The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.

Aiming to contribute toward the characterization of new, biotechnologically relevant cellulolytic enzymes, we report here the first crystal structure of the catalytic core domain of Cel7A (cellobiohydrolase I) from the filamentous fungus Trichoderma harzianum IOC 3844. Our structural studies and molecular dynamics simulations show that the flexibility of Tyr260, in comparison with Tyr247 from the homologous Trichoderma reesei Cel7A, is enhanced as a result of the short side‐chains of adjacent Val216 and Ala384 residues and creates an additional gap at the side face of the catalytic tunnel. T. harzianum cellobiohydrolase I also has a shortened loop at the entrance of the cellulose‐binding tunnel, which has been described to interact with the substrate in T. reesei Cel7A. These structural features might explain why T. harzianum Cel7A displays higher kcat and Km values, and lower product inhibition on both glucoside and lactoside substrates, compared with T. reesei Cel7A.

The statistical coupling analysis of 768 β-glucosidases from the GH1 family revealed 23 positions in which the amino acid frequencies are coupled. The roles of these covariant positions in terms of the properties of β-glucosidases were investigated by alanine-screening mutagenesis using the fall armyworm Spodoptera frugiperda β-glycosidase (Sfβgly) as a model. The effects of the mutations on the Sfβgly kinetic parameters (k cat/K m) for the hydrolysis of three different p-nitrophenyl β-glycosides and structural comparisons of several β-glucosidases showed that eleven covariant positions (54, 98, 143, 188, 195, 196, 203, 398, 451, 452 and 460 in Sfβgly numbering) form a layer surrounding the active site of the β-glucosidases, which modulates their catalytic activity and substrate specificity via direct contact with the active site residues. Moreover, the influence of the mutations on the transition temperature (T m) of Sfβgly indicated that nine of the coupled positions (49, 62, 143, 188, 223, 278, 309, 452 and 460 in Sfβgly numbering) are related to thermal stability. In addition to being preferentially occupied by prolines, structural comparisons indicated that these positions are concentrated at loop segments of the β-glucosidases. Therefore, due to these common biochemical and structural properties, these nine covariant positions, even without physical contacts among them, seem to jointly modulate the thermal stability of β-glucosidases.

The ability of basic leucine zipper transcription factors for homo- or heterodimerization provides a paradigm for combinatorial control of eukaryotic gene expression. It has been unclear, however, how facultative dimerization results in alternative DNA-binding repertoires on distinct regulatory elements. To unravel the molecular basis of such coupled preferences, we determined two high-resolution structures of the transcription factor MafB as a homodimer and as a heterodimer with c-Fos bound to variants of the Maf-recognition element. The structures revealed several unexpected and dimer-specific coiled-coil-heptad interactions. Based on these findings, we have engineered two MafB mutants with opposite dimerization preferences. One of them showed a strong preference for MafB/c-Fos heterodimerization and enabled selection of heterodimer-favoring over homodimer-specific Maf-recognition element variants. Our data provide a concept for transcription factor design to selectively activate dimer-specific pathways and binding repertoires.

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