Examination of the Polypeptide Substrate Specificity for <i>Escherichia coli</i> ClpA

Enzyme-catalyzed protein unfolding is essential for a large array of biological functions, including microtubule severing, membrane fusion, morphogenesis and trafficking of endosomes, protein disaggregation, and ATP-dependent proteolysis. These enzymes are all members of the ATPases associated with various cellular activity (AAA+) superfamily of proteins. <i>Escherichia coli</i> ClpA is a hexameric ring ATPase responsible for enzyme-catalyzed protein unfolding and translocation of a polypeptide chain into the central cavity of the tetradecameric <i>E. coli</i> ClpP serine protease for proteolytic degradation. Further, ClpA also uses its protein unfolding activity to catalyze protein remodeling reactions in the absence of ClpP. ClpA recognizes and binds a variety of protein tags displayed on proteins targeted for degradation. In addition, ClpA binds unstructured or poorly structured proteins containing no specific tag sequence. Despite this, a quantitative description of the relative binding affinities for these different substrates is not available. Here we show that ClpA binds to the 11-amino acid SsrA tag with an affinity of 200 ± 30 nM. However, when the SsrA sequence is incorporated at the carboxy terminus of a 30–50-amino acid substrate exhibiting little secondary structure, the affinity constant decreases to 3–5 nM. These results indicate that additional contacts beyond the SsrA sequence are required for maximal binding affinity. Moreover, ClpA binds to various lengths of the intrinsically unstructured protein, α-casein, with an affinity of ∼30 nM. Thus, ClpA does exhibit modest specificity for SsrA when incorporated into an unstructured protein. Moreover, incorporating these results with the known structural information suggests that SsrA makes direct contact with the domain 2 loop in the axial channel and additional substrate length is required for additional contacts within domain 1

<i>Escherichia coli</i> DnaK Allosterically Modulates ClpB between High- and Low-Peptide Affinity States

ClpB and DnaKJE provide protection to <i>Escherichia coli</i> cells during extreme environmental stress. Together, this co-chaperone system can resolve protein aggregates, restoring misfolded proteins to their native form and function in solubilizing damaged proteins for removal by the cell’s proteolytic systems. DnaK is the component of the KJE system that directly interacts with ClpB. There are many hypotheses for how DnaK affects ClpB-catalyzed disaggregation, each with some experimental support. Here, we build on our recent work characterizing the molecular mechanism of ClpB-catalyzed polypeptide translocation by developing a stopped-flow FRET assay that allows us to detect ClpB’s movement on model polypeptide substrates in the absence or presence of DnaK. We find that DnaK induces ClpB to dissociate from the polypeptide substrate. We propose that DnaK acts as a peptide release factor, binding ClpB and causing the ClpB conformation to change to a low-peptide affinity state. Such a role for DnaK would allow ClpB to rebind to another portion of an aggregate and continue nonprocessive translocation to disrupt the aggregate

Multisubunit RNA Polymerase Cleavage Factors Modulate the Kinetics and Energetics of Nucleotide Incorporation: An RNA Polymerase I Case Study

All cellular RNA polymerases are influenced by protein factors that stimulate RNA polymerase-catalyzed cleavage of the nascent RNA. Despite divergence in amino acid sequence, these so-called “cleavage factors” appear to share a common mechanism of action. Cleavage factors associate with the polymerase through a conserved structural element of the polymerase known as the secondary channel or pore. This mode of association enables the cleavage factor to reach through the secondary channel into the polymerase active site to reorient the active site divalent metal ions. This reorientation converts the polymerase active site into a nuclease active site. Interestingly, eukaryotic RNA polymerases I and III (Pols I and III, respectively) have incorporated their cleavage factors as bona fide subunits known as A12.2 and C11, respectively. Although it is clear that A12.2 and C11 dramatically stimulate the polymerase’s cleavage activity, it is not known if or how these subunits affect the polymerization mechanism. In this work we have used transient-state kinetic techniques to characterize a Pol I isoform lacking A12.2. Our data clearly demonstrate that the A12.2 subunit profoundly affects the kinetics and energetics of the elementary steps of Pol I-catalyzed nucleotide incorporation. Given the high degree of conservation between polymerase–cleavage factor interactions, these data indicate that cleavage factor-modulated nucleotide incorporation mechanisms may be common to all cellular RNA polymerases

Metallathiacrown Ethers: Synthesis and Characterization of Transition-Metal Complexes Containing α,ω-Bis(phosphite)-Polythioether Ligands and an Evaluation of Their Soft Metal Binding Capabilities

The metallathiacrown ethers <i>cis</i>-Mo­(CO)₄{2,2′-(C₁₂H₈O₂)­PO­(CH₂CH₂S)_<i>n</i>CH₂CH₂OP­(2,2′-(O₂H₈C₁₂))} (<i>n</i> = 2, 3) and <i>cis</i>-Mo­(CO)₄{2,2′-(C₁₂H₈O₂)­POCH₂CH₂S-1-(C₆H₄)-2-SCH₂CH₂OP­(2,2′-(O₂H₈C₁₂))} have been prepared as soft metal selective molecular receptors. Multinuclear NMR spectroscopy and X-ray crystallography have been used to show that byproducts formed during the syntheses of the metallathiacrown ethers <i>cis</i>-Mo­(CO)₄{2,2′-(C₁₂H₈O₂)­PO­(CH₂CH₂S)_<i>n</i>CH₂CH₂OP­(2,2′-(O₂H₈C₁₂))} (<i>n</i> = 2, 3) are homobinuclear complexes with <i>cis</i>-Mo­(CO)₄(P-donor)­(S-donor) centers. The abilities of the metallathiacrown ethers to bind PdCl₂ and PtCl₂ have been assessed using ³¹P­{¹H} NMR spectroscopy and X-ray crystallography. The complexes showed null results with PdCl₂; however, the PtCl₂ experiments showed that the complexes <i>cis</i>-Mo­(CO)₄{2,2′-(C₁₂H₈O₂)­PO­(CH₂CH₂S)_<i>n</i>CH₂CH₂OP­(2,2′-(O₂H₈C₁₂))} (<i>n</i> = 2, 3) formed heterobinuclear <i>cis,cis</i>-{[Mo­(CO)₄{2,2′-(C₁₂H₈O₂)­PO­(CH₂CH₂S)_<i>n</i>CH₂CH₂OP­(2,2′-(O₂H₈C₁₂))}]­PtCl₂} (<i>n</i> = 2, 3) complexes. The ³¹P­{¹H} NMR spectra of these complexes suggest a <i>cis</i>-PtS₂Cl₂ coordination environment in each, which leads to asymmetric binding in the latter complex. Binding of HgCl₂ by the complexes <i>cis</i>-Mo­(CO)₄{2,2′-(C₁₂H₈O₂)­PO­(CH₂CH₂S)_<i>n</i>CH₂CH₂OP­(2,2′-(O₂H₈C₁₂))} (<i>n</i> = 2, 3) has been studied using ³¹P­{¹H} NMR spectroscopy. Each complex was titrated with HgCl₂, and the quantitative shifts in the ³¹P­{¹H} NMR spectra were fit to a binding mechanism. The <i>n</i> = 2 metallathiacrown ether binds HgCl₂ to form a 1:1 complex with <i>K</i>_a = 12.0(0.2) M^–1. In contrast, interaction of HgCl₂ with the <i>n</i> = 3 metallathiacrown ether results in isomerization to the trans complex, and HgCl₂ binds to both isomers. A model has been adapted to fit the titration data of this complex and to extract equilibrium constants for each step in the cycle: the cis–trans equilibrium of the free, <i>K</i>_free (0.570 (0.004)), and bound, <i>K</i>_bound (0.16 (0.03)), metallathiacrown ethers, as well as the association binding constants for the cis, <i>K</i>_cis ([4.1(0.3)] × 10² M^–1) and trans, <i>K</i>_trans ([1.1(0.2)] × 10² M^–1), metallathiacrown ethers. The equilibrium constants demonstrate that the cis metallathiacrown ether binds more strongly to the HgCl₂ than does the trans metallathiacrown ether and that the cis–trans equilibrium favors the cis metallathiacrown ether

Correlating the Activity of Rhodium(I)-Phosphite-Lariat Ether Styrene Hydroformylation Catalysts with Alkali Metal Cation Binding through NMR Spectroscopic Titration Methods

Alkali metal salts can affect both the activities and regioselectivities of alkene hydroformylation catalysts containing polyether-functionalized phosphorus-donor ligands; however, it is unclear whether these effects arise from direct alkali metal cation binding to the active catalysts. To gain more insight into these effects, a series of phosphite-lariat ether ligands derived from the alkali metal cation binding agents 2-hydroxymethyl-12-crown-4 and 2-hydroxymethyl-15-crown-5 have been prepared. Rhodium­(I) complexes of these ligands have been evaluated as styrene hydroformylation catalysts in the absence and presence of a variety of alkali metal salts. The activities of catalysts containing phosphites derived from 2,2′-biphenol or 1,1′-binaphthol increased significantly (up to 92%) in the presence of alkali metal cations that are “moderately oversized” for archetypal binding to the crown cavity. When this criterion are not met, a decrease in the catalytic activity is observed upon addition of an alkali metal salt. NMR titrations (³¹P­{¹H} and ¹H) of two model <i>cis</i>-Mo­(CO)₄(phosphite-lariat)₂ complexes in which the phosphite was derived from 2,2′-biphenol were carried out to gain insight into the manner in which the alkali metal cations interact with the ligands. Both model complexes bind Li⁺ through a 2:1 two-site binding mechanism, and the model complex with the larger crown ether also binds Na⁺ in this fashion. In contrast, 1:1 complexes are formed upon Na⁺ and K⁺ binding to the model complex containing the smaller crown ether and upon K⁺ binding to the model complex containing the larger crown ether. Correlation between increases in catalyst activity and binding mode in complexes containing cations “moderately oversized” for archetypal binding to the crown cavity strongly suggests that the increases are due to a specific type of alkali metal cation binding by the lariat ether groups in these catalysts

Characterization of Calmodulin–Fas Death Domain Interaction: An Integrated Experimental and Computational Study

The Fas death receptor-activated death-inducing signaling complex (DISC) regulates apoptosis in many normal and cancer cells. Qualitative biochemical experiments demonstrate that calmodulin (CaM) binds to the death domain of Fas. The interaction between CaM and Fas regulates Fas-mediated DISC formation. A quantitative understanding of the interaction between CaM and Fas is important for the optimal design of antagonists for CaM or Fas to regulate the CaM–Fas interaction, thus modulating Fas-mediated DISC formation and apoptosis. The V254N mutation of the Fas death domain (Fas DD) is analogous to an identified mutant allele of Fas in <i>lpr</i>-cg mice that have a deficiency in Fas-mediated apoptosis. In this study, the interactions of CaM with the Fas DD wild type (Fas DD WT) and with the Fas DD V254N mutant were characterized using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and molecular dynamics (MD) simulations. ITC results reveal an endothermic binding characteristic and an entropy-driven interaction of CaM with Fas DD WT or with Fas DD V254N. The Fas DD V254N mutation decreased the association constant (<i>K</i>_a) for CaM–Fas DD binding from (1.79 ± 0.20) × 10⁶ to (0.88 ± 0.14) × 10⁶ M^–1 and slightly increased a standard state Gibbs free energy (Δ<i>G</i>°) for CaM–Fas DD binding from −8.87 ± 0.07 to −8.43 ± 0.10 kcal/mol. CD secondary structure analysis and MD simulation results did not show significant secondary structural changes of the Fas DD caused by the V254N mutation. The conformational and dynamical motion analyses, the analyses of hydrogen bond formation within the CaM binding region, the contact numbers of each residue, and the electrostatic potential for the CaM binding region based on MD simulations demonstrated changes caused by the Fas DD V254N mutation. These changes caused by the Fas DD V254N mutation could affect the van der Waals interactions and electrostatic interactions between CaM and Fas DD, thereby affecting CaM–Fas DD interactions. Results from this study characterize CaM–Fas DD interactions in a quantitative way, providing structural and thermodynamic evidence of the role of the Fas DD V254N mutation in the CaM–Fas DD interaction. Furthermore, the results could help to identify novel strategies for regulating CaM–Fas DD interactions and Fas DD conformation and thus to modulate Fas-mediated DISC formation and thus Fas-mediated apoptosis