The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.

Post-translational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CaaX sequence motifs. Isoprenylation is followed by cleavage of the aaX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CaaX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active-site and substrate binding groove. The cavity is accessible to the external milieu via gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds via a processive mechanism of substrate insertion, translocation and ejection.

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AmrZ is a putative ribbon-helix-helix (RHH) transcriptional regulator. RHH proteins utilize residues within the ␤-sheet for DNA binding, while the ␣-helices promote oligomerization. AmrZ is of interest due to its dual roles as a transcriptional activator and as a repressor, regulating genes encoding virulence factors associated with both chronic and acute Pseudomonas aeruginosa infection. In this study, cross-linking revealed that AmrZ forms oligomers in solution but that the amino terminus, containing an unordered region and a ␤-sheet, were not required for oligomerization. The first 12 unordered residues (extended amino terminus) contributed minimally to DNA binding. Mutagenesis of the AmrZ ␤-sheet demonstrated that residues 18, 20, and 22 were essential for DNA binding at both activation and repressor sites, suggesting that AmrZ utilizes a similar mechanism for binding to these sites. Mice infected with amrZ mutants exhibited reduced bacterial burden, morbidity, and mortality. Direct in vivo competition assays showed a 5-fold competitive advantage for the wild type over an isogenic amrZ mutant. Finally, the reduced infection phenotype of the amrZ-null strain was similar to that of a strain expressing a DNA-binding-deficient AmrZ variant, indicating that DNA binding and transcriptional regulation by AmrZ is responsible for the in vivo virulence defect. These recent infection data, along with previously identified AmrZ-regulated virulence factors, suggest the necessity of AmrZ transcriptional regulation for optimal virulence during acute infection.The Pseudomonas aeruginosa transcriptional regulator AmrZ is a proposed member of the ribbon-helix-helix (RHH) family of DNA-binding proteins, sharing structural similarity with the Arc and Mnt repressors of Salmonella enterica serovar Typhimurium bacteriophage P22. This family is grouped by structural similarity and includes several transcriptional regulators found in prokaryotes, archaea and their viruses, and other bacteriophages (2,7,12,20,24,26,34,46). Based on amino acid identity as well as secondary-structure prediction models, AmrZ likely possesses a ribbon-helix-helix motif (i.e., one ␤-strand and two ␣-helices) (Fig. 1B) responsible for DNA-binding activity in this family of proteins (44). RHH proteins function through the oligomerization of the ␣-helices, which allows the two ␤-strands to form an antiparallel ␤-sheet that recognizes and binds in the major groove of the operator site (31, 38). Arc exists as a dimer in solution, while Mnt utilizes an extra carboxy-terminal ␣-helical domain to maintain a tetramer configuration (43). When binding DNA, these oligomers are maintained, and the inhibition of oligomerization negatively impacts DNA-binding activity (44). To facilitate higher-order oligomers at the RHH binding site, operator sites often contain sequences in either a direct repeat or palindromic orientation (34,46). Because there are specific contacts between residues of the DNA-binding ␤-sheet and bases in the operator site, mutations of critical base...

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation.