Structural and Biochemical Characterization of Compounds Inhibiting Mycobacterium tuberculosis Pantothenate Kinase

Mycobacterium tuberculosis, the bacterial causative agent of tuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemical characterizations of two new classes of compounds that inhibit pantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenate and phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for any pantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.     

Testing the Sulfotransferase Molecular Pore Hypothesis

Human cytosolic sulfotransferases (SULTs) regulate the activities of hundreds of signaling metabolites via transfer of the sulfuryl moiety (-SO₃) from activated sulfate (3′-phosphoadenosine 5′-phosphosulfate) to the hydroxyls and primary amines of xeno- and endobiotics. How SULTs select substrates from the scores of competing ligands present in a cytosolic milieu is an important issue in the field. Selectivity appears to be sterically controlled by a molecular pore that opens and closes in response to nucleotide binding. This point of view is fostered by structures showing nucleotide-dependent pore closure and the fact that nucleotide binding induces an isomerization that restricts access to the acceptor-binding pocket. Molecular dynamics models underscore the importance of pore isomerization in selectivity and predict that specific molecular linkages stabilize the closed pore in response to nucleotide binding. To test the pore model, these linkages were disrupted in SULT2A1 via mutagenesis, and the effects on selectivity were determined. The mutations uncoupled nucleotide binding from selectivity and produced enzymes that no longer discriminated between large and small substrates. The mutations did not affect the affinity or turnover of small substrates but resulted in a 183-fold gain in catalytic efficiently toward large substrates. Models predict that an 11-residue “flap” covering the acceptor-binding pocket can open and admit large substrates when nucleotide is bound; a mutant structure demonstrated that this is so. In summary, the model was shown to be a robust, accurate predictor of SULT structure and selectivity whose general features will likely apply to other members of the SULT family.     

A Structural View on the Functional Importance of the Sugar Moiety and Steroid Hydroxyls of Cardiotonic Steroids in Binding to Na,K-ATPase

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgP_i or in the E2BeF_x conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the β-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, digitoxin, and gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na⁺ ions to K⁺ binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid β-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.     

Discovery of a small-molecule inhibitor and cellular probe of Keap1–Nrf2 protein–protein interaction

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1–Nrf2 protein–protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure–activity relationships support its use as a lead for our ongoing optimization     

Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

The concomitant production of formic acid and pterin compounds from guanosine-5′-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, l-threo-neopterin–the major Crithidia factor in S. indica–, a cyclic phosphate of neopterin (cNP), d-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^?bm. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and d-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. l-threo-neopterin and cNP, from GTP in S. indica is also discussed.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

Hydroxylated chalcones with dual properties: Xanthine oxidase inhibitors and radical scavengers

In this study, we evaluated the abilities of a series of chalcones to inhibit the activity of the enzyme xanthine oxidase (XO) and to scavenge radicals. 20 mono- and polyhydroxylated chalcone derivatives were synthesized by Claisen–Schmidt condensation reactions and then tested for inhibitory potency against XO, a known generator of reactive oxygen species (ROS). In parallel, the ability of the synthesized chalcones to scavenge a stable radical was determined. Structure–activity relationship analysis in conjunction with molecular docking indicated that the most active XO inhibitors carried a minimum of three hydroxyl groups. Moreover, the most effective radical scavengers had two neighboring hydroxyl groups on at least one of the two phenyl rings. Since it has been proposed previously that XO inhibition and radical scavenging could be useful properties for reduction of ROS-levels in tissue, we determined the chalcones’ effects to rescue neurons subjected to ROS-induced stress created by the addition of β-amyloid peptide. Best protection was provided by chalcones that combined good inhibitory potency with high radical scavenging ability in a single molecule, an observation that points to a potential therapeutic value of this compound class.     

Substantial Influence of ERAP2 on the HLA-B*40:02 Peptidome: Implications for HLA-B*27-Negative Ankylosing Spondylitis

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Highlights

• •HLA-B*40:02 and ERAP2 are risk factors for ankylosing spondylitis.
• •The effects of ERAP2 on the B*40:02 peptidome are defined.
• •ERAP2 has a major influence mainly due to alterations of N-terminal residues.
• •These effects provide a basis for the association of ERAP2 with disease.