Assessing the Integrity of Designed Homomeric Parallel Three-Stranded Coiled Coils in the Presence of Metal Ions

De novo design of α-helical peptides that self-assemble to form helical coiled coils is a powerful tool for studying molecular recognition between peptides/proteins and determining the fundamental forces involved in their folding and structure. These amphipathic helices assemble in aqueous solution to generate the final coiled coil motif, with the hydrophobic residues in the interior and the polar/hydrophilic groups on the exterior. Considerable effort has been devoted to investigate the forces that determine the overall stability and final three-dimensional structure of the coiled coils. One of the major challenges in coiled coil design is the achievement of specificity in terms of the oligomeric state, with respect to number (two, three, four, or higher), nature (homomers vs heteromers), and strand orientation (parallel vs antiparallel). As seen in nature, metal ions play an important role in this self-organization process, and the overall structure of metalloproteins is primarily the result of two driving forces:  the metal coordination preference and the fold of the polypeptide backbone. Previous work in our group has shown that metal ions such as As(III) and Hg(II) can be used to enforce different aggregation states in the Cys derivatives of the designed homotrimeric coiled-coil TRI family [Ac-G(LKALEEK)4G-CONH2]. We are now interested in studying the interplay between the metal ion and peptide preferences in controlling the specificity and relative orientation of the α-helices in coiled coils. For this objective, two derivatives of the TRI family, TRi L2WL9C and TRi L2WL23C, have been synthesized. Along with those two peptides, two derivatives of Coil-Ser, CSL9C and CSL19C (CS = Ac-EWEALEKKLAALESKLQALEKKLEALEHG-CONH2), a similar de novo designed three-stranded coiled coil that has the potential to form antiparallel coiled coils, have also been used. Circular dichroism, UV−vis, and 199Hg and 113Cd NMR spectroscopy results reveal that the addition of Hg(II) and Cd(II) to the different mixtures of these peptides forms preferentially homotrimeric coiled coils, over a statistical population of heterotrimeric parallel and antiparallel coiled coils

Structure−Activity Studies on the Cleavage of an RNA Analogue by a Potent Dinuclear Metal Ion Catalyst: Effect of Changing the Metal Ion

Dinuclear Cd(II), Cu(II), and Zn(II) complexes of L2OH (L2OH = 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane) are compared as catalysts for cleavage of the RNA analogue HpPNP (HpPNP = 2-hydroxypropyl 4-nitrophenyl phosphate) at 25 °C, I = 0.10 M (NaNO3). Zn(II) and Cu(II) readily form dinuclear complexes at millimolar concentrations and a 2:1 ratio of metal ion to L2OH at neutral pH. The dinuclear Zn2(L2O) and Cu2(L2O) complexes have a bridging alkoxide group that brings together the two cations in close proximity to facilitate cooperative catalysis. Under similar conditions, the dinuclear complex of Cd(II) is a minor species in solution; only at high pH values (pH 10.4) does the Cd2(L2O) complex become the predominant species in solution. Analysis of the second-order rate constants for cleavage of HpPNP by Zn2(L2O) is straightforward because a linear dependence of pseudo-first-order rate constant on dinuclear complex is observed over a wide pH range. In contrast, plots of pseudo-first-order rate constants for cleavage of HpPNP by solutions containing a 2:1 ratio of Cd(II) to L2OH as a function of increasing L2OH are curved, and second-order rate constants are obtained by fitting the kinetic data to an equation for the formation of the dinuclear Cd(II) complex as a function of pH and [L2OH]. Second-order rate constants for cleavage of HpPNP by these dinuclear complexes at pH 9.3 and 25 °C vary by 3 orders of magnitude in the order Cd2(L2O) (2.8 M-1 s-1) > Zn2(L2O) (0.68 M-1 s-1) > Cu2(L2O) (0.0041 M-1 s-1). The relative reactivity of these complexes is discussed in terms of the different geometric preferences and Lewis acidity of the dinuclear Zn(II), Cu(II), and Cd(II) complexes, giving insight into the importance of these catalyst properties in the cleavage of phosphate diesters resembling RNA

Controlling and Fine Tuning the Physical Properties of Two Identical Metal Coordination Sites in De Novo Designed Three Stranded Coiled Coil Peptides

Herein we report how de novo designed peptides can be used to investigate whether the position of a metal site along a linear sequence that folds into a three-stranded α-helical coiled coil defines the physical properties of Cd(II) ions in either CdS3 or CdS3O (O-being an exogenous water molecule) coordination environments. Peptides are presented that bind Cd(II) into two identical coordination sites that are located at different topological positions at the interior of these constructs. The peptide GRANDL16PenL19IL23PenL26I binds two Cd(II) as trigonal planar 3-coordinate CdS3 structures whereas GRANDL12AL16CL26AL30C sequesters two Cd(II) as pseudotetrahedral 4-coordinate CdS3O structures. We demonstrate how for the first peptide, having a more rigid structure, the location of the identical binding sites along the linear sequence does not affect the physical properties of the two bound Cd(II). However, the sites are not completely independent as Cd(II) bound to one of the sites (113Cd NMR chemical shift of 681 ppm) is perturbed by the metalation state (apo or [Cd(pep)(Hpep)2]+ or [Cd(pep)3]−) of the second center (113Cd NMR chemical shift of 686 ppm). GRANDL12AL16CL26AL30C shows a completely different behavior. The physical properties of the two bound Cd(II) ions indeed depend on the position of the metal center, having pKa2 values for the equilibrium [Cd(pep)(Hpep)2]+ → [Cd(pep)3]− + 2H+ (corresponding to deprotonation and coordination of cysteine thiols) that range from 9.9 to 13.9. In addition, the L26AL30C site shows dynamic behavior, which is not observed for the L12AL16C site. These results indicate that for these systems one cannot simply assign a “4-coordinate structure” and assume certain physical properties for that site since important factors such as packing of the adjacent Leu, size of the intended cavity (endo vs exo) and location of the metal site play crucial roles in determining the final properties of the bound Cd(II)

Constant-pH MD Simulations Portray the Protonation and Structural Behavior of Four Decapeptides Designed to Coordinate Cu²⁺

The cyclic decapeptide C-Asp, containing one Asp residue and three His residues, was designed by Fragoso et al. (<i>Chem. Eur. J.</i> <b>2013</b>, <i>19</i>, 2076) to bind Cu²⁺ exclusively through the side chain groups and mimic copper coordination in metalloproteins. A variant of the cyclodecapeptide where Asp is substituted by Asn (C-Asn) has also been synthesized in addition to the linear (“open”) counterparts of both forms (O-Asp and O-Asn), testing the importance of cyclization and the presence of Asp in Cu²⁺ coordination (<i>Chem. Eur. J.</i> <b>2013</b>, <i>19</i>, 2076; <i>Dalton Trans.</i> <b>2013</b>, <i>42</i>, 6182). All peptides formed a major species at neutral pH that was able to coordinate Cu²⁺ exclusively through the neutral imidazole groups and the Asp side chain, when present, with C-Asp being the most effective. A detailed description of the protonation behavior of each histidine could help understanding the coordination species being formed in the pH range and eventually further optimizing the peptide’s design. However, the standard current methods (NMR titrations) are not very suited for proximal groups titrating in the same pH range. In this work, we used the stochastic titration constant-pH molecular dynamics method to calculate the protonation curves and p<i>K</i>_a of each titrable residue in the four decapeptides, in the absence of Cu²⁺ ions. The global protonation curves obtained in our simulations are in very good agreement with the existing potentiometric titration curves. The histidines are titrating very closely, and the Asp forms abundant salt bridges with the basic residues, displaying an unusually low p<i>K</i>_a value. In addition, we could observe that the four peptides are very unstructured in the absence of copper, and not even the cyclic forms exhibit a significant β-sheet, unlike what could be expected from the presence of β-turn inducer units in this type of scaffold

Cooperativity between Metal Ions in the Cleavage of Phosphate Diesters and RNA by Dinuclear Zn(II) Catalysts

A series of ligands containing linked 1,4,7-triazacyclononane macrocycles are studied for the preparation of dinuclear Zn(II) complexes including 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (L2OH), 1,5-bis(1,4,7-triazacyclonon-1-yl)pentane (L3), 2,9-bis(1-methyl-1,4,7-triazacyclonon-1-yl)-1,10-phenanthroline (L4), and α,α‘-bis(1,4,7-triazacyclonon-1-yl)-m-xylene (L5). The titration of these ligands with Zn(NO3)2 was monitored by 1H NMR. Each ligand was found to bind two Zn(II) ions with a very high affinity at near neutral pH under conditions of millimolar ligand and 2 equiv of Zn(NO3)2. In contrast, a stable mononuclear complex was formed in solutions containing 5.0 mM L2OH and 1 equiv of Zn(NO3)2. 1H and 13C NMR spectral data are consistent with formation of a highly symmetric mononuclear complex Zn(L2OH) in which a Zn(II) ion is sandwiched between two triazacyclononane units. The second-order rate constant kZn for the cleavage of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP) at pH 7.6 and 25 °C catalyzed by Zn2(L2O) is 120-fold larger than that for the reaction catalyzed by the closely related mononuclear complex Zn(L1) (L1 = 1,4,7-triazacyclononane). By comparison, the observation that the values of kZn determined under similar reaction conditions for cleavage of HPNP catalyzed by the other Zn(II) dinuclear complexes are only 3−5-fold larger than values of kZn for catalysis by Zn(L1) provides strong evidence that the two Zn(II) cations in Zn2(L2O) act cooperatively in the stabilization of the transition state for cleavage of HPNP. The extent of cleavage of an oligoribonucleotide by Zn(L1), Zn2(L5), and Zn2(L2O) at pH 7.5 and 37 °C after 24 h incubation is 4,10, and 90%. The rationale for the observed differences in catalytic activity of these dinuclear Zn(II) complexes is discussed in terms of the mechanism of RNA cleavage and the structure and speciation of these complexes in solution

Solvent Deuterium Isotope Effects on Phosphodiester Cleavage Catalyzed by an Extraordinarily Active Zn(II) Complex

The effect of increasing pL on the extraordinary catalytic activity of a dinuclear Zn2+ complex toward cleavage of uridine 3‘-4-nitrophenyl phosphate (UpPNP) in H2O and D2O was determined. This change from H2O to D2O causes an increase from 7.8 to 8.4 in the apparent pKa of a catalytic functional group, but has little effect on the activity of the active form of the catalyst toward cleavage of UpPNP, so that there is no primary kinetic SDIE on the cleavage reaction from movement of a proton at the rate-determining transition state. It is concluded that essentially all of the rate acceleration for this catalyst is due to electrostatic stabilization of the transition state by interactions between opposing cationic and anionic charges

Physical and Kinetic Analysis of the Cooperative Role of Metal Ions in Catalysis of Phosphodiester Cleavage by a Dinuclear Zn(II) Complex

A dinuclear metal ion complex Zn2(L2O) and its mononuclear analogue Zn(L1OH) were synthesized and studied as catalysts of the cleavage of the phosphate diester 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). X-ray crystal structure data, potentiometric titrations, and 1H NMR spectra obtained over a wide range of pH values provide strong evidence that the alcohol linker in the complex Zn2(L2O) is ionized below pH 6.0, while the alcohol group in the complex Zn(L1OH) remains protonated even at high pH. The ionizations observed at high pH correspond to the formation of the monohydroxo complexes, Zn2(L2O)(OH) and Zn(L1OH)(OH), with pKa's of 8.0 and 9.2, respectively. The pH-rate profiles of second-order rate constants for metal-ion complex-catalyzed cleavage of HPNP are reported. These show downward curvature centered at the pKa's for the respective zinc-bound waters, and limiting second-order rate constants at high pH of kc = 0.71 M-1 s-1 for Zn2(L2O) and 0.061 M-1 s-1 for Zn(L1OH). The larger catalytic activity of Zn2(L2O) compared with Zn(L1OH) is due to the cooperative role of the metal ions in facilitating the formation of the ionized zinc-bound water at close to neutral pH and in providing additional stabilization of the rate-limiting transition state for phosphodiester cleavage. Zn2(L2O) complex (1 M) at pH 7.6 stabilizes the transition state for the uncatalyzed reaction by 9.3 kcal/mol. Assuming that the dissociation constant determined for a diethyl phosphate inhibitor is similar to that for substrate, then ca. 2.4 kcal/mol of these stabilizing interactions are expressed in the ground-state Michaelis complex, while the bulk of these interactions are only expressed as the reaction approaches the transition state for phosphodiester cleavage

Physical and Kinetic Analysis of the Cooperative Role of Metal Ions in Catalysis of Phosphodiester Cleavage by a Dinuclear Zn(II) Complex

A dinuclear metal ion complex Zn2(L2O) and its mononuclear analogue Zn(L1OH) were synthesized and studied as catalysts of the cleavage of the phosphate diester 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). X-ray crystal structure data, potentiometric titrations, and 1H NMR spectra obtained over a wide range of pH values provide strong evidence that the alcohol linker in the complex Zn2(L2O) is ionized below pH 6.0, while the alcohol group in the complex Zn(L1OH) remains protonated even at high pH. The ionizations observed at high pH correspond to the formation of the monohydroxo complexes, Zn2(L2O)(OH) and Zn(L1OH)(OH), with pKa's of 8.0 and 9.2, respectively. The pH-rate profiles of second-order rate constants for metal-ion complex-catalyzed cleavage of HPNP are reported. These show downward curvature centered at the pKa's for the respective zinc-bound waters, and limiting second-order rate constants at high pH of kc = 0.71 M-1 s-1 for Zn2(L2O) and 0.061 M-1 s-1 for Zn(L1OH). The larger catalytic activity of Zn2(L2O) compared with Zn(L1OH) is due to the cooperative role of the metal ions in facilitating the formation of the ionized zinc-bound water at close to neutral pH and in providing additional stabilization of the rate-limiting transition state for phosphodiester cleavage. Zn2(L2O) complex (1 M) at pH 7.6 stabilizes the transition state for the uncatalyzed reaction by 9.3 kcal/mol. Assuming that the dissociation constant determined for a diethyl phosphate inhibitor is similar to that for substrate, then ca. 2.4 kcal/mol of these stabilizing interactions are expressed in the ground-state Michaelis complex, while the bulk of these interactions are only expressed as the reaction approaches the transition state for phosphodiester cleavage

Experimental and Theoretical Evaluation of Multisite Cadmium(II) Exchange in Designed Three-Stranded Coiled-Coil Peptides

An important factor that defines the toxicity of elements such as cadmium­(II), mercury­(II), and lead­(II) with biological macromolecules is metal ion exchange dynamics. Intriguingly, little is known about the fundamental rates and mechanisms of metal ion exchange into proteins, especially helical bundles. Herein, we investigate the exchange kinetics of Cd­(II) using <i>de novo</i> designed three-stranded coiled-coil peptides that contain metal complexing cysteine thiolates as a model for the incorporation of this ion into trimeric, parallel coiled coils. Peptides were designed containing both a single Cd­(II) binding site, <b>Grand</b>L12AL16C [<b>Grand</b> = AcG-(LKALEEK)₅-GNH₂], <b>Grand</b>L26AL30C, and <b>Grand</b>L26AE28QL30C, as well as <b>Grand</b>L12AL16CL26AL30C with two Cd­(II) binding sites. The binding of Cd­(II) to any of these sites is of high affinity (<i>K</i>_A > 3 × 10⁷ M^–1). Using ¹¹³Cd NMR spectroscopy, Cd­(II) binding to these designed peptides was monitored. While the Cd­(II) binding is in extreme slow exchange regime without showing any chemical shift changes, incremental line broadening for the bound ¹¹³Cd­(II) signal is observed when excess ¹¹³Cd­(II) is titrated into the peptides. Most dramatically, for one site, L26AL30C, all ¹¹³Cd­(II) NMR signals disappear once a 1.7:1 ratio of Cd­(II)/(peptide)₃ is reached. The observed processes are not compatible with a simple “free-bound” two-site exchange kinetics at any time regime. The experimental results can, however, be simulated in detail with a multisite binding model, which features additional Cd­(II) binding site(s) which, once occupied, perturb the primary binding site. This model is expanded into differential equations for five-site NMR chemical exchange. The numerical integration of these equations exhibits progressive loss of the primary site NMR signal without a chemical shift change and with limited line broadening, in good agreement with the observed experimental data. The mathematical model is interpreted in molecular terms as representing binding of excess Cd­(II) to surface Glu residues located at the helical interfaces. In the absence of Cd­(II), the Glu residues stabilize the three-helical structure though salt bridge interactions with surface Lys residues. We hypothesize that Cd­(II) interferes with these surface ion pairs, destabilizing the helical structure, and perturbing the primary Cd­(II) binding site. This hypothesis is supported by the observation that the Cd­(II)-excess line broadening is attenuated in <b>Grand</b>L26AE28QL30C, where a surface Glu(28), close to the metal binding site, was changed to Gln. The external binding site may function as an entry pathway for Cd­(II) to find its internal binding site following a molecular rearrangement which may serve as a basis for our understanding of metal complexation, transport, and exchange in complex native systems containing α-helical bundles

Physical and Kinetic Analysis of the Cooperative Role of Metal Ions in Catalysis of Phosphodiester Cleavage by a Dinuclear Zn(II) Complex

A dinuclear metal ion complex Zn2(L2O) and its mononuclear analogue Zn(L1OH) were synthesized and studied as catalysts of the cleavage of the phosphate diester 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP). X-ray crystal structure data, potentiometric titrations, and 1H NMR spectra obtained over a wide range of pH values provide strong evidence that the alcohol linker in the complex Zn2(L2O) is ionized below pH 6.0, while the alcohol group in the complex Zn(L1OH) remains protonated even at high pH. The ionizations observed at high pH correspond to the formation of the monohydroxo complexes, Zn2(L2O)(OH) and Zn(L1OH)(OH), with pKa's of 8.0 and 9.2, respectively. The pH-rate profiles of second-order rate constants for metal-ion complex-catalyzed cleavage of HPNP are reported. These show downward curvature centered at the pKa's for the respective zinc-bound waters, and limiting second-order rate constants at high pH of kc = 0.71 M-1 s-1 for Zn2(L2O) and 0.061 M-1 s-1 for Zn(L1OH). The larger catalytic activity of Zn2(L2O) compared with Zn(L1OH) is due to the cooperative role of the metal ions in facilitating the formation of the ionized zinc-bound water at close to neutral pH and in providing additional stabilization of the rate-limiting transition state for phosphodiester cleavage. Zn2(L2O) complex (1 M) at pH 7.6 stabilizes the transition state for the uncatalyzed reaction by 9.3 kcal/mol. Assuming that the dissociation constant determined for a diethyl phosphate inhibitor is similar to that for substrate, then ca. 2.4 kcal/mol of these stabilizing interactions are expressed in the ground-state Michaelis complex, while the bulk of these interactions are only expressed as the reaction approaches the transition state for phosphodiester cleavage