Structural and Biochemical Studies of a Eukaryotic Transcription Factor and a Bacterial Quorum Quenching Enzyme

In the first part of this thesis, the crystal structure of the T-box DNA binding domain (DBD) of T-bet (T-box expressed in T-cells) in complex with a 24 base pair palindromic consensus DNA recognition sequence is presented. The transcription factor T-bet is a master regulator that plays a central role in T helper cell lineage commitment, where it controls the Th1 immune response. T-bet is a member of the T-box family of transcription factors, a family that includes the mammalian Tbx1, Tbx3, Tbx5, and Brachyury proteins, which are involved in early embryonic development and organogenesis. T-bet is distinct from the other T-box proteins in that it coordinately regulates the expression of many more genes. A central unresolved question is how T-bet is able to potentiate distal transcription regulatory DNA elements or modules, which are located thousands of base pairs away from the promoter recognition element of the gene it regulates. The structure presented here shows a novel quaternary structure for the T-bet DBD dimer in which the two subunits have their DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the palindromic consensus DNA sequence. All other T-box proteins whose DNA complex structures have been determined appear to be monomers in solution and in the crystalline state bind DNA in a classical one T-box dimer-one palindromic site stoichiometry. In contrast, T-bet DBD as a dimer binds simultaneously to identical half-sites on two independent DNA oligomers. A fluorescence-based assay confirms that T-bet dimers exist in solution with the protein able to bring two independent DNA molecules into close juxtaposition. Evidence is also presented for the existence of T-bet dimers in vivo. Furthermore, data from chromosome conformation capture (3C) assays firmly established that T-bet functions in the direct formation of chromatin loops. We present a DNA looping/tethering model for transcriptional regulation by T-bet in which a single dimer of the transcription factor recognizes distinct genetic elements, including a promoter site plus a distant regulatory site or promoter sites on two different genes. This model suggests that the unique structure of the T-bet DBD dimer allows it to coalesce multiple regulatory sites, as has been suggested for so-called “transcriptional factories”.;In the second part of this thesis, crystal structures and steady-state kinetics of AiiA (Autoinducer Inactivator A) mutant enzymes are presented. AiiA is a di-metallo hydrolase that inactivates quorum-sensing N-acyl homoserine lactone (HSL) molecules. It is an enzyme with broad substrate specificity showing preference for substrates with long N-acyl sidechains. A central unresolved question is what is the molecular mechanism that controls substrate specificity in AiiA. Co-crystal structures of AiiA mutants with either substrate C10-HSL or product C10-Hse, both of which contains a long 10-carbon acyl sidechain, reveal a new ligand binding conformation. The 10-carbon acyl sidechain is bound in a hydrophobic channel clamped by two phenylalanine residues F64 and F68. Steady-state kinetics using substrates of various lengths with AiiA bearing mutations at the phenylalanine clamp, including the insertion of a redox sensitive cysteine pair, substantiate the importance of this hydrophobic motif for substrate specificity. A thorough understanding of the molecular mechanism by which AiiA determines substrate preference will aid in the engineering of quorum-quenching enzymes with specific substrate preference properties and as potential antibiotics against pathogenic bacteria that utilize quorum sensing.