Design of HIV Coreceptor Derived Peptides That Inhibit Viral Entry at Submicromolar Concentrations.

HIV/AIDS continues to pose an enormous burden on global health. Current HIV therapeutics include inhibitors that target the enzymes HIV protease, reverse transcriptase, and integrase, along with viral entry inhibitors that block the initial steps of HIV infection by preventing membrane fusion or virus-coreceptor interactions. With regard to the latter, peptides derived from the HIV coreceptor CCR5 were previously shown to modestly inhibit entry of CCR5-tropic HIV strains, with a peptide containing residues 178-191 of the second extracellular loop (peptide 2C) showing the strongest inhibition. Here we use an iterative approach of amino acid scanning at positions shown to be important for binding the HIV envelope, and recombining favorable substitutions to greatly improve the potency of 2C. The most potent candidate peptides gain neutralization breadth and inhibit CXCR4 and CXCR4/CCR5-using viruses, rather than CCR5-tropic strains only. We found that gains in potency in the absence of toxicity were highly dependent on amino acid position and residue type. Using virion capture assays we show that 2C and the new peptides inhibit capture of CD4-bound HIV-1 particles by antibodies whose epitopes are located in or around variable loop 3 (V3) on gp120. Analysis of antibody binding data indicates that interactions between CCR5 ECL2-derived peptides and gp120 are localized around the base and stem of V3 more than the tip. In the absence of a high-resolution structure of gp120 bound to coreceptor CCR5, these findings may facilitate structural studies of CCR5 surrogates, design of peptidomimetics with increased potency, or use as functional probes for further study of HIV-1 gp120-coreceptor interactions.

A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold-improved potency against a range of laboratory and clinical bacterial strains which we named P10. Whole-genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds, including fluoroquinolone antibiotics.

Rapid kinetics of dehalogenation promoted by iodotyrosine deiodinase from human thyroid.

Reductive dehalogenation such as that catalyzed by iodotyrosine deiodinase (IYD) is highly unusual in aerobic organisms but necessary for iodide salvage from iodotyrosine generated during thyroxine biosynthesis. Equally unusual is the dependence of this process on flavin. Rapid kinetics have now been used to define the basic processes involved in IYD catalysis. Time-dependent quenching of flavin fluorescence was used to monitor halotyrosine association to IYD. The substrates chloro-, bromo-, and iodotyrosine bound with similar rate constants (kon) ranging from 1.3 × 10(6) to 1.9 × 10(6) M(-1) s(-1). Only the inert substrate analogue fluorotyrosine exhibited a significantly (5-fold) slower kon (0.3 × 10(6) M(-1) s(-1)). All data fit a standard two-state model and indicated that no intermediate complex accumulated during closure of the active site lid induced by substrate. Subsequent halide elimination does not appear to limit reactions of bromo- and iodotyrosine since both fully oxidized the reduced enzyme with nearly equivalent second-order rate constants (7.3 × 10(3) and 8.6 × 10(3) M(-1) s(-1), respectively) despite the differing strength of their carbon-halogen bonds. In contrast to these substrates, chlorotyrosine reacted with the reduced enzyme approximately 20-fold more slowly and revealed a spectral intermediate that formed at approximately the same rate as the bromo- and iodotyrosine reactions.

Perturbations in intracellular Ca2+ handling in skeletal muscle in the G93A*SOD1 mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by skeletal muscle atrophy and weakness, ultimately leading to respiratory failure. The purpose of this study was to assess changes in skeletal muscle excitation-contraction (E-C) coupling and intracellular Ca(2+) handling during disease progression in the G93A*SOD1 ALS transgenic (ALS Tg) mouse model. To assess E-C coupling, single muscle fibers were electrically stimulated (10-150 Hz), and intracellular free Ca(2+) concentration was assessed using fura-2. There were no differences in peak fura-2 ratio at any stimulation frequency at 70 days (early presymptomatic). However, at 90 days (late presymptomatic) and 120-140 days (symptomatic), fura-2 ratio was increased at 10 Hz in ALS Tg compared with wild-type (WT) fibers (0.670 ± 0.02 vs. 0.585 ± 0.02 for 120-140 days; P < 0.05). There was also a significant increase in resting fura-2 ratio at 90 days (0.351 ± 0.008 vs. 0.390 ± 0.009 in WT vs. ALS Tg; P < 0.05) and 120-140 days (0.374 ± 0.001 vs. 0.415 ± 0.003 in WT vs. ALS Tg; P < 0.05). These increases in intracellular Ca(2+) in ALS Tg muscle were associated with reductions in the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase proteins SERCA1 (to 54% and 19% of WT) and SERCA2 (to 56% and 11% of WT) and parvalbumin (to 80 and 62% of WT) in gastrocnemius muscle at 90 and 120-140 days, respectively. There was no change in dihydropyridine receptor/l-type Ca(2+) channel at any age. Overall, these data demonstrate minimal changes in electrically evoked Ca(2+) transients but elevations in intracellular Ca(2+) attributable to decreased Ca(2+)-clearance proteins. These data suggest that elevations in cellular Ca(2+) could contribute to muscle weakness during disease progression in ALS mice.

Evidence in support of lysine 77 and histidine 96 as acid-base catalytic residues in saccharopine dehydrogenase from Saccharomyces cerevisiae.

Saccharopine dehydrogenase (SDH) catalyzes the final reaction in the α-aminoadipate pathway, the conversion of l-saccharopine to l-lysine (Lys) and α-ketoglutarate (α-kg) using NAD⁺ as an oxidant. The enzyme utilizes a general acid-base mechanism to conduct its reaction with a base proposed to accept a proton from the secondary amine of saccharopine in the oxidation step and a group proposed to activate water to hydrolyze the resulting imine. Crystal structures of an open apo form and a closed form of the enzyme with saccharopine and NADH bound have been determined at 2.0 and 2.2 Å resolution, respectively. In the ternary complex, a significant movement of domain I relative to domain II that closes the active site cleft between the two domains and brings H96 and K77 into the proximity of the substrate binding site is observed. The hydride transfer distance is 3.6 Å, and the side chains of H96 and K77 are properly positioned to act as acid-base catalysts. Preparation of the K77M and H96Q single-mutant and K77M/H96Q double-mutant enzymes provides data consistent with their role as the general acid-base catalysts in the SDH reaction. The side chain of K77 initially accepts a proton from the ε-amine of the substrate Lys and eventually donates it to the imino nitrogen as it is reduced to a secondary amine in the hydride transfer step, and H96 protonates the carbonyl oxygen as the carbinolamine is formed. The K77M, H976Q, and K77M/H96Q mutant enzymes give 145-, 28-, and 700-fold decreases in V/E(t) and >10³-fold increases in V2/K(Lys)E(t) and V2/K(α-kg)E(t) (the double mutation gives >105-fold decreases in the second-order rate constants). In addition, the K77M mutant enzyme exhibits a primary deuterium kinetic isotope effect of 2.0 and an inverse solvent deuterium isotope effect of 0.77 on V2/K(Lys). A value of 2.0 was also observed for (D)(V2/K(Lys))(D2O) when the primary deuterium kinetic isotope effect was repeated in D2O, consistent with a rate-limiting hydride transfer step. A viscosity effect of 0.8 was observed on V2/K(Lys), indicating the solvent deuterium isotope effect resulted from stabilization of an enzyme form prior to hydride transfer. A small normal solvent isotope effect is observed on V, which decreases slightly when repeated with NADD, consistent with a contribution from product release to rate limitation. In addition, V2/K(Lys)E(t) is pH-independent, which is consistent with the loss of an acid-base catalyst and perturbation of the pK(a) of the second catalytic group to a higher pH, likely a result of a change in the overall charge of the active site. The primary deuterium kinetic isotope effect for H96Q, measured in H2O or D2O, is within error equal to 1. A solvent deuterium isotope effect of 2.4 is observed with NADH or NADD as the dinucleotide substrate. Data suggest rate-limiting imine formation, consistent with the proposed role of H96 in protonating the leaving hydroxyl as the imine is formed. The pH-rate profile for V2/K(Lys)E(t) exhibits the pK(a) for K77, perturbed to a value of ∼9, which must be unprotonated to accept a proton from the ε-amine of the substrate Lys so that it can act as a nucleophile. Overall, data are consistent with a role for K77 acting as the base that accepts a proton from the ε-amine of the substrate lysine prior to nucleophilic attack on the α-oxo group of α-ketoglutarate, and finally donating a proton to the imine nitrogen as it is reduced to give saccharopine. In addition, data indicate a role for H96 acting as a general acid-base catalyst in the formation of the imine between the ε-amine of lysine and the α-oxo group of α-ketoglutarate.

Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae.

The amino acid L-lysine is synthesized in Saccharomyces cerevisiae via the α-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of α-aminoadipate from α-ketoadipate in the L-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses L-tyrosine, L-phenylalanine, α-ketoadipate, and L-α-aminoadipate as substrates. The UV-visible spectrum of the aminotransferase exhibits maxima at 280 and 343 nm at pH 7.5. As the pH is decreased the peak at 343 nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400 nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of α-ketoadipate to free enzyme leads to disappearance of the absorbance at 343 nm and appearance of peaks at 328 and 424 nm. The V/E(t) and V/K(α-ketoadipate)E(t) pH profiles are pH independent from pH 6.5 to 9.6, while the V/K(L-tyrosine) pH-rate profile decreases below a single pK(a) of 7.0 ± 0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a α-aminoadipate aminotransferase.

The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH.

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate L-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E(t) and V/KE(t) at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V1/E(t) and V1/K(NAD)Et at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK(a) of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.

Glutamates 78 and 122 in the active site of saccharopine dehydrogenase contribute to reactant binding and modulate the basicity of the acid-base catalysts.

Saccharopine dehydrogenase catalyzes the NAD-dependent oxidative deamination of saccharopine to give l-lysine and alpha-ketoglutarate. There are a number of conserved hydrophilic, ionizable residues in the active site, all of which must be important to the overall reaction. In an attempt to determine the contribution to binding and rate enhancement of each of the residues in the active site, mutations at each residue are being made, and double mutants are being made to estimate the interrelationship between residues. Here, we report the effects of mutations of active site glutamate residues, Glu(78) and Glu(122), on reactant binding and catalysis. Site-directed mutagenesis was used to generate E78Q, E122Q, E78Q/E122Q, E78A, E122A, and E78A/E122A mutant enzymes. Mutation of these residues increases the positive charge of the active site and is expected to affect the pK(a) values of the catalytic groups. Each mutant enzyme was completely characterized with respect to its kinetic and chemical mechanism. The kinetic mechanism remains the same as that of wild type enzymes for all of the mutant enzymes, with the exception of E78A, which exhibits binding of alpha-ketoglutarate to E and E.NADH. Large changes in V/K(Lys), but not V, suggest that Glu(78) and Glu(122) contribute binding energy for lysine. Shifts of more than a pH unit to higher and lower pH of the pK(a) values observed in the V/K(Lys) pH-rate profile of the mutant enzymes suggests that the presence of Glu(78) and Glu(122) modulates the basicity of the catalytic groups.

Conformational flexibility in the multidrug efflux system protein AcrA.

Intrinsic resistance to multiple drugs in many gram-negative bacterial pathogens is conferred by resistance nodulation cell division efflux pumps, which are composed of three essential components as typified by the extensively characterized Escherichia coli AcrA-AcrB-TolC system. The inner membrane drug:proton antiporter AcrB and the outer membrane channel TolC export chemically diverse compounds out of the bacterial cell, and require the activity of the third component, the periplasmic protein AcrA. The crystal structures of AcrB and TolC have previously been determined, and we complete the molecular picture of the efflux system by presenting the structure of a stable fragment of AcrA. The AcrA fragment resembles the elongated sickle shape of its homolog Pseudomonas aeruginosa MexA, being composed of three domains: beta-barrel, lipoyl, and alpha-helical hairpin. Notably, unsuspected conformational flexibility in the alpha-helical hairpin domain of AcrA is observed, which has potential mechanistic significance in coupling between AcrA conformations and TolC channel opening.