Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.

The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding. Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.     

Co-repressor induced order and biotin repressor dimerization: a case for divergent followed by convergent evolution

BirA catalyzes the adenylation and subsequent covalent attachment of biotin to the biotin carboxyl carrier protein (BCCP). In the absence of apo-BCCP, biotin-5'-AMP acts as a co-repressor that induces BirA dimerization and binding to the bio operator to repress biotin biosynthesis. The crystal structures of apo-BirA, and BirA in complex with biotin have been reported. We here describe the 2.8A resolution crystal structure of BirA in complex with the co-repressor analog biotinol-5'-AMP. It was previously shown that the structure of apo-BirA is monomeric and that binding of biotin weakly induces a dimeric structure in which three disordered surface loops become organized to form the dimer interface. The structure of the co-repressor complex is also a dimer, clearly related to the BirA.biotin structure, but with several significant conformational changes. A hitherto disordered "adenylate binding loop" forms a well-defined structure covering the co-repressor. The co-repressor buttresses the dimer interface, resulting in improved packing and a 12 degrees change in the hinge-bending angle along the dimer interface relative to the BirA.biotin structure. This helps explain why the binding of the co-repressor is necessary to optimize the binding of BirA to the bioO operator. The structure reveals an unexpected use of the nucleotide-binding motif GXGXXG in binding adenylate and controlling the repressor function. Finally, based on structural analysis we propose that the class of adenylating enzymes represented by BirA, lipoate protein ligase and class II tRNA synthetases diverged early and were selected based on their ability to sequester co-factors or amino acid residues, and adenylation activity arose independently through functional convergence.     

Role of residue 147 in the gene regulatory function of the Escherichia coli purine repressor.

The crystal structures of corepressor-bound and free Escherichia coli purine repressor (PurR) have delineated the roles of several residues in corepressor binding and specificity and the intramolecular signal transduction (allosterism) of this LacI/GalR family member. From these structures, residue W147 was implicated as a key component of the allosteric response, but in many members of the LacI/GalR family, position 147 is occupied by an arginine. To understand the role of this tryptophan at position 147, three proteins, substituted by phenylalanine (W147F), alanine (W147A), or arginine (W147R), were constructed and characterized in vivo and in vitro, and their structures were determined. W147F displays a decreased affinity for corepressor and is a poor repressor in vivo. W147A and W147R, on the other hand, are super repressors and bind corepressor 13.6 and 7.9 times more tightly, respectively, than wild-type. Each mutant PurR-hypoxanthine-purF operator holo complex crystallizes isomorphously to wild-type. Whereas the apo corepressor binding domain (CBD) of W147F crystallizes under those conditions used for the wild-type protein, neither the apo CBD of W147R nor W147A crystallizes, although screened extensively for new crystal forms. Structures of the holo repressor mutants have been solved to resolutions between 2.5 and 2.9 A, and the structure of the apo CBD of W147F has been solved to 2.4 A resolution. These structures provide insight into the altered biochemical properties and physiological functions of these mutants, which appear to depend on the sometimes subtle preference for one conformation (apo vs holo) over the other.     

Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding

The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system. Two repressor monomers bind to the 40 base-pair biotin operator sequence. In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site. Here, rapid kinetic methods have been used to directly determine the binding mechanism. Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps. Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds. Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator. This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.     

Allosteric signaling in the biotin repressor occurs via local folding coupled to global dampening of protein dynamics

The biotin repressor is an allosterically regulated, site-specific DNA-binding protein. Binding of the small ligand bio-5′-AMP activates repressor dimerization, which is a prerequisite to DNA binding. Multiple disorder-to-order transitions, some of which are known to be important for the functional allosteric response, occur in the vicinity of the ligand-binding site concomitant with effector binding to the repressor monomer. In this work, the extent to which these local changes are coupled to additional changes in the structure/dynamics of the repressor was investigated using hydrogen/deuterium exchange coupled to mass spectrometry. Measurements were performed on the apo-protein and on complexes of the protein bound to four different effectors that elicit a range of thermodynamic responses in the repressor. Global exchange measurements indicate that binding of any effector to the intact protein is accompanied by protection from exchange. Mass spectrometric analysis of pepsin-cleavage products generated from the exchanged complexes reveals that the protection is distributed throughout the protein. Furthermore, the magnitude of the level of protection in each peptide from hydrogen/deuterium exchange correlates with the magnitude of the functional allosteric response elicited by a ligand. These results indicate that local structural changes in the binding site that occur concomitant with effector binding nucleate global dampening of dynamics. Moreover, the magnitude of dampening of repressor dynamics tracks with the magnitude of the functional response to effector binding.     

trp repressor arms contribute binding energy without occupying unique locations on DNA

The amino-terminal arms of dimeric Escherichia coli trp repressor were removed by chymotryptic cleavage of the first 6 residues from each subunit. The role of the arms in structure and function of the repressor was probed by comparing the properties of intact and proteolyzed forms. The armless protein retains the ability to form stable dimers and to bind the corepressor L-tryptophan, but its affinity for both operator and nonoperator DNA is reduced by about 50-fold. Footprinting analysis shows that the intact repressor makes contacts with nucleotides on only one face of operator DNA. Thus, the arms do not wrap around the DNA as is the case for several other DNA binding proteins. All the contact sites identified by footprinting using the intact repressor are preserved in the complex formed with the armless repressor. This result indicates that the arms do not occupy a unique position on the DNA although they contribute substantially to the energy of DNA binding.     

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.     

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.     

Role of the purine repressor hinge sequence in repressor function.

A protease-hypersensitive hinge sequence in Escherichia coli purine repressor (PurR) connects an N-terminal DNA-binding domain with a contiguous corepressor-binding domain. Binding of one molecule of dimeric repressor to operator DNA protects the hinge against proteolytic cleavage. Mutations in the hinge region impair repressor function in vivo. Several nonfunctional hinge mutants were defective in low-affinity binding to operator DNA in the absence of corepressor as well as in high-affinity corepressor-dependent binding to operator DNA, although binding of corepressor was similar to binding of the wild-type repressor. These results establish a role for the hinge region in operator binding and lead to a proposal for two routes to form the holoPurR-operator complex.     

The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.