Isolation of mutations that disrupt cooperative DNA binding by the Drosophila bicoid protein.

Cooperative DNA binding is thought to contribute to the ability of the Drosophila melanogaster protein, Bicoid, to stimulate transcription of target genes in precise sub-domains within the embryo. As a first step toward testing this idea, we devised a genetic screen to isolate mutations in Bicoid that specifically disrupt cooperative interactions, but do not disrupt DNA recognition or transcription activation. The screen was carried out in Saccharomyces cerevisiae and 12 cooperativity mutants were identified. The mutations map across most of the Bicoid protein, with some located within the DNA-binding domain (homeodomain). Four homeodomain mutants were characterized in yeast and shown to activate a single-site reporter gene to levels comparable to that of wild-type, indicating that DNA binding per se is not affected. However, these mutants failed to show cooperative coupling between high and low-affinity sites, and showed reduced activation of a reporter gene carrying a natural Drosophila enhancer. Homology modeling indicated that none of the four mutations is in residues that contact DNA. Instead, these residues are likely to interact with other DNA-bound Bicoid monomers or other parts of the Bicoid protein. In vitro, the isolated homeodomains did not show strong cooperativity defects, supporting the idea that other regions of Bicoid are also important for cooperativity. This study describes the first systematic screen to identify cooperativity mutations in a eukaryotic DNA-binding protein.

The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations.

Global conformational transitions are of central functional importance for many enzymes and binding proteins. It is not known, however, how much variability can exist in such structural-functional linkages. We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography. We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery.

Cooperative non-specific DNA binding by octamerizing lambda cI repressors: a site-specific thermodynamic analysis.

Relationships between dimerization and site-specific binding have been characterized previously for wild-type and mutant cI repressors at the right operator (OR) of bacteriophage lambda DNA. However, the roles of higher-order oligomers (tetramers and octamers) that are also formed from these cI molecules have remained elusive. In this study, a clear correlation has been established between repressor oligomerization and non-specific DNA-binding activity. A modification of the quantitative DNase I footprint titration technique has been used to evaluate the degree of saturation of non-specific, OR-flanking lambda DNA by cI repressor oligomers. With the exception of one mutant, only those repressors capable of octamerizing were found to exhibit non-specific DNA-binding activity. The non-specific interaction was accurately modeled using either a one-dimensional, univalent, site-specific Ising lattice approximation, or a more traditional, multivalent lattice approach. It was found that non-specific DNA-binding by repressor oligomers is highly cooperative and energetically independent from site-specific binding at OR. Furthermore, the coupling free energy resolved for non-specific binding was similar to that of site-specific binding for each repressor, suggesting that similar structural elements may mediate the cooperative component of both binding processes. It is proposed that the state of assembly of the repressor molecule modulates its relative affinity for specific and non-specific DNA sequences. These specificities are allosterically regulated by the transmission of assembly-state information from the C-terminal domain, which mediates self-association and cooperativity, to the N-terminal domain, which primarily mediates DNA-binding. While dimers have a high affinity for their cognate sites within OR, tetramers and octamers may preferentially recognize non-specific DNA sequences. The concepts and findings developed in this study may facilitate quantitative characterization of the relationships between specific, and non-specific binding in other systems that utilize multiple modes of DNA-binding cooperativity.

Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo.

The Bicoid morphogen directs pattern formation along the anterior-posterior (A-P) axis of the Drosophila embryo. Bicoid is distributed in a concentration gradient that decreases exponentially from the anterior pole, however, it transcribes target genes such as hunchback in a step-function-like pattern; the expression domain is uniform and has a sharply defined posterior boundary. A 'gradient-affinity' model proposed to explain Bicoid action states that (i) cooperative gene activation by Bicoid generates the sharp on/off switch for target gene transcription and (ii) target genes with different affinities for Bicoid are expressed at different positions along the A-P axis. Using an in vivo yeast assay and in vitro methods, we show that Bicoid binds DNA with pairwise cooperativity; Bicoid bound to a strong site helps Bicoid bind to a weak site. These results support the first aspect of the model, providing a mechanism by which Bicoid generates sharp boundaries of gene expression. However, contrary to the second aspect of the model, we find no significant difference between the affinity of Bicoid for the anterior gene hunchback and the posterior gene knirps. We propose, instead, that the arrangement of Bicoids bound to the target gene presents a unique signature to the transcription machinery that, in combination with overall affinity, regulates the extent of gene transcription along the A-P axis.

Cooperativity mutants of bacteriophage lambda cI repressor: temperature dependence of self-assembly.

Analytical ultracentrifugation was used to study higher order self-assembly of lambda cI repressors, including eight mutants whose monomer to dimer reactions were recently characterized [Burz et al. (1994) Biochemistry 33, 8399]. Six of the mutants were found to remain dimeric up to 50 microM total protein; the remaining mutants (EK102 and PT158) were found to undergo higher order oligomerization, as does wild-type cI. For these three repressors, we determined the stoichiometries and energetics of higher order assembly over the temperature range 5-40 degrees C. Weak dimerization exhibited by two other mutants, SN228 and SR228, was also evaluated by sedimentation equilibrium over this same temperature range. The end-state for higher order assembly of wild-type cI was determined to be octameric, in agreement with Senear et al. [(1993) Biochemistry 32, 6179-6189]. The assembly free energies resolved by the sedimentation analysis program NONLIN [Johnson, M. L., et al. (1981) Biophys. J. 36, 575-588] leads to the prediction that tetramers may contribute significantly to the intermediate populations during assembly. This analysis of the species populations is in accord with recent conclusions from fluorescence anisotropy studies [Banik et al. (1993) J. Biol. Chem. 268, 3938]. It was found that two of the mutant repressors (EK102 and PTI58) assemble into octamers, but with differing possible intermediates. PT158 satisfies the stoichiometry 8M1 <--> 2M4 <--> M8, while the EK102 data conforms to a 4M2 <--> 2M4 <--> M8 model, similar to WT (both the EK102 and WT data could also be described by a dimer-octamer model with no intermediates). Of the six repressors found in this study to remain dimeric, three exhibit non-cooperative DNA binding (GD147, KN192, YH210), two express intermediate cooperativity (EK188, SR228), and one is fully cooperative (SN228). The three octamerizing repressors are fully cooperative [Burz & Ackers (1994) Biochemistry 33, 8399], suggesting a correlation between their ability to form higher order assemblies and to engage in cooperative DNA binding. Linear van't Hoff plots were obtained for overall assembly of wild-type and EK102 dimers, while that of PT158 monomers was curved, indicating a negative heat capacity change. The van't Hoff analyses of dimerization constants for SN228 and SR228 were distinctly different from each other and also from that of wild type; such differences might be related to the disparate cooperative behavior found previously for these mutants (Burz & Ackers, 1994).

Self-assembly of bacteriophage lambda cI repressor: effects of single-site mutations on the monomer-dimer equilibrium.

Dimerization of lambda cI repressor monomers is required for high-affinity binding to bacteriophage lambda operator DNA and is known to involve protein-protein contacts between C-terminal domains of the repressor monomers. In order to address the importance of the C-terminal domain in mediating the oligomeric properties of dimerization and cooperative binding to operator DNA, eight single-site mutant repressors were screened for possible deficiencies in cooperative interactions; all but one of the amino acid substitutions are located within the C-terminal domain. As a prelude to binding studies and the complete characterization of cooperativity mutants of lambda cI repressor (Burz, D. S., & Ackers, G. K. (1994) Biochemistry 33, 8406-8416), the thermodynamics of self-assembly of seven of these mutants was examined from 10(-11) to 10(-5) M total repressor using analytical gel chromatography. Results show that the structural perturbation accompanying single amino acid replacement does not significantly affect the monomer-dimer equilibrium with the exception of that accompanying replacements of serine 228; mutations at that site weaken, by 2-4 kcal/mol, the protein-protein interactions responsible for self-association. An additional mutant repressor, Pro158-->Thr, was also examined and found to associate reversibly from monomers to a species with stoichiometry greater than 2. All mutations increase the apparent Stokes radius of the monomeric form by 2-4.5 A and that of dimers by 1 or 3 A.

Single-site mutations in the C-terminal domain of bacteriophage lambda cI repressor alter cooperative interactions between dimers adjacently bound to OR.

Wild-type cI repressor dimers bind with 2.5-3 kcal/mol of cooperative free energy to the tripartite right operator region (OR) of bacteriophage lambda [Johnson, A. D., et al. (1981) Nature 294, 217-223; Brenowitz, M., et al. (1986) Methods Enzymol. 130, 132-181]. Quantitative modeling has suggested that cooperativity is required for maintenance of the lysogenic state and for the efficient switch from lysogenic to lytic growth [Ackers, G. K., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1129-1133; Shea, M. A., & Ackers, G. K. (1985) J. Mol. Biol. 181, 211-230]. Cooperativity and self-association are thought to involve protein-protein contacts between C-terminal domains of the repressor molecule [Pabo, C. O., et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1608-1612]. To address the importance of the C-terminal domain in mediating the cooperativity exhibited by lambda cI repressor, a number of single-site mutant candidates were screened for possible deficiencies in cooperative interactions [Beckett, D., et al. (1993) Biochemistry 32, 9073-9079; Burz, D. S., et al. (1994) Biochemistry 33, 8399-8405]. Since repressor dimerization and binding to operator sites are coupled processes, elucidation of the energetic basis of regulation in this system requires that the equilibrium dimerization constants and the intrinsic and cooperative free energies of binding be measured. In this work we evaluate the interaction of eight mutant repressors with OR DNA: Gly147-->Asp (GD147), Pro158-->Thr (PT158), Glu188-->Lys (EK188), Lys192-->Asn (KN192), Tyr210-->His (YH210), Ser228-->Arg (SR228), and Ser228-->Asn (SN228), each with an amino acid substitution in the C-terminal domain, and Glu102-->Lys (EK102) where the substitution lies in the "linker sequence" between domains. Self-assembly properties of six of these mutant repressors are presented in the preceding paper (Burz et al., 1994). In this work, the binding of mutant cI repressors to OR was examined using quantitative DNAse I footprinting. This technique monitors individual site occupancy concurrent with binding at the other sites within a multisite complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of lambda repressor mutants with defects in cooperative operator binding.

A hybrid operator-promoter region was designed to aid in a screen for cooperativity mutants of the lambda repressor. In this system, lambda repressor mutants with defects in pairwise cooperative binding are unable to act as efficient transcriptional repressors. Four single amino acid substitutions in the C-terminal domain of the repressor were isolated. Studies of the DNA binding properties of the purified mutant proteins show that a repressor bearing the Gly147-->Asp mutation binds with normal affinity to single operator sites but is defective in pairwise cooperative site binding. Quantitative footprinting studies show that the free energy of interaction between repressor dimers bound at operator sites OR1 and OR2 is reduced from -2.4 kcal/mol for the wild-type repressor to 0 kcal/mol for the GD147 mutant.

Differential scanning calorimetric studies of E. coli aspartate transcarbamylase. III. The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain.

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.

Use of analytical gel chromatography to analyze tertiary and quaternary structural changes in E. coli aspartate transcarbamylase.

E. coli aspartate transcarbamylase (ATCase) is a large (310 kDa) protein that undergoes major changes in quaternary structure when substrates and regulatory nucleotides bind. We have used analytical gel chromatography to detect quaternary structure changes in both the holoenzyme and its catalytic subunit (c3), to characterize the quaternary structure of single site mutant proteins and to monitor urea-induced dissociation and unfolding of c3. Binding of the bisubstrate analog PALA (N-(phosphonacetyl)-L-aspartate) to ATCase and c3 has been shown to alter s20.w by -3.3% and + 1.4%, respectively [Howlett, G.J. and Schachman, H.K. (1977), Biochemistry 23, 5077-5083]. The corresponding changes in the chromatographic partition coefficient (sigma) are -2.6 +/- 0.3% and 5.5 +/- 1.9% on Sephacryl S400HR and S200, respectively. Partition coefficients of mutant ATCases with single site mutations in the c chain differ from those of the wild-type protein by +/- 0.5% in small zone experiments; for example, mutations Arg 269----Gly and Glu 239----Gln alter the partition coefficient by 0.4% and -0.5%, respectively. The partition coefficient of mutant Glu 50----Gln is identical to the wild type enzyme. In the presence of saturating PALA, partition coefficients of Glu 50----Gln and Arg 269----Gly, but not Glu 239----Gln are identical to those of the wild type. Results for Glu 239----Gln are consistent with measurements of activity, small angle X-ray scattering and sedimentation coefficient that indicate that mutations at this site shift the quaternary structure towards the R state [Ladjimi and Kantrowitz (1988), Biochemistry 27, 276-83; Vachette and Hervé, cited by Kantrowitz and Lipscomb (1988), Science 241, 669-674; Newell and Schachman (1988), FASEB J. 2, A551]. Results for Glu 50----Gln are also consistent with measurements of activity (Ladjimi et al. (1988), Biochemistry 27, 268-276). The changes in tertiary and quaternary structure that result from urea-induced denaturation of c3 result in larger changes in the partition coefficient. Dissociation into folded monomers in 1-1.75 M urea is accompanied by a 4.6% increase in partition coefficient, while denaturation at greater than 5 M urea gives rise to a 43% decrease on S-300 Sephacryl. The bisubstrate analog PALA suppresses dissociation and increases the cooperativity of the unfolding reaction.

Effects of ATP and CTP on the conformation of the regulatory subunit of Escherichia coli aspartate transcarbamylase in solution: a medium-resolution hydrogen exchange study.

Medium-resolution hydrogen exchange methods have been used to examine the solvent accessibility of seven peptides in the regulatory subunit (r2) of Escherichia coli aspartate transcarbamylase in the presence and absence of ATP and CTP. Both nucleotides are allosteric effectors of the holoenzyme; binding of ATP increases the affinity of the holoenzyme for the substrate L-Asp, while CTP has the opposite effect. Following Rosa and Richards (1979, 1981, 1982) and Englander et al. (1983, 1985), exchange-out curves for individual peptides were generated by adjusting the pH to 2.7 to quench exchange-out, digesting the protein with pepsin, separating peptides by reverse-phase HPLC, determining their radioactivity, and correcting for radioactivity lost during the analysis. Sixteen peptides from segments 1-11 and 76-153 were identified by amino acid and N-terminal analysis. Nine fell in regions where background was too high or were present at too low concentrations for exchange to be monitored. The number of protons whose exchange could be followed in peptides 1-11, 76-91, 78-90, 84-101, 93-112, 108-114, and 115-125 ranged from approximately 1 (1-11, 108-114) to 10 (84-101) and 11 (93-112). The pattern of results obtained suggests that the structure of r2 in solution is similar to that of the regulatory subunits in crystalline ATCase. Both CTP and ATP reduce rates of exchange from all seven peptides except 115-125. Although CTP slows exchange more than ATP, the effect is small except for peptides 76-91 and 78-90 which are near the nucleotide binding site.(ABSTRACT TRUNCATED AT 250 WORDS)