Elucidation of the molecular details of allosteric communication between distant sites in a protein is key to understanding and manipulating many biological regulatory processes. Although protein disorder is acknowledged to play an important thermodynamic role in allostery, the molecular mechanisms by which this disorder is harnessed for long distance communication are known for a limited number of systems. Transcription repression by the Escherichia coli biotin repressor, BirA, is allosterically activated by binding of the small molecule effector biotinoyl-5′-AMP. The effector acts by promoting BirA dimerization, which is a prerequisite for sequence-specific binding to the biotin biosynthetic operon operator sequence. A 30 Å distance separates the effector binding and dimerization surfaces in BirA, and previous studies indicate that allostery is mediated, in part, by disorder-to-order transitions on the two coupled sites. In this work, combined experimental and computational methods have been applied to investigate the molecular basis of allosteric communication in BirA. Double-mutant cycle analysis coupled with thermodynamic measurements indicates functional coupling between residues in disordered loops on the two distant surfaces. All atom molecular dynamics simulations reveal that this coupling occurs through long distance reciprocal modulation of the structure and dynamics of disorder-to-order transitions on the two surfaces.

Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5′-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein’s dimerization free energy by −4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5′-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5′-AMP binding at the distant effector binding site.

Solvent reorganization can contribute significantly to the energetics of protein–protein interactions. However, our knowledge of the magnitude of the energetic contribution is limited, in part, by a dearth of quantitative experimental measurements. The biotin repressor forms a homodimer as a prerequisite to DNA binding to repress transcription initiation. At 20 °C, the dimerization reaction, which is thermodynamically coupled to binding of a small ligand, bio-5′-AMP, is characterized by a Gibbs free energy of −7 kcal/mol. This modest net dimerization free energy reflects underlying, very large opposing enthalpic and entropic driving forces of 41 ± 3 and −48 ± 3 kcal/mol, respectively. The thermodynamics have been interpreted as indicating coupling of solvent release to dimerization. In this work, this interpretation has been investigated by measuring the effect of replacing H2O with D2O on the dimerization thermodynamics. Sedimentation equilibrium measurements performed at 20 °C reveal a solvent isotope effect of −1.5 kcal/mol on the Gibbs free energy of dimerization. Analysis of the temperature dependence of the reaction in D2O indicates enthalpic and entropic contributions of 28 and −37 kcal/mol, respectively, considerably smaller than the values measured in H2O. These large solvent isotope perturbations to the thermodynamics are consistent with a significant contribution of solvent release to the dimerization reaction.

One emphasis of the Gibbs Conference on Biothermodynamics is the value of thermodynamic measurements for understanding behaviors of biological systems. In this study, the correlation between thermodynamic measurements of in vitro DNA binding affinity with in vivo transcription repression was investigated for two transcription repressors. In the first system, which comprised an engineered LacI/GalR homolog, mutational changes altered the equilibrium constant for binding DNA. Changes correlated with altered repression, but estimates of in vivo repressor concentration suggest a ≥ 25-fold discrepancy with in vitro conditions. In the second system, changes in ligand binding to BirA altered dimerization and subsequent DNA occupancy. Again, these changes correlate with altered in vivo repression, but comparison with in vitro measurements reveals a ~ 10-fold discrepancy. Further analysis of each system suggests that the observed discrepancies between in vitro and in vivo results reflect the contributions of additional equilibria to the transcription repression process.Research highlights► Biothermodynamic measurement has more value when used to predict behavior of biological systems. ► For a LacI/GalR repressor binding DNA, mutational changes in Kd correlate with altered repression. ► For BirA, changes in ligand binding alter DNA occupancy and correlate with altered repression. ► Analysis of each system suggests that additional equilibria contribute to the biological function.

Biotin protein ligases constitute a family of enzymes that catalyze the linkage of biotin to biotin-dependent carboxylases. In bacteria, these enzymes are functionally divided into two classes: the monofunctional enzymes that catalyze only biotin addition and the bifunctional enzymes that also bind to DNA to regulate transcription initiation. Biochemical and biophysical studies of the bifunctional Escherichia coli ligase suggest that several properties of the enzyme have evolved to support its additional regulatory role. Included among these properties are the order of substrate binding and linkage between the oligomeric state and ligand binding. To test this hypothesized relationship between functionality and biochemical properties in ligases, we have conducted studies of the monofunctional ligase from Pyrococcus horikoshii. Sedimentation equilibrium measurements to determine the effect of ligand binding on oligomerization indicate that the enzyme exists as a dimer regardless of liganded state. Measurements performed using isothermal titration calorimetry and fluorescence spectroscopy indicate that, in contrast to the bifunctional E. coli enzyme, substrate binding does not occur by an obligatorily ordered mechanism. Finally, thermodynamic signatures of ligand binding to the monofunctional enzyme differ significantly from those measured for the bifunctional enzyme. These results indicate a correlation between the functional complexity of biotin protein ligases and their detailed biochemical characteristics.

The ability of the Escherichia coli protein BirA to function as both a metabolic enzyme and a transcription repressor relies on the use of a single surface for two distinct protein:protein interactions. BirA forms a heterodimer with the biotin acceptor protein of acetyl-coenzyme A carboxylase and catalyzes posttranslational biotinylation. Alternatively, it forms a homodimer that binds sequence-specifically to DNA to repress transcription initiation at the biotin biosynthetic operon. Several surface loops on BirA, two of which exhibit sequence conservation in all biotin protein ligases and the remainder of which are highly variable, are located at the two interfaces. The function of these loops in both homodimerization and biotin transfer was investigated by characterizing alanine-substituted variants at 18 positions of one constant and three variable loops. Sedimentation equilibrium measurements reveal that 11 of the substitutions, which are distributed throughout conserved and variable loops, significantly alter homodimerization energetics. By contrast, steady-state and single-turnover kinetic measurements indicate that biotin transfer to biotin carboxyl carrier protein is impacted by seven substitutions, the majority of which are in the constant loop. Furthermore, constant loop residues that function in biotin transfer also support homodimerization. The results reveal clues about the evolution of a single protein surface for use in two distinct functions.Graphical AbstractDownload high-res image (150KB)Download full-size imageHighlights► A single surface on the protein BirA participates in two distinct protein:protein interactions that support its transcription regulatory and posttranslational biotin addition functions. ► Alanine replacement of loop residues on the surface reveals their significance for the two interactions. ► The data support a model for coevolution of surface loop sequences to preserve one essential function while acquiring a second.

The ability of a single protein to interact with multiple protein partners is central to many biological processes. However, the physical–chemical and structural basis of the multispecificity is not understood. In Escherichia coli, the protein BirA can self-associate to a homodimer or form a heterodimer with the biotin carboxyl carrier protein of the biotin-dependent carboxylase, acetyl coenzyme A carboxylase. The first interaction results in binding of BirA to the biotin operator sequence to repress transcription initiation at the biotin biosynthetic operon and the second is a prerequisite to posttranslational biotin addition to the carrier protein for use in metabolism. A single surface on BirA is used for both interactions and previous studies indicate that, despite the structural differences between the alternative partners, the two dimerization reactions are isoenergetic. In this work, the underlying thermodynamic driving forces and the sequence determinants of the two interactions were investigated in order to elucidate the energetic and structural underpinnings of the dual specificity. Combined measurements of the temperature and salt dependencies of heterodimerization indicate a modest unfavorable enthalpy and no dependence on salt concentration. By contrast, homodimerization is characterized by a very large unfavorable enthalpy and a modest dependence on salt concentration. Measurements of the function of BirA variants with single amino acid replacements in the alternative dimerization reactions indicate that although considerable overlap in structural determinants for both interactions exists, hotspots specific for one but not the other were detected.

Proteins can perform completely distinct functions in response to the particular partners that they bind to. Consequently, determination of the mechanism of functional regulation in such systems requires elucidation of the mechanism switching between binding partners. The central protein of the Escherichia coli biotin regulatory system, BirA, switches between its function as a metabolic enzyme or a transcriptional repressor in response to binding either the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase or a second BirA monomer. These two protein–protein interactions are structurally mutually exclusive. The results of earlier studies suggest that the system is regulated by kinetic partitioning between the two protein–protein interactions. In this work, sedimentation velocity was employed to monitor the partitioning directly. The results indicate similar equilibrium parameters governing formation of the two protein–protein interactions. Kinetic analysis of the sedimentation velocity data indicated that holoBirA dimerization is governed by very slow forward and reverse rate constants. The slow kinetics of holoBirA dimerization combined with fluctuations in the intracellular apoBCCP pool are critical determinants in partitioning BirA between its distinct biological functions.

•Although disorder maximizes allosteric coupling in proteins, mechanisms by which disorder contributes to allostery remain to be determined.•Corepressor binding and protein dimerization are coupled in the E. coli biotin repressor.•The structure of the G142A BirA variant, in which coupling is abolished, reveals accompanying loss of disorder-to-order transitions on distant corepressor binding and dimerization surfaces.•Allosteric coupling in BirA is achieved through communication of disorder-to-order transitions over a 33-Å distance.Intrinsic disorder provides a means of maximizing allosteric coupling in proteins. However, the mechanisms by which the disorder functions in allostery remain to be elucidated. Small ligand, bio-5′-AMP, binding and dimerization of the Escherichia coli biotin repressor are allosterically coupled. Folding of a disordered loop in the allosteric effector binding site is required to realize the full coupling free energy of − 4.0 ± 0.3 kcal/mol observed in the wild-type protein. Alanine substitution of a glycine residue on the dimerization surface that does not directly contribute to the dimerization interface completely abolishes this coupling. In this work, the structure of this variant, solved by X-ray crystallography, reveals a monomeric corepressor-bound protein. In the structure loops, neither of which contains the alanine substitution, on both the dimerization and effector binding surfaces that are folded in the corepressor-bound wild-type protein are disordered. The structural data combined with functional measurements indicate that allosteric coupling between ligand binding and dimerization in BirA (E. coli biotin repressor/biotin protein ligase) is achieved via reciprocal communication of disorder-to-order transitions on two distant functional surfaces.Download high-res image (143KB)Download full-size image

•Protein partner swapping controls many biological switches.•Partner exchange can be subject to kinetic and/or equilibrium control.•BirA switches between enzymatic and transcription repression functions.•Direct and inhibition footprint titrations were used to investigate the switch.•The switch is subject to a hierarchy of kinetic and equilibrium control.Protein partner exchange plays a key role in regulating many biological switches. Although widespread, the mechanisms dictating protein partner identity and, therefore, the outcome of a switch have been determined for a limited number of systems. The Escherichia coli protein BirA undergoes a switch between posttranslational biotin attachment and transcription repression in response to cellular biotin demand. Moreover, the functional switch reflects formation of alternative mutually exclusive protein:protein interactions by BirA. Previous studies provided a set of alanine-substituted BirA variants with altered kinetic and equilibrium parameters of forming these interactions. In this work, DNase I footprinting measurements were employed to investigate the consequences of these altered properties for the outcome of the BirA functional switch. The results support a mechanism in which BirA availability for DNA binding and, therefore, transcription repression is controlled by the rate of the competing protein:protein interaction. However, occupancy of the transcriptional regulatory site on DNA by BirA is exquisitely tuned by the equilibrium constant governing its homodimerization.Download high-res image (60KB)Download full-size image

The Escherichia coli biotin repressor BirA is an allosteric transcription regulatory protein to which binding of the small ligand corepressor biotinyl-5′-AMP promotes homodimerization and subsequent DNA binding. Structural data indicate that the apo or unliganded repressor is characterized by four partially disordered loops that are ordered in the ligand-bound dimer. While three of these loops participate directly in the dimerization, the fourth, consisting of residues 212–234 is distal to the interface. This loop, which is ordered around the adenine ring of the adenylate moiety in the BirAadenylate structure, is referred to as the adenylate-binding loop (ABL). Although residues in the loop do not interact directly with the ligand, a hydrophobic cluster consisting of a tryptophan and two valine side-chains assembles over the adenine base. Results of previous measurements suggest that folding of the ABL is integral to the allosteric response. This idea and the role of the hydrophobic cluster in the process were investigated by systematic replacement of each side-chain in the cluster with alanine and analysis of the mutant proteins for small ligand binding and dimerization. Isothermal titration calorimetry measurements indicate defects in adenylate binding for all ABL variants. Additionally, sedimentation equilibrium measurements reveal that coupling between adenylate binding and dimerization is compromised in each mutant. Partial proteolysis measurements indicate that the mutants are defective in ligand-linked folding of the ABL. These results indicate that the hydrophobic cluster is critical to the ligand-induced disorder-to-order transition in the ABL and that this transition is integral to the allosteric response in the biotin repressor.

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.