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Superrepression through Altered CorepressorActivated Protein:protein Interactions Chenlu He, Gregory S Custer, Jingheng Wang, Silvina Matysiak, and Dorothy Beckett Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01122 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Biochemistry

Superrepression through Altered Corepressor-Activated Protein:protein Interactions Chenlu He1, Gregory Custer2, Jingheng Wang1, Silvina Matysiak2 & Dorothy Beckett1* Department of Chemistry & Biochemistry1, Fischell Department of Bioengineering2, University of Maryland, College Park, MD 20742 *Corresponding author

Contact: email [email protected]

Running title: Protein:protein interactions in superrepression

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Abstract

Small molecules regulate transcription in both eukaryotes and prokaryotes

by either enhancing or decreasing assembly of transcription regulatory complexes. For allosteric transcription repressors superrepressor mutants can exhibit increased sensitivity to small molecule corepressors. However, since many transcription regulatory complexes assemble in multiple steps, the superrepressor phenotype can reflect changes in any or all of the individual assembly steps. E. coli biotin operon repression complex assembly, which responds to input biotin concentration, occurs via three coupled equilibria including corepressor binding, holorepressor dimerization and dimer binding to DNA. A genetic screen has yielded superrepressor mutants that repress biotin operon transcription in vivo at biotin concentrations much lower than required by the wild type repressor. In this work, isothermal titration calorimetry and sedimentation measurements were used to determine the superrepressor biotin binding and homodimerization properties. The results indicate that, although all variants exhibit biotin binding affinities similar to that measured for BirAwt, five of the six superrepressors show altered homodimerization energetics. Molecular dynamics simulations suggest that the altered dimerization results from perturbation of an electrostatic network that contributes to allosteric activation of BirA for dimerization. Modeling of the multi-‐ step repression complex assembly for these proteins reveals that the altered sensitivity of the transcription response to biotin concentration is readily explained solely by the altered superrepressor homodimerization energetics. These results highlight how coupled equilibria enable alterations in a transcription regulatory response to input signal through an indirect mechanism. Key words: Transcription repression, protein:protein interactions, analytical ultracentrifugation, isothermal titration calorimetry, All Atom MD simulations

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Introduction

Communication between metabolism and transcription regulation provides a

mechanism for adjusting gene expression in response to an organism's demand for nutrients and metabolites. In bacteria this communication is frequently achieved through binding of a small molecule effector to a transcription regulatory protein to allosterically alter its sequence-‐specific binding to DNA. In negative feedback systems such as the bacterial tryptophan biosynthetic pathway, the end product of the pathway, tryptophan, binds to the repressor to promote operator binding, thus repressing synthesis of the biosynthetic genes when metabolic demand for tryptophan is satisfied 1. For an inducible system exemplified by the lactose operon, the substrate for the metabolic pathway, allolactose, binds to the lactose repressor to relieve repression, thereby allowing synthesis of gene products required for lactose transport and catabolism 2. In these two classic systems the allosteric effector, corepressor for TrpR and inducer for LacI, binds to a repressor oligomer to alter its affinity for DNA. In other transcription regulatory systems, effector binding can change DNA occupancy by altering the regulatory protein oligomeric state 3; 4. Linkage between regulatory protein self-‐association and effector binding yields a steep response of transcription level to small changes in effector concentration.

The bifunctional E. coli biotin repressor/ligase links biotin utilization to its

biosynthesis by functioning as both an enzyme and a sequence specific DNA binding protein 5; 6. In its enzymatic function, obligatorily ordered biotin followed by ATP binding leads to biotinoyl-‐5'-‐adenylate (bio-‐5'-‐AMP) synthesis. The resulting highly stable enzyme-‐adenylate complex, holoBirA, has two possible fates (Figure 17). First, it can bind to the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase to catalyze biotin linkage to the epsilon amino group of a single lysine residue on the protein. This post-‐translational biotin addition activates carboxylase-‐ catalyzed synthesis of malonyl-‐CoA, the substrate in fatty acid biosynthesis 5. Alternatively, holoBirA can homodimerize and bind to the biotin operator sequence to regulate transcription of the biotin biosynthetic genes (Figure 1,8; 9).

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Figure 1: The Biotin Regulatory System :Biotin binding, followed by ATP, results in bio-‐5'-‐AMP synthesis to form holoBirA. HoloBirA can interact with apoBCCP and transfer the biotin. Alternatively, it can dimerize and bind to the biotin operator sequence, bioO, to repress transcription initiation.

Partitioning of holoBirA between its two functions is regulated by the

relative rates of forming the alternative protein:protein interactions. When the demand for biotin is relatively high, the elevated apoBCCP concentration favors rapid heterodimerization between holoBirA and the acceptor protein 10; 11; 12. In conditions of low biotin demand, e.g. slow growth, the relatively low apoBCCP concentration allows holoBirA accumulation and relatively slow homodimerization. The resulting homodimer binds to the biotin operator (bioO) to repress transcription initiation at the biotin biosynthetic operon. Thus the system is subject to kinetic control with high apoBCCP concentrations resulting in rapid formation of the heterodimeric complex between BirA and the carboxylase subunit and low acceptor protein concentrations permitting slow homodimerization.

In addition to the control exerted by apoBCCP concentration, intracellular

biotin concentration influences BirA function 13. HoloBirA dimerization is a prerequisite to bioO binding and repression. Moreover, bio-‐5'-‐AMP binding

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regulates the homodimerization energetics, enhancing it by -‐4 kcal/mol or 1000-‐ fold in the equilibrium constant 14. Since adenylate synthesis occurs by an obligatorily ordered mechanism, with biotin binding first, biotin concentration regulates repression by regulating adenylate synthesis and the resulting holoBirA concentration 15.

The complex set of inputs into the biotin regulatory system allows for several

possible levels of modulating BirA function. Three factors must be considered in transcription repression complex assembly starting from apoBirA (Figure 1 16). First, the holoBirA availability is limited by biotin affinity and intracellular concentration. Second, the dimerization free energy dictates the fraction of holoBirA that is dimer, the active species in bioO binding. Finally, the affinity of the holoBirA dimer for bioO limits the fractional saturation of the operator and the resulting level of transcription repression. Perturbation of the parameters that govern any of these three steps should alter the transcriptional response to biotin concentration.

Chakravartty and Cronan recently employed a genetic screen to identify BirA

superrepressors that repress transcription at the biotin operon promoters in vivo at lower biotin concentrations than required by the wild type protein17. In in vitro measurements a subset of the purified BirA superrepressor proteins showed higher overall affinity, which reflects dimerization plus bioO binding, for the biotin operator sequence. The single amino acid substitutions that yield a superrepressor phenotype are located in the central or catalytic/dimerization domain of the BirA protein, not the DNA binding domain. Furthermore, although a subset of the substitutions are of amino acids that are in the vicinity of the dimerization surface in the holoBirAwt structure, these amino acids do not directly participate in noncovalent interactions at the dimer interface 18. Taking into account the three inputs into the transcription regulatory complex assembly, the superrepressor phenotype could, in principle, reflect altered biotin binding, dimerization or sequence-‐specific binding of the resulting holoBirA dimer. However, previous results suggest that the final possibility is unlikely since holoBirA variant dimers

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with alanine substitutions on the dimerization surface all bind to bioO with the same affinity, despite exhibiting a broad range of self-‐association energetics 19.

In this work we have subjected six superrepressor BirA variants to biotin

binding and homodimerization measurements. Isothermal titration calorimetry (ITC) measurements reveal that all variant proteins bind to biotin with affinities similar to that measured for BirAwt. However, sedimentation equilibrium measurements indicate that the bio-‐5'-‐AMP-‐bound proteins are altered in dimerization. Results of molecular dynamics simulations suggest that the altered dimerization of some variants reflects changes in an electrostatic network that links the corepressor binding site to the remainder of the BirA protein structure. Modeling of the biotin concentration dependence of repression complex assembly for the variants indicates that altered sensitivities of BirA mutants to biotin input can be readily explained solely by altered dimerization properties. These results illustrate that in transcriptional regulatory systems governed by linked equilibria the response to input can be significantly changed by mutations that affect protein:protein interactions.

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Materials and Methods Chemicals and Biochemicals All chemicals and biochemicals were at least reagent grade. Biotin (Acros) stock solutions were prepared in Standard Buffer (10 mM TrisHCl [pH=7.5 at 20 °C], 200 mM KCl, 2 mM MgCl2) and stored at −80 °C. Solutions of bio-‐5′-‐AMP, which was synthesized and purified as previously described3; 20, were prepared in Milli-‐Q H2O and stored at −80 °C in 1ml aliquots. The bio-‐5’-‐AMP concentration was determined by UV absorbance at 259 nm using molar extinction coefficient of 15,400 M-‐1cm-‐1. Protein Purification The overexpression strains for BirA suprerepressors were obtained from Dr. John Cronan, University of Illinois. The recombinant plasmids are pET19b derivatives that encode C-‐terminal hexahistidine-‐tagged BirA variants 17. The E. coli strain BL21(λDE3) transformed with each of these plasmids was grown in LB media containing 100 μg/ml ampicillin. When the culture had reached an OD600 of 0.8 protein expression was induced by adding isopropyl β-‐D-‐1-‐thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Induction was carried out for 4 hours at 30°C for all variant except G154D, which was expressed for 17 hours at 20°C. The cells were disrupted by sonication in lysis buffer (100 mM sodium phosphate [pH 6.5], 200 mM NaCl, 5% glycerol). Polyethylene Immine (PEI) was added to the lysis supernatant to a final concentration of 0.2% to precipitate nucleic acids. After centrifugation, the protein in the resulting supernatant was precipitated by adding saturated (NH4)2SO4 solution to a final concentration of 60% w/v. Following centrifugation the protein pellet was resuspended in and dialyzed against buffer containing 50 mM NaH2PO4 [pH 8.0], 300 mM NaCl, 10 mM imidazole, 5% glycerol, then subjected to affinity chromatography on Ni-‐NTA (Qiagen & Thermo Scientific) . Non-‐specifically bound proteins were removed by washing the column with the same buffer containing 20 mM imidazole. The protein was eluted with an increasing imidazole concentration (up to 135 mM), and fractions containing BirA were dialyzed against buffer containing 50 mM TrisHCl [pH 7.5 at 4oC], 50 mM KCl, 5% glycerol and 0.1 mM 1,4-‐dithiothreitol (DTT). Further purification was carried out

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by cation-‐exchange chromatography on SP Sepharose fast flow resin (GE Healthcare). The column was washed with the above buffer and protein was eluted with a gradient of 50-‐375 mM KCl. For G154D, a 2nd SP Sepharose column was required using a gradient of 50-‐312.5 mM KCl to elute the pure protein. The purified protein was dialyzed against storage buffer (50 mM TrisHCl [pH 7.5 at 4oC], 200 mM KCl, 5% glycerol, and 0.1 mM DTT) and stored at −80 °C in 1 mL aliquots. The protein concentration was determined by UV absorbance at 280 nm using the extinction coefficient of 47,510 M-‐1cm-‐1, which was calculated from amino acid composition21. All variant proteins were at least 95% pure based on the band intensities of SDS-‐PAGE gel. Isothermal Titration Calorimetry

Biotin-‐binding measurements were carried out using a VP-‐ITC

microcalorimeter (Microcal) in Standard Buffer (10 mM TrisHCl [pH=7.5 at 20 °C], 200 mM KCl, and 2.5 mM MgCl2). The protein was dialyzed against Standard Buffer and then filtered through a 0.22 μm PES syringe filter (SIMSII) to remove any precipitate. The protein concentration in the resulting sample was determined by UV absorbance at 280 nm. The dialysate was degassed for 8 minutes in ThermoVac (Microcal) and protein and ligand were both gravimetrically diluted into this buffer to the working concentrations. The resulting solutions were degassed for 8 minutes immediately before use. For all measurements, the sample cell was filled with the protein solution at a concentration of 2 μM and the reference cell was filled with Milli-‐Q H2O. The injection syringe contained biotin solution at a concentration of 20 μM. All titrations were performed at 20 °C with 23 injections in total, with a 2 μL volume for the first injection and 13 μL for the remaining 22. The stirring speed was 310 rpm for all titrations.

Data analysis was performed in Origin 7.0 (MicroCal, Inc.). Following

integration of the raw titration peaks, the ligand injection heat, which was calculated from the average heat of last five injections, was subtracted from the integrated heat associated with each ligand injection. After calculating the molar enthalpy per injection the resulting data were analyzed using a single-‐site binding model to

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obtain the stoichiometry, n, the equilibrium association constant KA, and the molar enthalpy ΔH. At least two independent biotin titrations were performed for each variant. Sedimentation Equilibrium

The homodimerization constants of all bio-‐5’-‐AMP-‐bound superrepressor

proteins were determined in Standard Buffer by sedimentation equilibrium using an Optima XL-‐1 analytical ultracentrifuge (Beckman Coulter). In the case of Y178C, 1mM TCEP-‐HCl (PIERCE) was added to the buffer as a reducing agent. The protein was dialyzed exhaustively against Standard Buffer at 4°C and filtered through a 0.22 μm PES syringe filter (SIMSII) to remove any precipitation. After determining protein concentration by UV absorption, the samples were prepared at three protein concentrations. The bio-‐5’-‐AMP was added to each sample at a 1:1.5 protein:ligand molar ratio under stoichiometric conditions. For all measurements, the samples were centrifuged at 20 °C at three speeds (18000, 21000 and 24000 rpm) in standard 12-‐mm six-‐hole cells in a four-‐hole An-‐60 rotor (Beckman Coulter). After eight hours at each speed, absorbance scans were acquired at 290, 295, or 300 nm with a step size of 0.001 cm and five replicates per step. The wavelength was chosen based on the total protein concentrations in the samples so that the absorbance falls within the detection limit of the spectrophotometer. Overlaying of scans acquired at eight and nine hours of centrifugation at each speed indicated that equilibrium had been achieved. At least three independent sedimentation equilibrium measurements were performed for each variant. Sedimentation Velocity

Sedimentation velocity measurements were performed using Optima XL-‐1

analytical ultracentrifuge (Beckman Coulter) equipped with a four-‐hole An-‐60 rotor (Beckman Coulter). The protein was dialyzed against Standard Buffer, filtered through a 0.22 μm PES syringe filter (SIMSII) to remove precipitate, and its concentration was determined by uv absorption. The resulting protein was diluted to the working concentration and, if appropriate, bio-‐5’-‐AMP was added to the

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sample at a 1:1.5 protein:ligand molar ratio. Standard two-‐channel charcoal-‐filled Epon centerpieces cells with a 3mm or 12 mm pathlength were used. Prior to centrifugation the cells and rotor were equilibrated at 20 °C under vacuum for at least 2 hours. The centrifugation was run at 20 °C at a single speed of 50,000 rpm. Continuous scans with a step size of 0.003 cm and one replicate were acquired at 290 nm. Data Analysis Sedimentation Equilibrium The absorbance versus radius profiles were globally analyzed using Nonlin22 in HeteroAnalyisis version 1.01.0060 (http://core.uconn.edu/resources/biophysics). A monomer-‐dimer model was used to obtain the best-‐fit equilibrium dissociation constant governing the homodimerization from the following equation:

c(r) = δ + c(ro

σ m (r 2 −ro2 ) 2 )e

1

2

+ c(ro ) (

K dim

2σ m (r 2 −ro2 ) 2 )e

(1)

where c(r) is the protein concentration at any radial position , c(rO) is the protein concentration at reference radial position rO, δ is the baseline offset, Kdim is the € equilibrium dissociation constant governing dimerization and σm is the reduced molecular weight of BirA monomer, which was obtained using the following relationship:

σ=

(1− v ρ)ω 2 RT

where M is the molecular weight of BirA monomer,

(2)

is the partial specific volume

of the protein, €ρ the buffer density, ω is the angular velocity of the rotor, R is the gas constant and T is absolute temperature. The molecular weight of His-‐tagged BirA is 36,100 g/mol, the partial specific volume of 0.755 ml/g was experimentally determined8, and the buffer density was calculated from its composition at the working temperature using Sednterp (http://sednterp.unh.edu/). In all analyses, the reduced molecular weight of the dimer was assumed to be twice that of the monomer. The quality of each fit was assessed by the magnitude of the square root of variance and the distribution of the residuals.

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Sedimentation Velocity Data analysis was carried out using DCDT+ version 2.4.3.2297623; 24. A subset of scans (20-‐30 scans) was used to produce the g(s*) distribution. The data were analyzed using the appropriate model to obtain the sedimentation coefficient at standard condition s(20,w). The partial specific volume value of 0.755 cm3/g was experimentally determined8 and the solvent density of 1.008 g/ml and the solvent viscosity of 1.002 cp were calculated using Sednterp (http://sednterp.unh.edu/). Molecular Dynamics (MD) Simulations and Analysis

MD simulations were performed for wild type BirA and four variants, P143T,

G154D, Y178C, and M310L, all in their monomeric form in complex with the corepressor analogue biotinol-‐5’-‐AMP (btnOH-‐AMP). Starting coordinates for these simulations were taken from chain A of the BirA dimer found in Protein Data Bank (PDB) entry 2EWN 18. This structure contains BirAwt bound to btnOH-‐AMP, and was chosen because it is the most complete of the available BirAwt structures, having all residues modeled in both the dimerization and ligand binding regions. Amino acid substitutions for simulations of variants were made in PyMOL 25. For each simulation the protein model was placed in a rhombic dodecahedral box with walls extending ~1nm beyond the protein. The protein was then solvated with ~20300 SPC/E 26 water molecules. Na+ counterions were added to the system as needed to render the system neutral, replacing randomly chosen water molecules. For the wt, P143T, Y178C, and M310L variants one Na+ counterion was required, while for the G154D variant two Na+ counterions were required. The energy of each system was minimized using the steepest descent method, prior to production runs. Energy minimization was then followed by NVT and NPT equilibration runs with a duration of 100 ps each, using position restraints with a force constant of 1000 kJ mol-‐1nm-‐2 on the protein. Production runs were then conducted without position restraints, using an NPT ensemble with a pressure of 1 bar and a temperature of 300 K. Production runs had a duration of 1 μs, with the last 500 ns of the simulation used

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for analysis, as we have shown in a previous publication that the simulations take no longer than 500 ns to equilibrate 27.

MD simulations were performed using the GROMACS 4.6 simulator (28; 29; 30;

31) and the OPLS-‐AA force field 32. Parameters for btnOH-‐AMP were kept identical to

those used for our previous publication, and are available upon request 27. The time step for simulations was set to 2 fs, with neighbor list updates occurring every five steps. Bond lengths were constrained using the LINCS algorithm. The protein and water temperatures were regulated independently using the V-‐rescale algorithm 33 with a time constant of 0.1 ps. The ligand btnOH-‐AMP was grouped with protein for temperature coupling, while Na+ counterions were grouped with water. Isotropic pressure coupling was used in all simulations, with the Parrinello-‐Rahman barostat 34, using a time constant of 2 ps and a compressibility of 4.5 x 10-‐5 bar-‐1.

Images of structures from MD simulations were generated using VMD (41).

The structures represent the average calculated from the final 500 ns of each trajectory with 6,250 and 10,000 frames used for the wild type and variants, respectively. These average structures were calculated using the GROMACS tool g_covar (33-‐36). Alignments were performed in VMD (41) using the backbone alpha-‐carbon atoms.

The helicity of residues 140-‐152 was quantified by measuring the per-‐

residue average RMSD from an ideal helix (RMSDhx). As both 310-‐ and α-‐helices form in this residue range, both states were considered in this calculation. For the purposes of the calculation, ideal helices were constructed using only heavy backbone atoms (N, CA, C, and O), setting the φ and ψ angles to -‐57° and -‐47°, respectively, for the 310-‐helix and to -‐49° and -‐26°, respectively, for the α-‐helix 35. A five-‐residue ideal α-‐helix or 310-‐helix was used to calculate RMSDDα or RMSD310, respectively. For each residue, i, in the residue range 140-‐152, these ideal helix segments were aligned to a five-‐residue segment centered on residue i, and RMSD was calculated between the structures. The smaller of either RMSDα or RMSD310 was taken as the RMSDhx for residue i. This calculation was performed for each

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frame in the final 500 ns of the MD simulation trajectory, with the average per-‐ residue RMSDhx reported.

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Results Biotin binding is similar for all superrepressors

The BirA superrepressors repress transcription in vivo at lower biotin

concentrations than that required for wtBirA. A potential source of this increased sensitivity is an increased affinity for biotin, which was investigated by measuring the biotin affinities of the variants using isothermal titration calorimetry (ITC). The titration data for the M310L variant are well-‐described by a single site model (Figure 2A). Moreover, the binding parameters resolved from the data analysis are similar in magnitude to those measured for wtBirA (Table 1).

Figure 2. BirAM310L function in biotin binding and dimerization. In titration of BirAM310L with biotin a 20µM ligand solutionwas injected into a 2µM protein solution (top). The resulting binding data (squares) were analyzed using a single site binding model (solid line) to obtain the equilibrium association constant, the enthalpy and the stoichiometry of binding. B. Sedimentation equilibrium measurement of the monomer-‐dimer equilibrium for holoBirAM310L. Data from samples prepared at three protein concentrations (left, middle, right), and acquired at three rotor speeds (red, blue, black) were subjected to global analysis using a

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monomer-‐dimer model to obtain the equilibrium dissociation constant. The residuals of the fit are shown in the bottom panel.

Biotin binding measurements performed on the remaining superrepressors

variants indicate that they bind with energetics similar to those measured for wtBirA. The quality of titration data obtained for these variants was similar to that shown for BirAM310L (Figure 2A). Within error the equilibrium constants for biotin binding differ from that measured for the wild type protein by less than 2-‐fold, with the largest difference observed for BirAI187T. The binding enthalpies are all large and negative with the P143T variant showing the largest difference of 2.6 kcal/mol from the value obtained for BirAwt (Table 1). Analysis of titrations obtained for the majority of the variants yielded binding stoichiometries (n values) ranging from 0.8-‐ 1.0. The only exception is BirAG154D, for which the relatively low value of 0.73±0.02 may be related to instability. Nevertheless, this variant shows binding parameters similar to those measured for BirAwt. Table 1: Biotin Binding Thermodynamics of BirA Superrepressor Proteins Protein n Ka (M-1) ΔH (kcal/mol) -TΔS (kcal/mol) WT 0.84±0.07 2.4(±0.1)×107 -21.1±0.4 11.2±0.4 7 I187T 0.84±0.02 1.9(±0.1)×10 -19.5±0.4 9.8±0.4 K267M 0.83±0.03 2.7(±0.1)×107 -20.3±0.1 10.3±0.1 M310L 0.90±0.02 2.8(±0.3)×107 -20.2±0.1 10.23±0.04 P143T 0.867±0.004 2.4(±0.4)×107 -18.5±0.7 8.6±0.8 7 Y178C 0.84±0.04 2.4(±0.3)×10 -18.8±0.2 8.90±0.2 G154D 0.73±0.02 2.2(±0.3)×107 -20.2±0.3 10.3±0.4 o All measurements were conducted in Standard Buffer at 20 C. The errors represent the standard deviation resulting from averaging the results of at least three independent measurements. Several superrepressors dimerize more tightly than holoBirAwt

A second possible source of the enhanced sensitivity of the variants to biotin

concentration is altered dimerization of the holo-‐repressors. This was tested by measuring the self-‐association of the holo-‐variants using equilibrium analytical ultracentrifugation. For each variant, measurements were performed on samples

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prepared at three holo-‐repressor concentrations that were centrifuged at three rotor speeds. Global nonlinear least squares analysis of the resulting nine data sets was performed using both a single species and a monomer-‐dimer model. Measurements performed on holoBirAM310L variant indicate that it dimerizes with a Gibbs free energy that is -‐0.7±0.2 kcal/mol more favorable than does holoBirAwt (Figure 2B, Table 2). Table 2: Homodimerization Properties of BirA Superrepressor Proteins Protein Kdim, holo (M) ΔG°dim, holo (kcal/mol) s20,w (Svedbergs) -6 WT 11(±3)×10 -6.7±0.2 -6 I187T 3.4(±0.9)×10 -7.4±0.1 K267M 7(±2)×10-6 -7.0±0.2 M310L 3(±1)×10-6 -7.4±0.2 -6 P143T 3(±1)×10 -7.4±0.2 Y178C 7(±3)×10-4 -4.3±0.2 -8 G154D 7(±4)×10 -9.6±0.3 4.30±0.05 Measurements were conducted in Standard Buffer at 20oC and for each protein 9 data sets were globally analyzed using a monomer-dimer model. The errors represent the standard deviation resulting from averaging the results of at least three independent measurements.

The remaining variants, which yielded sedimentation equilibrium data

similar in quality to that obtained for holoBirAM310L, exhibit a range of dimerization energetics (Table 2). The holoBirAP143T and holoBirAI187T variants dimerize with free energies similar in magnitude to that measured for the M310L variant. By contrast, holoBirAK267M exhibits dimerization energetics similar to those of wtBirA. Finally, the holoBirAY178C unexpectedly dimerizes with a Gibbs free energy that is significantly less favorable than that measured for holoBirAwt.

The G154D variant, which shows the strongest superrepressor phenotype in

vivo, also exhibits the tightest dimerization. Indeed, although an equilibrium constant for holoBirAG154D dimerization of 7.1x10-‐8 M was obtained from the sedimentation equilibrium data (Table 2), the species populations obtained from the data analysis indicate that the protein is nearly 100% dimer. Unfortunately, the detection limits of the absorption optics precluded measurements at lower protein concentrations. Results of sedimentation velocity measurements performed at 10µM holoBirAG154D indicate that 90% of the signal is associated with a single

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species characterized by a sedimentation coefficient of 4.30±0.05S (Table 2), consistent with the protein being primarily in the dimer state. The sedimentation coefficient obtained for the holoBirAG154D dimer is significantly larger than the 3.9S value determined for holoBirAwt dimer 12. This variant has previously been shown to migrate in SDS-‐PAGE at an apparently higher molecular weight than do BirAwt and other variants 17 and preparations used in this work showed the same aberrant migration.

Coupling free energies for two of the variants are similar to that measured

for the wild type protein. Corepressors bio-‐5'-‐AMP binding to BirAwt enhances its dimerization free energy by -‐4.0 kcal/mol, which reflects a 1000-‐fold decrease in the dimerization equilibrium dissociation constant. Although four of the superrepressors dimerize more tightly than holoBirAwt, it is not known if these proteins also dimerize more tightly in their apo forms. In other words, the magnitude of the coupling between effector binding and dimerization for the variants is not known. However, assuming a coupling free energy of -‐4.0 kcal/mol, it is possible to calculate the dimerization free energies of the apo species for any superrepressor. For example, the Gibbs free energy of holoBirAP143T is -‐7.4 kcal/mol, which yields an expected dimerization free energy for the apo-‐form of -‐3.4 kcal/mol. The equilibrium constant calculated from this free energy can be used to predict the fraction dimer as a function of total protein concentration. These calculations yield predictions of 6% and 4% dimer, respectively, at total protein concentrations of 200 and 125uM for BirAP143T and BirAM310L (Table 3). Table 3. Sedmentation Velocity Analysis of apoBirA Variants Predicted % Experimental Experimental BirA variant Predicted s20,w a dimer s20,w %dimerb WT (100µM) 1 4 2.732 2.762±0.003 P143T(200µM) 5 10 2.783 2.84±0.02 M310L(125µM) 4 6 2.762 2.79±0.02 a. Prediction is based on the assumption that the coupling free energy for bio-‐5'-‐ AMP binding and dimerization is -‐4 kcal/mol for all proteins. b. The percent dimer was calculated from the experimentally-‐determined average sedimentation coefficient assuming that it represents the weight average of contributions from the monomer and dimer species.

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Sedimentation velocity provides a sensitive method to detect oligomeric

species. For a protein that rapidly equilibrates between monomer and a single oligomer, the average sedimentation coefficient should deviate from that of the monomer and increase as the total protein concentration is increased36. Sedimentation velocity measurements were performed on apoBirAP143T and apoBirAM310L, at total concentrations of 200 µM and 125 µM, respectively, and the resulting data were analyzed to identify the number of species and their sedimentation coefficients (Table 3). The analysis indicates that both proteins yield a single peak with sedimentation coefficients that are in reasonable agreement with that of apoBirAwt monomer. The absence of a significant amount of dimer for either protein at the concentrations employed indicates that the coupling free energy for these two variants is at least as favorable as the -‐4 kcal/mol value measured for BirAwt. Perturbations to dimerization reflect altered structures of the holo-variant monomers

Based on the structure of the holoBirA dimer, none of the amino acid

residues that are substituted in the superrepressors directly participate in the dimer interface in the wild type protein. Consequently, the energetic effects of the substitutions on dimerization are difficult to rationalize. This is particularly true for the superrepressors with substitutions at positions far from the dimer interface (Figure 3A). In the absence of structural data, atomistic simulations were used to probe the molecular origins of these energetic changes. Simulations previously performed on apo and holoBirAwt monomers indicate good agreement with experiment 27. For example, the structural differences between the apo and holo species, including folding of the biotin binding and adenylate binding loop (BBL, ABL) around the corepressor bio-‐5'-‐AMP, are observed in simulations (Figure 3B). The simulations also reproduce the disorder-‐to-‐order transitions that the dimerization surface loops comprised of residues 140-‐146 and 193-‐199 undergo as well as the interloop packing. Additionally, the conversion of residues 142-‐145

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from coil to helix to extend the helix formed by residues 146-‐165 in apoBirA was recapitulated in simulations. Based on the excellent agreement between experiment and simulation observed for the wild type protein, MD simulations were used to investigate the molecular origins of the altered dimerization properties of superrepressor variants.

Figure 3: A. Locations of single amino acid substitution in the holoBirA dimer structure that yield the superrepressor phenotype. Yellow:P143, Pink:G154, Orange: Y178, Cyan:I187, Green:K267, Blue: M310. B. Apo and holoBirAwt structures illustrating the disorder-‐to-‐order transitions that occur on the ligand binding and dimerization surfaces concomitant with bio-‐5'-‐AMP binding. Dashed lines in apoBirA indicate disorder. The BBL and ABL refer to the biotin and adenylate binding loops, respectively, that fold around bio-‐5'-‐AMP in holoBirA C. Root mean

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square deviations of backbone alpha carbons for each average holoBirA variant from that of the average holoBirAwt structure. Models were created in PyMol 25 using pdb files 1BIA or 2EWN as input.

Simulations performed on the superrepressor variants predict that the single

amino acid substitutions yield complex changes in the holoBirA monomer structure. Computations were carried out on four of the variants including BirAP143T and BirAM310L with substitutions located on the dimerization surface, and BirAG154D and BirAY178C with amino acid substitutions in the central domain core. The simulations were performed on the holo-‐monomer species of each protein for a total of 1µs and analysis was performed on the final 500 ns of each trajectory, at which time each simulation had reached equilibrium. An overview of the effects of each amino acid substitution on the protein structure was first obtained by calculating the root mean square deviations (RMSD) of the C backbone atoms for the average structures of α

each variant relative to holoBirAwt structure, both obtained from simulations (Figure 3C). Because the N-‐terminal DNA binding domain has previously been shown to exhibit considerable flexibility both internally and relative to the central domain, simulation results for only the central and carboxyl terminal domains are shown 18. For the dimerization surface variants, the holoBirAP143T structure is similar to that of the wild type protein, but holoBirAM310L variant shows large structural deviations in both the 140-‐146 and 193-‐199 loops. The holoBirAG154D and holoBirAY178C variants, which have substitutions in the central domain core, are both characterized by large RMSD values in the C-‐terminal domain.

Structures of the dimerization loops segments observed in simulations vary

for the supperrepressors. HoloBirAP143T is structurally identical to holoBirAwt with respect to the helical conformation of residues 140-‐152 as well as the interaction between the 140-‐146 and 193-‐199 loops (Figures 3C, 4A). By contrast, residues 142-‐150 in holoBirAM310L deviate significantly from their structure in the wild type protein (Figure 4A). However, the two loops are more closely packed in this variant

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than they are in the wild type protein (Figure 4B). Like the P143T variant, holoBirAY178C and holoBirAG154D show dimerization surface loop structures identical to those observed in the wild type protein (Figure 4A).

Figure 4. A. Average per-‐residue RMSD from an ideal helix for residues 140-‐152 calculated from simulations performed on the holoBirA variants. Error bars indicate 95% confidence intervals. B. Dimerization surface loops 140-‐146 and 193-‐199 for holoBirAwt (green) and holoBirAM310L (blue). *Results for BirAwt were originally published in Wang, et al. 27.

Comparison of the experimentally determined apo and holoBirAwt structures

reveals differences in the C-‐terminal domain that may contribute to allosteric activation of dimerization. The comparison reveals an electrostatic network in holoBirAwt that incorporates both central and C-‐terminal domain residues (Figure 5). Although a subset of these interactions is present in apoBirAwt, a number of them can form only in the holo-‐protein because BBL residues R118, R119 and R121, which are disordered in the absence of ligand, participate in the network. Importantly, the average holoBirAwt structure obtained from molecular dynamics simulations reveals a network that is nearly identical to that observed in the experimentally determined structure (Figure 5).

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Figure 5. Electrostatic networks in BirA variants: Network shown on the holoBirA structure (upper left) and cartoon representations of the network in BirAwt and super repressor variants. The positively charged residues are shown in blue, negatively charged in read and neutral polar residues in grey. The model was created in PyMol25 using 2EWN as the input file.

The electrostatic network is altered in the simulated holoBirA variant

structures. The network in the holoBirAP143A variant shows a loss of two interactions and a gain of an interaction between R119 and E313 (Figure 5). The holoBirAM310L variant network is nearly identical to that of holoBirAwt, with loss of only one charged hydrogen bond between Y178 and K272. For the holoBirAG154D variant the insertion of the additional charged residue is accompanied by both disruption of

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some interactions and addition of others. By contrast, the holoBirAY178C variant shows loss of multiple electrostatic interactions that are present in holoBirAwt. The differences in the network observed for holoBirAG154D and holoBirAY178C correlate with changes in the orientation of C-‐terminal domain relative to the central domain (Figure 6). In each panel the C-‐terminal domain alignment was obtained from pairwise alignment of the central domain backbone C atoms of the relevant α

holoBirAvariant with those of holoBirAwt. Notably, the backbone C alignments of the α

isolated C-‐terminal domain of each variant with that of the wild type protein indicate close overall agreement. Thus the differences observed in Figure 6 reflect differences in the C-‐terminal domain orientation relative to the central domain.

Figure 6: C-‐terminal domain structures are altered in some superrepressor variants. Alignments of holoBirAwt:(grey) C-‐terminal domain with that of (A. ) holoBirAP143T, (B.) holoBirAM310L, (C.) holoBirAG154D, and (D) holoBirAY178C. Alignment of the central

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domain (residues 80 to 269) of the average structure of each variant with that of holoBirAwt was performed using MultiSeq in VMD37.

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Discussion

The BirA superrepressors repress transcription in vivo at biotin

concentrations lower than those required by BirAwt. Nevertheless, ITC measurements indicate that all of the proteins have biotin binding properties similar to those measured for BirAwt. It is possible that the proteins have an increased affinity for bio-‐5'-‐AMP. However, their tight dimerization precludes measurement of adenylate binding because the ligand binding cannot be deconvoluted from the coupled dimerization process. The biotin binding data indicate that altered sensitivity to a transcription regulatory system input can be achieved in the absence of any change in repressor affinity for the input ligand.

With a few notable exceptions the superrepressor dimerization free energies

agree with the results reported by Chakravartty and Cronan 17. First, consistent with results of in vivo gene expression measurements, the G154D variant shows the tightest dimerization (Table 4). Although the P143T, M310L and I187T variants all dimerize with identical Gibbs free energies, BirAI187T exhibits slightly greater repression of β-‐galactosidase expression in bioF::lacZ fusion strains. The K267M variant, which exhibits a superrepressor phenotype in vivo, dimerizes with a Gibbs free energy identical, within error, to that of the wild type protein. Finally, despite displaying a superrepressor phenotype similar to that of the M310L, P143T and I187T variants, the Y178C variant dimerizes much more weakly than BirAwt. Similar results were obtained on multiple preparations of the protein in the absence and presence of the reducing agent TCEP. Moreover, the sequence of the entire coding sequence for the variant in the expression plasmid was verified. Thus, the origin of the discrepancy between in vivo and in vitro measurements for this protein is currently unknown.

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Table 4: Comparison of in vivo and in vitro properties of BirA superrepressors Biotinylation bioF::lacZY bioF::lacZY ΔG°biotin ΔG°dim, holo activitiesc, Variant a b expression derepression kcat/Km (kcal/mol) (kcal/mol) (M-1s-1) wt 17.3 7.36 1.6±0.3x105 -9.90±0.02 -6.7±0.2 I187T 1.6 3.04 1.16±0.02x105 -9.76±0.03 -7.4±0.4 K267M 5.4 4.14 + -9.97±0.02 -7.0±0.2 5 M310L 4.9 2.61 1.4±0.1x10 -9.98±0.03 -7.6±0.4 P143T 4.2 2.24 + -9.90±0.03 -7.6±0.4 Y178C 4.1 3.04 + -9.90±0.03 -4.5±0.4 G154D 0.3 0.86 n.d. -9.84±0.04 -9.6±0.3 a

The bioF::lacZY expression was determined using β-galactosidase activity assays in cell culture containing 1.6nM biotin17. b Fold bioF::lacZY derepression in response to overexpression of the biotin acceptor protein (AccB-AccC) measured in 40nM biotin. c The symbol + represents the ability to ligate biotin to AccB-87 in vitro, whereas n.d. represents no detectable biotin transfer.

Given the complexity of the E. coli Biotin Regulatory System, multiple

potential sources of the observed discrepancy between the in vivo and in vitro results obtained for the BirAY178C variant were considered. For example, this variant is altered in its competitive interaction with apoBCCP in biotin transfer. However, in vivo measurements of derepression resulting from BCCP overexpression and in vitro measurements of biotin transfer indicate that, with the exception of the G154D variant, all superrepressors are similar to BirAwt in their biotin transfer properties (Table 4) 17. Moreover, in the strain used for identifying superrepressor mutants the biotin transfer function was supplied by a plasmid born heterologous yeast biotin protein ligase17, which rendered the superrepressor phenotype independent of biotin transfer function. A second potential source of the discrepancy is that the affinity of the holoBirAY178C dimer for bioO is significantly higher than that of the holoBirAwt dimer. However, previous studies reveal that the energetics of the holoBirA dimer:bioO binding interaction is independent of dimerization free energy19. A final consideration that was not addressed in in vivo studies of the

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superrepressors is the intracellular concentrations of the variant proteins. However, overexpression of the BirAY178C variant for purification revealed no difference in its levels relative to those observed for the wild type protein.

The holoBirAwt structure combined with results of MD simulations provides

predictions of the molecular origins of the altered superrepressor dimerization properties. In BirAP143T and BirAM310L, the substituted residues are located in dimerization surface loops (Figure 3A). The P143 residue participates in the coil to helix transition that residues 142-‐145 undergo upon bio-‐5'-‐AMP binding. Previous studies of the holoBirAP143A variant, which dimerizes less tightly than does holoBirAwt, indicate that it does not undergo this transition27. Consistent with its relatively strong dimerization, simulations performed on holoBirAP143T indicate preservation of the helical structure. The M310L substitution, which enhances dimerization, is located in the BirA C-‐terminal domain and simulations suggest that this substitution alters dimerization through enhancing the packing between the 140-‐146 and 193-‐199 loops on the dimerization surface (Figure 4B).

In the G154D and Y178C variants with substitutions in the central domain

core, shifts in the relative orientation of the central and C-‐terminal domains may be responsible for altering BirA dimerization. Bio-‐5'-‐AMP binding to BirAwt is accompanied by formation of an electrostatic network involving BBL residues R118, R119 and R121, which are disordered in the absence of ligand18; 38. Comparison of the apo and holoBirAwt structures indicates that bio-‐5'-‐AMP binding is accompanied by rotation of the C-‐terminal domain relative to the central domain 18. Since the electrostatic network physically connects the BBL to the C-‐terminal domain, this rotation is likely associated with network formation. Simulations predict that the electrostatic network is altered in both holoBirAG154D and holoBirAY178C. Moreover, although backbone C alignments indicate that the structure of the domain itself is α

similar for the wild type and variant proteins, BirAG154D and BirAY178C show a shift in the orientation of the C-‐terminal domain relative to the central domain (Figure 6).

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Future structural studies will provide tests of this predicted coupling between electrostatic network formation and the domain shifts in BirA.

Modeling of the biotin concentration dependence of transcription repression

complex assembly reveals that it is highly sensitive to holoBirA dimerization energetics. As indicated in the Introduction (Figure 1), biotin operon transcription repression complex assembly occurs in multiple steps. First, biotin followed by ATP binding results in bio-‐5'-‐AMP synthesis and accumulation of holoBirA monomer. This obligatorily ordered mechanism renders the fractional saturation of BirA by bio-‐5'-‐AMP dependent only on biotin binding. Therefore, holoBirA concentration can be predicted using the following equation: [holoBirA] = [BirA]total Ybiotin = [BirA]total

K A,biotin [biotin] (3) 1+ K A,biotin [biotin]

in which [holoBirA] is the BirA•bio-‐5’-‐AMP concentration, [BirA]total is the total BirA €concentration, Y biotin is the fractional saturation of BirA with biotin, KA,biotin is the

equilibrium association constant governing biotin binding to BirA and [biotin] is the free biotin concentration. HoloBirA assembly onto the biotin operator sequence, € bioO, occurs through coupled dimerization and holoBirA dimer binding to bioO. Thus, the fractional saturation of bioO by holoBirA,

, is expressed by the

following equation:

YbioO =

K DIM K bioO [holoBirA]2 1 +K DIM K bioO [holoBirA]2

(4)

€in which KDIM and KbioO are the equilibrium association constants governing

holoBirA dimerization and dimer binding to bioO, respectively. Combined Equations (3) and (4) allow predictions of the dependence of biotin operator occupancy on biotin concentration at a single total BirA concentration16. These predicted curves have previously been shown to agree reasonably well with in vivo measurements of the dependence of transcription level

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on biotin concentration16. Predicted fractional saturation curves for four BirA variants are shown in Figure 7. The KA,biotin and KDIM values used in the simulations were obtained from the ITC and sedimentation equilibrium measurements, respectively, reported in this work. The magnitude of the KbioO parameter used for all variants was the value obtained for holoBirAwt binding using DNaseI footprint titration19. The total BirA concentration of 6.6x10-‐7M was calculated from the absolute protein synthesis rate obtained by ribosome-‐profiling 39 (http://ecoliwiki.net/tools/proteome/) and cell volume for E. coli estimated as 1.3 µm3 (http://book.bionumbers.org/how-‐big-‐is-‐an-‐e-‐coli-‐cell-‐and-‐what-‐is-‐its-‐mass/).

Figure 7: Modeling sensitivity of the dependence of bioO fractional saturation by BirA on biotin concentration for BirAG154D:Pink:, BirAM3!0L Blue, BirAwt:Black; BirAY178C:Orange. See text for a description of the equations and parameters used in generating the curves. The steep dependence of bioO saturation on biotin concentration observed in all curves reflects the coupling between biotin binding, which triggers bio-‐5'-‐AMP synthesis, dimerization and DNA binding in repression complex assembly (Figure

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7). The tighter dimerizing protein, BirAM310L, occupies the operator at lower biotin concentration than does BirAwt. BioO saturation by the G154D variant is sensitive to even lower biotin concentration, with a midpoint or 0.5 fractional saturation occurring at nanomolar biotin concentration. By contrast, the weakly dimerizing Y178C variant requires significantly higher biotin concentration to achieve bioO occupancy. Thus, the altered dimerization energetics of the variants are predicted to result in a two orders of magnitude range of sensitivities of occupancy/repression to biotin concentration. Direct comparison of the predicted dependence of bioO occupancy on biotin concentration will require detailed in vivo measurements.16

In general several factors can contribute to the sensitivity of any

transcriptional response to the input stimulus. Two levels of sensitivity to input can be considered. The first refers to the concentration span of input molecule over which the response occurs, with a narrow span indicating high sensitivity to changes in input concentration. Coupled reactions are typically responsible for a steep response. For example, in the bacteriophage lambda lysogeny to lysis switch the steepness of the transcription response to changing input cI repressor concentration reflects coupling between protein:protein and protein:DNA interactions 40. The steepness or cooperativity of the transcription response to tetracycline concentration mediated by the tetracycline repressor is correlated with the arrangement and juxtaposition of regulatory sites as well as the promoter sequences associated with a particular regulon 41. For the biotin biosynthetic operon the steep transcription response to biotin concentration reflects the three-‐coupled equilibria that dictate the fractional occupancy of the bioO DNA sequence by holoBirA. A second measure of sensitivity is the minimum amount of input signal required to achieve a response. Altering the affinity of the regulatory protein for the input signal molecule biotin provides a direct route to changing this sensitivity. The results provided in this work indicate that a broad range of sensitivities of a transcriptional response to input signal concentration can also be achieved by modulating the self-‐association energetics of the transcription regulatory protein. Further, the BirA dimerization free energy is subject to significant changes resulting

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from single amino acid substitutions at amino acid positions that are distributed throughout the protein structure.

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Acknowledgements: This work was supported in part by NIH Grant S10 RR15899 to DB. The authors thank Dr. John E. Cronan for the plasmids used for protein expression. References 1.

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Rose, J. K., Squires, C. L., Yanofsky, C., Yang, H. L. & Zubay, G. (1973). Regulation of in vitro transcription of the tryptophan operon by purified RNA polymerase in the presence of partially purified repressor and tryptophan. Nature (London), New Biol. 245, 133-‐137. Barkley, M. D., Riggs, A. D., Jobe, A. & Burgeois, S. (1975). Interaction of effecting ligands with lac repressor and repressor-‐operator complex. Biochemistry 14, 1700-‐1712. Lane, M. D., Rominger, K. L., Young, D. L., and Lynen, F. (1964). The enzymatic synthesis of holotranscarboxylase from apotranscarboxylase and (+)-‐biotin. J. Biol. Chem. 239, 2865-‐2871. Semavina, M., Beckett, D. & Logan, T. M. (2006). Metal-‐linked dimerization in the iron-‐dependent regulator from Mycobacterium tuberculosis. Biochemistry 45, 12480-‐12490. Barker, D. F., and Campbell, A. M. (1981). The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase. J. Mol. Biol 146, 451-‐467. Barker, D. F. & Campbell, A. M. (1981). Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli. J. Mol. Biol. 146, 469-‐492. Cronan, J. E., Jr. (1989). The E.coli bio operon:transcriptional repression by an essential protein modification enzyme. Cell 58, 427-‐429. Eisenstein, E. & Beckett, D. (1999). Dimerization of the Escherichia coli biotin repressor: corepressor function in protein assembly. Biochemistry 38, 13077-‐ 13084. Streaker, E. D., and Beckett, D. (2003). Coupling of protein assembly and DNA binding:biotin repressor dimerization preceeds biotin operator binding. J. Mol. Biol. 325, 937-‐948. Li, S. J. & Cronan, J. E., Jr. (1993). Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis. J Bacteriol. 175, 332-‐340. Streaker, E. D. & Beckett, D. (2006). The biotin regulatory system: kinetic control of a transcriptional switch. Biochemistry 45, 6417-‐6425. Zhao, H. & Beckett, D. (2008). Kinetic partitioning between alternative protein-‐protein interactions controls a transcriptional switch. J Mol Biol 380, 223-‐236. Barker, D. F. & Campbell, A. M. (1980). Use of bio-‐lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli. J Bacteriol. 143, 789-‐800. Streaker, E. D., Gupta, A., and Beckett, D. (2002). The biotin repressor: thermodynamic coupling of corepressor binding, protein assembly, and sequence-‐specific DNA binding. Biochemistry 41, 14263-‐14271. 32


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