Functional studies of the Mycobacterium tuberculosis iron-dependent regulator

The iron-dependent regulator (IdeR) protein in Mycobacterium tuberculosis, and its better characterized homologue, the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae, are iron-dependent regulatory proteins that control gene expression in response to iron availability in bacteria. IdeR regulates several genes required for iron uptake and storage including those involved in the synthesis of transition metal chelators called siderophores that are linked to the M. tuberculosis virulence. In this study, the metal ion and binding affinities for IdeR binding to an fxbA operator duplex DNA were estimated using fluorescence assays. The Fe(2+), Co(2+), and Ni(2+) affinities of the two metal ion binding sites in IdeR that are involved in the activation of the regulator DNA binding process in vitro were independently estimated. Binding to the two metal ion binding sites is apparently cooperative and the two affinities differ significantly. Occupation of the first metal ion binding site causes dimerization of IdeR, and the metal ion affinity is about 4 microM for Ni(2+) and much less for Fe(2+) and Co(2+). Binding of the second metal ion fully activates IdeR for binding to the fxbA operator. The equilibrium metal ion dissociation constants for IdeR-fxbA operator binding are approximately 9 microM for Fe(2+), 13 microM for Ni(2+), and 23 microM for Co(2+). Interestingly, the natural IdeR cofactor, Fe(2+), shows high affinities toward both binding sites. These results provide insight into the possible roles for each metal binding site in IdeR activation.     

Characterization of the binding of Cu(II) and Zn(II) to transthyretin: effects on amyloid formation

Although metal ions can promote amyloid formation from many proteins, their effects on the formation of amyloid from transthyretin have not been previously studied. We therefore screened the effects of Cu(II), Zn(II), Al(III), and Fe(III) on amyloid formation from wild-type (WT) transthyretin as well as its V30M, L55P, and T119M mutants. Cu(II) and Zn(II) promoted amyloid formation from the L55P mutant of transthyretin at pH 6.5 but had little effect on amyloid formation from the other forms of the protein. Zn(II) promoted L55P amyloid formation at pH 7.4 but Cu(II) inhibited it. Cu(II) gave dose-dependent quenching of the tryptophan fluorescence of transthyretin and the fluorescence of 1-anilino-8-naphthalene sulfonate bound to it. Zn(II) gave dose-dependent quenching of the tryptophan but not the 1-anilino-8-naphthalene sulfonate fluorescence. Apparent dissociation constants for Cu(II) and Zn(II) binding at pH 7.4 of approximately 10 nM and approximately 1 microM (approximately 0.4 microM and approximately 5 microM at pH 6.5), respectively, were obtained from the quenching data. Zn(II) enhanced urea-mediated the dissociation of the L55P but not the WT transthyretin tetramer. Cu(II), depending on its concentration, either had no effect or stabilized the WT tetramer but could enhance urea-mediated dissociation of L55P.     

Coupled energetics of lambda cro repressor self-assembly and site-specific DNA operator binding II: cooperative interactions of cro dimers

The bacteriophage lambda relies on interactions of the cI and cro repressors which self assemble and bind the two operators (O(R) and O(L)) of the phage genome to control the lysogenic to lytic switch. While the self assembly and O(R) binding of cI have been investigated in detail, a more complete understanding of gene regulation by phage lambda also requires detailed knowledge of the role of cro repressor as it dimerizes and binds at O(R) sites. Since dimerization and operator binding are coupled processes, a full elucidation of the regulatory energetics in this system requires that the equilibrium constants for dimerization and cooperative binding be determined. The dimerization constant for cro has been measured as a prelude to these binding studies. Here, the energetics of cro binding to O(R) are evaluated using quantitative DNaseI footprint titration techniques. Binding data for wild-type and modified O(R) site combinations have been simultaneously analyzed in concert with the dimerization energetics to obtain both the intrinsic and cooperative DNA binding energies for cro with the three O(R) sites. Binding of cro dimers is strongest to O(R)3, then O(R)1 and lastly, O(R)2. Adjacently bound repressors exhibit positive cooperativity ranging from -0.6 to -1.0 kcal/mol. Implications of these, newly resolved, energetics are discussed in the framework of a dynamic model for gene regulation. This characterization of the DNA-binding properties of cro repressor establishes the foundation on which the system can be explored for other, more complex, regulatory elements such as cI-cro cooperativity.     

Metal ion binding to alpha-lactalbumin species

A strong cation (calcium) binding site has been demonstrated to exist in several alpha-lactalbumin species; bovine, goat, human, and guinea pig. A metal ion induced conformational change occurs, resulting in a unique (10-14-nm) blue shift and relative quenching of Trp fluorescence for all species. Calcium ion binding to the alpha-lactalbumins yielded dissociation constants (Kdiss consistently in the 10(-10)--10(-12) M range, while Mn(II) binding was in the 20-30 microM range. Independent determinations of these cation binding equilibria were made by ESR measurements of free unliganded Mn(II) in titrations with the bovine species. One strong site (Kdiss = 30.5 microM) was found, which correlated directly with the fluorescence-associated cation binding, plus three weaker sites (Kdiss = 1.1, 5.0, and 5.0 mM, respectively). Several lanthanides as well as Mg(II) were found to displace Mn(II) from the strong site on bovine alpha-lactalbumin (as monitored by ESR) and to cause the identical fluorescence changes as found for Ca(II) and Mn(II) above. The importance of measuring these equilibria by both fluorescence and ESR was borne out by demonstrating the potential errors in estimating dissociation equilibria by the fluorescence method alone. Also, the errors in estimating Kdiss for samples containing partially metal bound apo-alpha-lactalbumin are described as well as rapid, sensitive methods for estimating the extent of metal-free protein and correctly accounting for residual bound metal in equilibrium calculations.     

Reversible ligand-induced dissociation of a tryptophan-shift mutant of phosphofructokinase from Bacillus stearothermophilus

The biophysical properties of a tryptophan-shifted mutant of phosphofructokinase from Bacillus stearothermophilus (BsPFK) have been examined. The mutant, designated W179Y/Y164W, has kinetic and thermodynamic properties similar to the wild-type enzyme. A 2-fold decrease in kcat is observed, and the mutant displays a 3-fold smaller K(0.5) for the substrate, fructose-6-phosphate (Fru-6-P), as compared to the wild-type enzyme. The dissociation constant for the inhibitor, phospho(enol)pyruvate (PEP), increases 2-fold, and the coupling parameter, Q(ay), decreases 2-fold. This suggests that while the mutant displays a slightly decreased affinity for PEP, PEP is still an effective inhibitor once bound. The new position of the tryptophan in W179Y/Y164W is approximately 6 A from the Fru-6-P portion of the active site. A 25% decrease in fluorescence intensity is observed upon Fru-6-P binding, and an 80% decrease in fluorescence intensity is observed with PEP binding. In addition, the intrinsic fluorescence polarization increases from 0.327 +/- 0.001 to 0.353 +/- 0.001 upon Fru-6-P binding, but decreases to 0.290 +/- 0.001 when PEP binds. Most notably, the presence of PEP induces dissociation of the tetramer. Dissociation of the tetramer into dimers occurs along the active site interface and can be monitored by the loss in activity or the loss in tryptophan fluorescence that is observed when the enzyme is titrated with PEP. Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.     

Kinetics of the association/dissociation cycle of an ATP-binding cassette nucleotide-binding domain

Most ATP binding cassette (ABC) proteins are pumps that transport substrates across biological membranes using the energy of ATP hydrolysis. Functional ABC proteins have two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is unresolved. This is due in part to the limited kinetic information on NBD association and dissociation. Here, we show dimerization of a catalytically active NBD and follow in real time the association and dissociation of NBDs from the changes in fluorescence emission of a tryptophan strategically located at the center of the dimer interface. Spectroscopic and structural studies demonstrated that the tryptophan can be used as dimerization probe, and we showed that under hydrolysis conditions (millimolar MgATP), not only the dimer dissociation rate increases, but also the dimerization rate. Neither dimer formation or dissociation are clearly favored, and the end result is a dynamic equilibrium where the concentrations of monomer and dimer are very similar. We proposed that based on their variable rates of hydrolysis, the rate-limiting step of the hydrolysis cycle may differ among full-length ABC proteins.     

Cooperative binding of copper(I) to the metal binding domains in Menkes disease protein.

We have optimised the overexpression and purification of the N-terminal end of the Menkes disease protein expressed in Escherichia coli, containing one, two and six metal binding domains (MBD), respectively. The domain(s) have been characterised using circular dichroism (CD) and fluorescence spectroscopy, and their copper(I) binding properties have been determined. Structure prediction derived from far-UV CD indicates that the secondary structure is similar in the three proteins and dominated by beta-sheet. The tryptophan fluorescence maximum is blue-shifted in the constructs containing two and six MBDs relative to the monomer, suggesting more structurally buried tryptophan(s), compared to the single MBD construct. Copper(I) binding has been studied by equilibrium dialysis under anaerobic conditions. We show that the copper(I) binding to constructs containing two and six domains is cooperative, with Hill coefficients of 1.5 and 4, respectively. The apparent affinities are described by K(0.5), determined to be 65 microM and 19 microM for constructs containing two and six domains, respectively. Our data reveal a unique regulation of Menkes protein upon a change in copper(I) concentration. The regulation does not occur as an 'all-or-none' cooperativity, suggesting that the copper(I) binding domains have a basal low affinity for binding and release of copper(I) at low concentrations but are able to respond to higher copper levels by increasing the affinity, thereby contributing to prevent the copper concentration from reaching toxic levels in the cell.     

Protein interactions with immobilized transition metal ions: quantitative evaluations of variations in affinity and binding capacity

The interaction of proteins with immobilized transition-metal ions proceeds via mechanisms influenced by metal type and degree of coordination, variations in mobile phase constituents, and protein surface architecture at or near the metal binding site(s). The contributions each of these variables make toward the affinity of protein surfaces for immobilized metal ions remain empirical. We have used equilibrium binding analyses to evaluate the influence of pH and competitive binding reagents on the apparent equilibrium dissociation constant (Kd) and binding capacity of immobilized Cu(II) and Ni(II) ions for several model proteins of known three-dimensional structure. Linear Scatchard plots suggested that 8/13 of the proteins evaluated interacted with immobilized metal ions via a single class of operational (Kd = 10-700 microM) binding sites. Those proteins with the highest affinities for the immobilized Cu(II) ions (5/13) showed evidence of multiple, non-identical or nonindependent binding sites. The effects of altered metal type, pH, and concentration of competitive affinity reagents (e.g., imidazole, free metal ions) on the apparent Kd and binding capacity varied in magnitude for individual proteins. The presence of free Cu(II) ions did not detectably alter either the affinity or binding capacity of the proteins for immobilized Cu(II) ions. The expected relationship between the relative chromatographic elution sequence and calculated affinity constants was not entirely evident by evaluation under only one set of conditions. Our results demonstrate the utility of nonchromatographic equilibrium binding analyses for the quantitative evaluation of experimental variables affecting the relative affinity and capacity of immobilized metal ions for proteins. This approach affords the opportunity to improve understanding and to vary the contribution of interaction mechanisms involved.     

Binding of Cu(II) or Zn(II) in a de novo designed triple-stranded alpha-helical coiled-coil toward a prototype for a metalloenzyme.

We previously reported the IZ-3adH peptide, which formed a triple-stranded coiled-coil after binding Ni(II), Cu(II), or Zn(II). In this paper, we report the peptide, IZ-3aH, having a new metal binding specificity. The IZ-3aH peptide was found to bind Cu(II) and Zn(II) and form a triple-stranded coiled-coil. However, it did not bind Ni(II). Metal ion titrations monitored by circular dichroism revealed that the dissociation constants, K(d) were 9 microm for Zn(II) and 10 microm for Cu(II). The bound Cu(II) ion has a planar tetragonal geometry, where the coordination positions are three nitrogens of the His residues and one H(2)O.     

Induced fit and kinetic mechanism of adenylation catalyzed by Escherichia coli threonyl-tRNA synthetase

Threonyl-tRNA synthetase (ThrRS) must discriminate among closely related amino acids to maintain the fidelity of protein synthesis. Here, a pre-steady state kinetic analysis of the ThRS-catalyzed adenylation reaction was carried out by monitoring changes in intrinsic tryptophan fluorescence. Stopped flow fluorimetry for the forward reaction gave a saturable fluorescence quench whose apparent rate increased hyperbolically with ATP concentration, consistent with a two-step mechanism in which rapid substrate binding precedes an isomerization step. From similar experiments, the equilibrium dissociation constants for dissociation of ATP from the E.Thr complex (K(3) = 450 +/- 180 microM) and threonine from the E.ATP complex (K'(4) = 135 microM) and the forward rate constant for adenylation (k(+5) = 29 +/- 4 s(-1)) were determined. A saturable fluorescence increase accompanied the pyrophosphorolysis of the E.Thr - AMP complex, affording the dissociation constant for PP(i) (K(6) = 170 +/- 50 microM) and the reverse rate constant (k(-5) = 47 +/- 4 s(-1)). The longer side chain of beta-hydroxynorvaline increased the apparent dissociation constant (K(4[HNV]) = 6.8 +/- 2.8 mM) with only a small reduction in the forward rate (k'(+5[HNV]) = 20 +/- 3.1 s(-1)). In contrast, two nonproductive substrates, threoninol and the adenylate analogue 5'-O-[N-(L-threonyl)sulfamoyl]adenosine (Thr-AMS), exhibited linear increases in k(app) with ligand concentration, suggesting that their binding is slow relative to isomerization. The proposed mechanism is consistent with steady state kinetic parameters. The role of threonine binding loop residue Trp434 in fluorescence changes was established by mutagenesis. The combined kinetic and molecular genetic analyses presented here support the principle of induced fit in the ThrRS-catalyzed adenylation reaction, in which substrate binding drives conformational changes that orient substrates and active site groups for catalysis.     

Reversible loss of cooperative calcium ion binding by sarcoplasmic reticulum calcium adenosinetriphosphatase

Incubation of rabbit skeletal muscle sarcoplasmic reticulum vesicles in solutions of very low [Ca2+] caused Ca2+ to bind noncooperatively, as determined by the dependence of the intrinsic tryptophan fluorescence intensity on added increments of Ca2+. Cooperative Ca2+ binding was obtained if the ATPase was incubated in [Ca2+] high enough (25 microM) to saturate the two high-affinity Ca2+ binding sites and then titrated with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The cooperative binding had an apparent association constant of 6.3 X 10(6) M-1 and a Hill coefficient of 2.6; these constants for the noncooperative binding case were 5.0 X 10(5) M-1 and 1.2, respectively. The transitions from the noncooperative to the cooperative Ca2+ binding forms of the enzyme were slow compared to the time required for Ca2+ binding to reach equilibrium. Thus, it appears that sarcoplasmic reticulum CaATPase is a hysteretic enzyme. Intrinsic association constants for Ca2+ binding and equilibrium constants for the transitions between the two forms in low and high [Ca2+] were estimated from analyses of a general scheme for cooperative and noncooperative binding.     

Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrices

Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple metal ions drive DNA association by PvuII endonuclease

Restriction enzymes serve as important model systems for understanding the role of metal ions in phosphodiester hydrolysis. To this end, a number of laboratories have reported dramatic differences between the metal ion-dependent and metal ion-independent DNA binding behaviors of these systems. In an effort to illuminate the underlying mechanistic details which give rise to these differences, we have quantitatively dissected these equilibrium behaviors into component association and dissociation rates for the representative PvuII endonuclease and use these data to assess the stoichiometry of metal ion involvement in the binding process. The dependence of PvuII cognate DNA on Ca(II) concentration binding appears to be cooperative, exhibiting half-saturation at 0.6 mM metal ion and yielding an n(H) of 3.5 +/- 0.2 per enzyme homodimer. Using both nitrocellulose filter binding and fluorescence assays, we observe that the cognate DNA dissociation rate (k(-)(1) or k(off)) is very slow (10(-)(3) s(-)(1)) and exhibits a shallow dependence on metal ion concentration. DNA trap cleavage experiments with Mg(II) confirm the general irreversibility of DNA binding relative to cleavage, even at low metal ion concentrations. More dramatically, the association rate (k(1) or k(on)) also appears to be cooperative, increasing more than 100-fold between 0.2 and 10 mM Ca(II), with an optimum value of 2.7 x 10(7) M(-)(1) s (-)(1). Hill analysis of the metal ion dependence of k(on) indicates an n(H) of 3.6 +/- 0.2 per enzyme dimer. This value is consistent with the involvement in DNA association of two metal ions per subunit active site, a result which lends new strength to arguments for two-metal ion mechanisms in restriction enzymes.     

The effect of F-actin on the relay helix position of myosin II, as revealed by tryptophan fluorescence, and its implications for mechanochemical coupling

The fluorescence properties of Dictyostelium discoideum (Dd) myosin II constructs containing a single tryptophan residue have revealed detailed information regarding nucleotide binding and hydrolysis steps. Here we extend these studies to investigate the influence of actin on nucleotide-induced fluorescence transients. The fluorescence from native actin tryptophan residues is not significantly perturbed on binding to myosin, although an apparent signal is detected as a consequence of a light scatter artifact. Actin has a minor effect on the response of W129, located at the entrance to the nucleotide-binding pocket, and reduces the forward rate constants for the isomerization(s) associated with binding of ATP, ATPgammaS, and ADP by 3-fold or less. The isomerization detected by W129 clearly precedes the dissociation of actin in the case of ADP and ATPgammaS binding. The fluorescence from the conserved W501 residue, located at the distal end of the relay helix, is very sensitive to the switch 2 and/or lever arm disposition. Consequently, the observed fluorescence emission intensity can be used to estimate the equilibrium constant between the pre- and post-power stroke conformations. Actin modulates this equilibrium by no more than 2-fold in the presence of nucleoside triphosphate. These data have implications for the mechanism of product release and suggest that actin activates another process in the mechanism, such as switch 1 movement and Pi release, rather than influencing the switch 2 equilibrium and lever arm position directly.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: reduced nicotinamide adenine dinucleotide phosphate binding and formation and reduction of acetoacetyl-enzyme

The kinetics of reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding to fatty acid synthase from chicken liver and of the reduction of enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase) and the steps leading to formation of the acetoacetyl-enzyme have been studied in 0.1 M potassium phosphate-1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, at 25 degrees C by monitoring changes in NADPH fluorescence with a stopped-flow apparatus. Improved fluorescence detection has permitted the use of NADPH concentrations as low as 20 nM. The kinetics of the binding of NADPH to the enzyme is consistent with a simple bimolecular binding mechanism and four equivalent sites on the enzyme (presumably two beta-ketoacyl reductase sites and two enoyl reductase sites). The bimolecular rate constant is 12.7 X 10(6) M-1 s-1, and the dissociation rate constant is 76.7 s-1, which gives an equilibrium dissociation constant of 6.0 microM. The formation of the acetoacetyl-enzyme and its subsequent reduction by NADPH could be analyzed as two consecutive pseudo-first-order reactions by mixing enzyme-NADPH with acetyl-CoA and malonyl-CoA under conditions where [acetyl-CoA], [malonyl-CoA] much greater than [enzyme] much greater than [NADPH]. From the dependence of the rate of reduction of aceto-acetyl-enzyme by NADPH on enzyme concentration, an independent estimate of the equilibrium dissociation constant for NADPH binding to the enzyme of 5.9 microM is obtained, and the rate constant for the reduction is 17.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)