Anne Chapman-Smith

The mammalian basic helix-loop-helix/PAS family of transcriptional regulators.

Basic helix-loop-helix (bHLH)/PAS proteins are critical regulators of gene expression networks underlying many essential physiological and developmental processes. These include transcriptional responses to environmental pollutants and low oxygen tension, mediated by the aryl hydrocarbon (Dioxin) receptor and hypoxia inducible factors (HIF), respectively, and controlling aspects of neural development, mediated by the single minded (SIM) proteins. bHLH proteins must dimerise to form functional DNA binding complexes and bHLH/PAS proteins are distinguished from other members of the broader bHLH superfamily by the dimerisation specificity conferred by their PAS homology domains. bHLH/PAS proteins tend to be ubiquitous, latent signal-regulated transcription factors that often recognise variant forms of the classic E-box enhancer sequence bound by other bHLH proteins. Two closely related forms of each of the hypoxia inducible factors alpha and single minded proteins and the general partner protein, aryl hydrocarbon receptor nuclear translocator (ARNT), are present in many cell types. Despite high sequence conservation within their DNA binding and dimerisation domains, and having very similar DNA recognition specificities, the homologues are functionally non-redundant and biologically essential. While the mechanisms controlling partner choice and target gene activation that determine this functional specificity are poorly understood, interactions mediated by the PAS domains are essential. Information on structures and protein/protein interactions for members of the steroid hormone/nuclear receptor superfamily has contributed to our understanding of the way these receptors function and assisted the development of highly specific agonists and antagonists. Similarly, it is anticipated that developing a detailed mechanistic and structural understanding of bHLH/PAS proteins will ultimately facilitate drug design.

The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity

Biotin is a coenzyme essential to all life forms. The vitamin has biological activity only when covalently attached to certain key metabolic enzymes. Most organisms have only one enzyme for attachment of biotin to other proteins and the sequences of these proteins and their substrate proteins are strongly conserved throughout nature. Structures of both the biotin ligase and the biotin carrier protein domain from Escherichia coli have been determined. These, together with mutational analyses of biotinylated proteins, are beginning to elucidate the exceptional specificity of this protein modification.

In vivo enzymatic protein biotinylation

Biotin is biologically active only when protein-bound and is covalently attached to a class of important metabolic enzymes, the biotin carboxylases and decarboxylases. Biotinylation is a relatively rare modification, with between one and five biotinylated protein species found in different organisms. We discuss the mechanism and structures involved in this extraordinarily specific protein modification and its exploitation in tagging recombinant proteins.

Contribution of the Per/Arnt/Sim (PAS) Domains to DNA Binding by the Basic Helix-Loop-Helix PAS Transcriptional Regulators

The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-α) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-α and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Here, we have analyzed purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1α and evaluated the contribution of the PAS domains to DNA binding in vitro. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1α with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1α/Arnt heterodimers with their cognate DNA sequences required the PAS A domains and exhibited KD values of 0.4 nm and ∼50 nm, respectively. In contrast, the presence of the PAS domains of Arnt had little effect on DNA binding by Arnt homodimers, and these bound DNA with a KD of 45 nm. In the case of the DR, both high affinity DNA binding and dimer stability were specific to its native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generated a destabilized protein that bound DNA poorly.

Molecular Recognition in a Post-translational Modification of Exceptional Specificity MUTANTS OF THE BIOTINYLATED DOMAIN OF ACETYL-CoA CARBOXYLASE DEFECTIVE IN RECOGNITION BY BIOTIN PROTEIN LIGASE

We have used localized mutagenesis of the biotin domain of the Escherichia coli biotin carboxyl carrier protein coupled with a genetic selection to identify regions of the domain having a role in interactions with the modifying enzyme, biotin protein ligase. We purified several singly substituted mutant biotin domains that showed reduced biotinylation in vivo and characterized these proteins in vitro. This approach has allowed us to distinguish putative biotin protein ligase interaction mutations from structurally defective proteins. Two mutant proteins with glutamate to lysine substitutions (at residues 119 or 147) behaved as authentic ligase interaction mutants. The E119K protein was virtually inactive as a substrate for biotin protein ligase, whereas the E147K protein could be biotinylated, albeit poorly. Neither substitution affected the overall structure of the domain, assayed by disulfide dimer formation and trypsin resistance. Substitutions of the highly conserved glycine residues at positions 133 and 143 or at a key hydrophobic core residue, Val-146, gave structurally unstable proteins.

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity.

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl‐CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl‐5′‐AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site‐directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild‐type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C‐terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild‐type BirA. No function has been identified previously for the BirA C‐terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C‐terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25‐fold decrease in the affinity for ATP. Our data indicate that the C‐terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.

Biotin Protein Ligase from Saccharomyces cerevisiae THE N-TERMINAL DOMAIN IS REQUIRED FOR COMPLETE ACTIVITY

Catalytically active biotin protein ligase fromSaccharomyces cerevisiae (EC 6.3.4.15) was overexpressed inEscherichia coli and purified to near homogeneity in three steps. Kinetic analysis demonstrated that the substrates ATP, biotin, and the biotin-accepting protein bind in an ordered manner in the reaction mechanism. Treatment with any of three proteases of differing specificity in vitro revealed that the sequence between residues 240 and 260 was extremely sensitive to proteolysis, suggesting that it forms an exposed linker between an N-terminal 27-kDa domain and the C-terminal 50-kDa domain containing the active site. The protease susceptibility of this linker region was considerably reduced in the presence of ATP and biotin. A second protease-sensitive sequence, located in the presumptive catalytic site, was protected against digestion by the substrates. Expression of N-terminally truncated variants of the yeast enzyme failed to complement E. colistrains defective in biotin protein ligase activity. In vitro assays performed with purified N-terminally truncated enzyme revealed that removal of the N-terminal domain reduced BPL activity by greater than 3500-fold. Our data indicate that both the N-terminal domain and the C-terminal domain containing the active site are necessary for complete catalytic function.

Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR

The basic helix–loop–helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends in part on partner protein choices, where dimerization with common partner Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of protein interactions, we have identified 22 amino acids in the N-terminal PAS domain of Arnt that are involved in heterodimerization with aryl hydrocarbon receptor (AhR). Of these, Arnt E163 and Arnt S190 were selective for the AhR/Arnt interaction, since mutations at these positions had little effect on Arnt dimerization with other bHLH.PAS partners, while substitution of Arnt D217 affected the interaction with both AhR and hypoxia inducible factor-1α but not with single minded 1 and 2 or neuronal PAS4. Arnt uses the same face of the N-terminal PAS domain for homo- and heterodimerization and mutational analysis of AhR demonstrated that the equivalent region is used by AhR when dimerizing with Arnt. These interfaces differ from the PAS β-scaffold surfaces used for dimerization between the C-terminal PAS domains of hypoxia inducible factor-2α and Arnt, commonly used for PAS domain interactions.

Novel DNA Binding by a Basic Helix-Loop-Helix Protein THE ROLE OF THE DIOXIN RECEPTOR PAS DOMAIN

Central issues surrounding the basic helix-loop-helix (bHLH) superfamily of dimeric transcription factors concern how specificity of partner selection and DNA binding are achieved. bHLH proteins bind DNA through the basic sequence that is contiguous with a helix-loop-helix dimerization domain. For the two subgroups within the family, dimerization is further regulated by an adjacent Per-Arnt-Sim homology (PAS) or leucine zipper (LZ) domain. We provide evidence that for the bHLH·PAS transcription factors Dioxin Receptor (DR) and Arnt, the DR PAS A domain has a unique interaction with the bHLH region that underpins both dimerization strength and affinity for an atypical E-box DNA sequence. A PAS swap heterodimer, where the DR bHLH domain was fused to Arnt PAS A and the Arnt bHLH fused to DR PAS A, gave strong DNA binding, but dimerization was only effective with the native arrangement, suggesting the PAS A domain is critical for each process via distinct mechanisms. LZ domains, which regulate heterodimerization for the bHLH·LZ family members Myc and Max, could not replace the PAS domains for either dimerization or DNA binding in the DR/Arnt heterodimer. In vitro footprinting revealed that the PAS domains influence the conformation of target DNA in a manner consistent with DNA bending. These results provide the first insights for understanding mechanisms of selective dimerization and DNA interaction that distinguish bHLH·PAS proteins from the broader bHLH superfamily.

Covalent Modification of an Exposed Surface Turn Alters the Global Conformation of the Biotin Carrier Domain of Escherichia coli Acetyl-CoA Carboxylase

We have studied the apo (unbiotinylated) and holo (biotinylated) forms of BCCP87, an 87-residue COOH-terminal peptide comprising the biotin carrier domain of the biotin carboxyl carrier protein subunit of Escherichia coli acetyl-CoA carboxylase. The apo protein spontaneously formed disulfide-linked dimers and was modified readily by sulfhydryl reagents, whereas the holo protein remained monomeric and was unreactive toward sulfhydryl reagents unless a protein denaturant was present. These data indicated that the single cysteine residue of the domain (Cys-116) was much more reactive in the apo form of the protein. Incubation of apoBCCP87 with biotin ligase for different times, followed by reaction with fluorescein-5-maleimide, clearly showed that the loss of Cys-116 reactivity was the result of modification with biotin. In addition, reaction of Cys-116 with 5,5′-dithiobis(2-nitrobenzoic acid) showed that apoBCCP87 denatured at lower urea concentrations than holoBCCP87. We also found that apoBCCP87 was at least 10-fold more sensitive than the holo form to proteolysis by a range of proteases. Identification of the cleavage sites indicated that the differences in protease sensitivity could not be attributed to shielding of susceptible bonds by the biotin moiety of the holo protein. These data indicate that a conformational change accompanies biotinylation of the biotin domain. Thus, modification of a β-turn protruding from the protein surface results in alteration of the overall structure of this protein domain.