strand exchange, consists of a three-stranded beta-sheet, proposed to recognize the cognate DNA site, and an alpha-helix. We report here that a site on this alpha-helix is critical for both homomeric interactions between Int protomers and heteromeric interactions with Xis. The mutant E47A, which was identified by alanine-scanning mutagenesis, abolishes interactions between Int and Xis bound at adjacent binding sites and reduces interactions between Int protomers bound at adjacent arm-type sites. Concomitantly, this residue is essential for excisive recombination and contributes to the efficiency of the integrative reaction. NMR titration data with a peptide corresponding to Xis residues 57-69 strongly suggest that the carboxyl-terminal tail of Xis and the alpha-helix of the aminoterminal domain of Int comprise the primary interaction surface for these two proteins. The use of a common site on lambda Int for both homotypic and heterotypic interactions fits well with the complex regulatory patterns associated with this site-specific recombination reaction.

Bacteriophage lambda↗

PubMed2002

Regulation of directionality in bacteriophage lambda site-specific recombination: structure of the Xis protein.

Upon induction of a bacteriophage lambda lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual "winged"-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single alpha-helix and loop structure ("wing"), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase-Xis-DNA complex required for phage excision.

Amino Acid Motifs↗

PubMed2002

Arm-site binding by lambda -integrase: solution structure and functional characterization of its amino-terminal domain.

The integrase protein (Int) from bacteriophage lambda catalyzes the insertion and excision of the viral genome into and out of Escherichia coli. It is a member of the lambda-Int family of site-specific recombinases that catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria, and yeast and belongs to the subset of this family that possesses two autonomous DNA-binding domains. The heterobivalent properties of Int can be decomposed into a carboxyl-terminal domain that executes the DNA cleavage and ligation reactions and a smaller amino-terminal domain that binds to an array of conserved DNA sites within the phage arms, thereby arranging Int protomers within the higher-order recombinogenic complex. We have determined that residues Met-1 to Leu-64 of Int constitute the minimal arm-type DNA-binding domain (INT-DBD(1-64)) and solved the solution structure by using NMR. We show that the INT-DBD(1-64) is a novel member of the growing family of three-stranded beta-sheet DNA-binding proteins, because it supplements this motif with a disordered amino-terminal basic tail that is important for arm-site binding. A model of the arm-DNA-binding domain recognizing its cognate DNA site is proposed on the basis of similarities with the analogous domain of Tn916 Int and is discussed in relation to other features of the protein.

Arginine↗