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Conformationally flexible hexakis‐urea 1 was synthesized efficiently by condensing hexakis(aminomethyl)benzene with 4‐nitrophenyl‐(3,5‐di‐tert‐butylphenyl)carbamate. The hexakis‐urea 1 is unexpectedly soluble in organic solvents of low polarity due to intramolecular hydrogen bonding. The hexakis‐urea 1 recognizes chloride, bromide, and acetate in a 1:2 host‐guest ratio and in a positive allosteric manner in CDCl3. The ability of 1 to recognize dihydrogen phosphate is a unique outcome, and the structure of the associated complex, which contains four dihydrogen phosphate ions, was clarified by single‐crystal X‐ray structural analysis. However, in solution, a complex with three dihydrogen phosphate ions was identified. The dihydrogen phosphate association in CDCl3 proceeds in an amphoteric allosteric manner; in a positive allosteric manner (K1K3) in the subsequent step. [ABSTRACT FROM AUTHOR]

ATP binding cassette type-C (ABCC) transporters move molecules across cell membranes upon hydrolysis of ATP; however, their coupling of ATP hydrolysis to substrate transport remains elusive. Drosophila multidrug resistance-associated protein (DMRP) is the functional ortholog of human long ABCC transporters, with similar substrate and inhibitor specificity, but higher activity. Exploiting its high activity, we kinetically dissected the catalytic mechanism of DMRP by using E2-d-glucuronide (E2G), the physiologic substrate of human ABCC. We examined the DMRP-mediated interdependence of ATP and E2G in biochemical assays. We observed E2G-dependent ATPase activity to be biphasic at subsaturating ATP concentrations, which implies at least 2 E2G binding sites on DMRP. Furthermore, transport measurements indicated strong nonreciprocal cooperativity between ATP and E2G. In addition to confirming these findings, our kinetic modeling with the Complex Pathway Simulator indicated a 10-fold decrease in the E2G-mediated activation of ATP hydrolysis upon saturation of the second E2G binding site. Surprisingly, the binding of the second E2G allowed for substrate transport with a constant rate, which tightly coupled ATP hydrolysis to transport. In summary, we show that the second E2G binding--similar to human ABCC2--allosterically stimulates transport activity of DMRP. Our data suggest that this is achieved by a significant increase in the coupling of ATP hydrolysis to transport. [ABSTRACT FROM AUTHOR]

New strategies to potentially improve drug safety and efficacy emerge with allosteric programs. Biased allosteric modulators can be designed with high subtype selectivity and defined receptor signaling endpoints, however, selecting the most meaningful parameters for optimization can be perplexing. Historically, “potency hunting” at the expense of physicochemical and pharmacokinetic optimization has led to numerous tool compounds with excellent pharmacological properties but no path to drug development. Conversely, extensive physicochemical and pharmacokinetic screening with only post hoc bias and allosteric characterization has led to inefficacious compounds or compounds with on-target toxicities. This field is rapidly evolving with new mechanistic understanding, changes in terminology, and novel opportunities. The intent of this digest is to summarize current understanding and debates within the field. We aim to discuss, from a medicinal chemistry perspective, the parameter choices available to drive SAR. [ABSTRACT FROM AUTHOR]

An enzyme's inherent structural plasticity is frequently associated with substrate binding, yet detailed structural characterization of flexible proteins remains challenging. This study employs complementary biophysical methods to characterize the partially unfolded structure of substrate-free AAC(6′)-Ii, an N-acetyltransferase of the GCN5-related N-acetyltransferase ( GNAT) superfamily implicated in conferring broad-spectrum aminoglycoside resistance on Enterococcus faecium. The X-ray crystal structure of AAC(6′)-Ii is analyzed to identify relative motions of the structural elements that constitute the dimeric enzyme. Comparison with the previously elucidated crystal structure of AAC(6′)-Ii with acetyl coenzyme A (AcCoA) reveals conformational changes that occur upon substrate binding. Our understanding of the enzyme's structural plasticity is further refined with small-angle X-ray scattering and circular dichroism analyses, which together reveal how flexible structural elements impact dimerization and substrate binding. These results clarify the extent of unfolding that AAC(6′)-Ii undergoes in the absence of AcCoA and provide a structural connection to previously observed allosteric cooperativity of this enzyme. Database Structural data are available in the PDB database under the accession number . [ABSTRACT FROM AUTHOR]

The iron-containing hemoglobins (Hbs) are essential proteins to serve as oxygen transporters in the blood. The cooperative oxygen-binding is a distinct feature for them to load and unload oxygen efficiently. Among various kinds of hemoglobins, the earthworm hemoglobins are the champions in carrying oxygen due to not only their large size but also the unusually high cooperativity. Here we report the first study on the cooperative mechanism in the Lumbricus terrestris hemoglobin. The near atomic resolution of our cryo-EM structure also enables us to further visualize the additional sinusoidal bracelet, which accounts for a new assembly mechanism of this enormous structure. [ABSTRACT FROM AUTHOR]

Abstract: A study is presented on the effect of zinc binding at the matrix side, on the proton pump of purified liposome reconstituted bovine heart cytochrome c oxidase (COV). Internally trapped Zn2+ resulted in 50% decoupling of the proton pump at level flow. Analysis of the pH dependence of inhibition by internal Zn2+ of proton release in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase indicates that Zn2+ suppresses two of the four proton pumping steps in the cycle, those taking place when the 2 OH− produced in the reduction of O2 at the binuclear center are protonated to 2 H2O. This decoupling effect could be associated with Zn2+ induced conformational alteration of an acid/base cluster linked to heme a 3 . [Copyright &y& Elsevier]

Protein kinase A (cAMP dependent protein kinase catalytic subunit, EC 2.7.11.11) binds simultaneously ATP and a phosphorylatable peptide. These structurally dissimilar allosteric ligands influence the binding effectiveness of each other. The same situation is observed with substrate congeners, which reversibly inhibit the enzyme. In this review these allosteric effects are quantified using the interaction factor, which compares binding effectiveness of ligands with the free enzyme and the pre-loaded enzyme complex containing another ligand. This analysis revealed that the allosteric effect depends upon structure of the interacting ligands, and the principle “better binding: stronger allostery” observed can be formalized in terms of linear free-energy relationships, which point to similar mechanism of the allosteric interaction between the enzyme-bound substrates and/or inhibitor molecules. On the other hand, the type of effect is governed by ligand binding effectiveness and can be inverted from positive allostery to negative allostery if we move from effectively binding ligands to badly binding compounds. Thus the outcome of the allostery in this monomeric enzyme is the same as defined by classical theories for multimeric enzymes: making the enzyme response more efficient if appropriate ligands bind. [ABSTRACT FROM AUTHOR]

Allosteric cooperativity between peptide and ATP binding sites on cAMP-dependent protein kinase catalytic subunit was studied kinetically for the reaction of phosphorylation of seven peptide substrates. The allosteric effect was quantified in terms of the interaction factor α by comparing binding effectiveness of a substrate molecule with the free enzyme and with the enzyme complex with another substrate. It was discovered that the magnitude of the allosteric feedback between these binding sites was governed by the effectiveness of substrate binding, which was varied by using different peptides, and the principle 'better binding: stronger allostery' was formulated. This interrelationship was further formalized in terms of a linear-free-energy relationship pα =C+SpKb, holding between the free energy of the allosteric interaction, quantified by the negative logarithm of the interaction factor α (pα ) and the effectiveness of substrate binding quantified by pKb. For the peptide phosphorylation reaction C= -1.4 and S = 0.4 were obtained. The negative intercept C indicated that the positive cooperativity between the binding sites, characterized by α < 1 at sub-millimolar Kb values, changed into negative cooperativity with α > 1 at millimolar Kb values. This means that inversion of the cooperative effect was induced by substrate structure, and allostery was used by this enzyme as an additional mechanism to discriminate between substrates, facilitating phosphorylation of good substrates and providing additional protection against phosphorylation of bad substrates. Some implications of this allosteric mechanism on substrate specificity of protein kinases were discussed. [ABSTRACT FROM AUTHOR]

Cover Feature: Amphoteric Homotropic Allosteric Association between a Hexakis-Urea Receptor and Dihydrogen Phosphate (Chem. Eur. J. 71/2019) Keywords: allosteric cooperativity; anion recognition; host-guest system; hydrogen bonding; receptors Allosteric cooperativity, anion recognition, host-guest system, hydrogen bonding, receptors. [Extracted from the article]