Thermodynamics of Coupled Folding in the Interaction of Archaeal RNase P Proteins RPP21 and RPP29

We have used isothermal titration calorimetry (ITC) to identify and describe binding-coupled equilibria in the interaction between two protein subunits of archaeal ribonuclease P (RNase P). In all three domains of life, RNase P is a ribonucleoprotein complex that is primarily responsible for catalyzing the Mg²⁺-dependent cleavage of the 5′ leader sequence of precursor tRNAs during tRNA maturation. In archaea, RNase P has been shown to be composed of one catalytic RNA and up to five proteins, four of which associate in the absence of RNA as two functional heterodimers, POP5–RPP30 and RPP21–RPP29. Nuclear magnetic resonance studies of the <i>Pyrococcus furiosus</i> RPP21 and RPP29 proteins in their free and complexed states provided evidence of significant protein folding upon binding. ITC experiments were performed over a range of temperatures, ionic strengths, and pH values, in buffers with varying ionization potentials, and with a folding-deficient RPP21 point mutant. These experiments revealed a negative heat capacity change (Δ<i>C</i>_<i>p</i>), nearly twice that predicted from surface accessibility calculations, a strong salt dependence for the interaction, and proton release at neutral pH, but a small net contribution from these to the excess Δ<i>C</i>_<i>p</i>. We considered potential contributions from protein folding and burial of interfacial water molecules based on structural and spectroscopic data. We conclude that binding-coupled protein folding is likely responsible for a significant portion of the excess Δ<i>C</i>_<i>p</i>. These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies

Homotropic Cooperativity from the Activation Pathway of the Allosteric Ligand-Responsive Regulatory <i>trp</i> RNA-Binding Attenuation Protein

The <i>trp</i> RNA-binding attenuation protein (TRAP) assembles into an 11-fold symmetric ring that regulates transcription and translation of <i>trp</i>-mRNA in bacilli via heterotropic allosteric activation by the amino acid tryptophan (Trp). Whereas nuclear magnetic resonance studies have revealed that Trp-induced activation coincides with both microsecond to millisecond rigidification and local structural changes in TRAP, the pathway of binding of the 11 Trp ligands to the TRAP ring remains unclear. Moreover, because each of 11 bound Trp molecules is completely surrounded by protein, its release requires flexibility of Trp-bound (holo) TRAP. Here, we used stopped-flow fluorescence to study the kinetics of Trp binding by <i>Bacillus stearothermophilus</i> TRAP over a range of temperatures and observed well-separated kinetic steps. These data were analyzed using nonlinear least-squares fitting of several two- and three-step models. We found that a model with two binding steps best describes the data, although the structural equivalence of the binding sites in TRAP implies a fundamental change in the time-dependent structure of the TRAP rings upon Trp binding. Application of the two-binding step model reveals that Trp binding is much slower than the diffusion limit, suggesting a gating mechanism that depends on the dynamics of apo TRAP. These data also reveal that dissociation of Trp from the second binding mode is much slower than after the first Trp binding mode, revealing insight into the mechanism for positive homotropic allostery, or cooperativity. Temperature-dependent analyses reveal that both binding modes imbue increases in bondedness and order toward a more compressed active state. These results provide insight into mechanisms of cooperative TRAP activation and underscore the importance of protein dynamics for ligand binding, ligand release, protein activation, and allostery

pH Dependence of the Stress Regulator DksA

<div><p>DksA controls transcription of genes associated with diverse stress responses, such as amino acid and carbon starvation, oxidative stress, and iron starvation. DksA binds within the secondary channel of RNA polymerase, extending its long coiled-coil domain towards the active site. The cellular expression of DksA remains constant due to a negative feedback autoregulation, raising the question of whether DksA activity is directly modulated during stress. Here, we show that <i>Escherichia coli</i> DksA is essential for survival in acidic conditions and that, while its cellular levels do not change significantly, DksA activity and binding to RNA polymerase are increased at lower pH, with a concomitant decrease in its stability. NMR data reveal pH-dependent structural changes centered at the interface of the N and C-terminal regions of DksA. Consistently, we show that a partial deletion of the N-terminal region and substitutions of a histidine 39 residue at the domain interface abolish pH sensitivity in vitro. Together, these data suggest that DksA responds to changes in pH by shifting between alternate conformations, in which competing interactions between the N- and C-terminal regions modify the protein activity.</p></div

Δ<i>dksA</i> mutants are sensitive to acidic conditions.

<p>(A) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown overnight in rich medium at pH 7.8. Cultures were diluted 1:50 into LB medium at pH 2.5. At selected time points aliquots were taken and the percentage of survival of bacteria was determined using viable count. (B) WT and Δ<i>dksA E</i>. <i>coli</i> strains were grown at pH 7.8, followed by 2.5 hour adaptation at pH 6.5–4.5, and then diluted into LB medium at pH 3.5. Survival was determined using viable counts; the result after 2 hour incubation at pH 3.5 is shown. (C) DksA concentration remains relatively constant at low pH. Samples taken at different time points after a change in pH were analyzed using Western blotting with anti-DksA antibodies. Extract from the Δ<i>dksA</i> strain and purified DksA were loaded as controls.</p

DksA is sensitive to changes in pH.

<p>(A) DksA activity increases at low pH. Increasing concentrations of DksA were added to holo RNAP (30 nM), ApC dinucleotide (0.2 mM), UTP (0.2 mM), GTP (4 μM) and [α-³²P]-GTP (10 μCi of 3000 Ci mmol⁻¹) followed by incubation for 15 minutes in Transcription buffer (20 mM Tris-HCl pH 7.9, 20 mM NaCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, 0.1 mM EDTA). A linear DNA fragment containing the <i>rrnB</i> P1 promoter was added to initiate transcription and the formation of a 4 nucleotide RNA product was monitored on a denaturing 8% acrylamide gel. A dotted line marks the inhibition of 50% of transcription and is denoted as IC₅₀. The IC₅₀ values (calculated using a single-site binding equation from three independent repeats combined in a best-fit curve, in μM) were: pH 7.6 − 0.7 ± 0.28, pH 6.7 − 0.11 ± 0.016. (B) DksA affinity to core increases at lower pH. DksA binding to core RNAP was performed using the localized Fe²⁺ mediated cleavage assay at different pH. DksA concentrations were: 0, 25, 50, 100, 200 and 400 nM. FL—Full length protein, Cl—cleaved protein, Kd app—apparent Kd.</p

Substitution of His39 alters DksA sensitivity to pH.

<p>(A) The effect of pH on DksA variants. DksA activity and IC₅₀ calculations were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g003" target="_blank">Fig. 3A</a> with the <i>rrnB</i> P1 promoter. Experiments were performed at least three times at each pH. (B) Thermostability of DksA^H39A is low and relatively insensitive to pH. Thermostability was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120746#pone.0120746.g004" target="_blank">Fig. 4</a>.</p

DksA structure is sensitive to pH.

<p>Two-dimensional ¹H-¹⁵N HSQC spectra at pH 8 (black) and 6 (red) reveal large chemical shift changes at (A) Tyr23 and (B) many other residues.</p