Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid.

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported.

Bilirubin Links Heme Metabolism to Neuroprotection by Scavenging Superoxide.

Bilirubin is one of the most frequently measured metabolites in medicine, yet its physiologic roles remain unclear. Bilirubin can act as an antioxidant in vitro, but whether its redox activity is physiologically relevant is unclear because many other antioxidants are far more abundant in vivo. Here, we report that depleting endogenous bilirubin renders mice hypersensitive to oxidative stress. We find that mice lacking bilirubin are particularly vulnerable to superoxide (O2⋅-) over other tested reactive oxidants and electrophiles. Whereas major antioxidants such as glutathione and cysteine exhibit little to no reactivity toward O2⋅-, bilirubin readily scavenges O2⋅-. We find that bilirubin's redox activity is particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging O2⋅- during NMDA neurotransmission. Bilirubin's unique redox activity toward O2⋅- may underlie a prominent physiologic role despite being significantly less abundant than other endogenous and exogenous antioxidants.

Dynamic allostery can drive cold adaptation in enzymes.

Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, in which two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes that catalyse essential reactions 3 , with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures4,5. The sequence changes responsible for these adaptive differences, however, are often at surface-exposed sites distant from the substrate-binding site, leaving the active site of the enzyme structurally unperturbed6,7. How such changes are allosterically propagated to the active site, to modulate activity, is not known. Here we show that entropy-tuning changes can be engineered into distal sites of Escherichia coli adenylate kinase, allowing us to quantitatively assess the role of dynamics in determining affinity, turnover and the role in driving adaptation. The results not only reveal a dynamics-based allosteric tuning mechanism, but also uncover a spatial separation of the control of key enzymatic parameters. Fluctuations in one mobile domain (the LID) control substrate affinity, whereas dynamic attenuation in the other domain (the AMP-binding domain) affects rate-limiting conformational changes that govern enzyme turnover. Dynamics-based regulation may thus represent an elegant, widespread and previously unrealized evolutionary adaptation mechanism that fine-tunes biological function without altering the ground state structure. Furthermore, because rigid-body conformational changes in both domains were thought to be rate limiting for turnover8,9, these adaptation studies reveal a new model for understanding the relationship between dynamics and turnover in adenylate kinase.

Nudt19 is a renal CoA diphosphohydrolase with biochemical and regulatory properties that are distinct from the hepatic Nudt7 isoform.

CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism. Dynamic regulation of CoA in different tissues and organs supports metabolic flexibility. Two mammalian Nudix hydrolases, Nudt19 and Nudt7, degrade CoA in vitro Nudt19 and Nudt7 possess conserved Nudix and CoA signature sequences and specifically hydrolyze the diphosphate bond of free CoA and acyl-CoAs to form 3',5'-ADP and 4'-(acyl)phosphopantetheine. Limited information is available on these enzymes, but the relatively high abundance of Nudt19 and Nudt7 mRNA in the kidney and liver, respectively, suggests that they play specific roles in the regulation of CoA levels in these organs. Here, we analyzed Nudt19-/- mice and found that deletion of Nudt19 elevates kidney CoA levels in mice fed ad libitum, indicating that Nudt19 contributes to the regulation of CoA in vivo Unlike what was observed for the regulation of Nudt7 in the liver, Nudt19 transcript and protein levels in the kidney did not differ between fed and fasted states. Instead, we identified chenodeoxycholic acid as a specific Nudt19 inhibitor that competed with CoA for Nudt19 binding but did not bind to Nudt7. Exchange of the Nudix and CoA signature motifs between the two isoforms dramatically decreased their kcat Furthermore, substitutions of conserved residues within these motifs identified amino acids playing different roles in CoA binding and hydrolysis in Nudt19 and Nudt7. Our results reveal that the kidney and liver each possesses a distinct peroxisomal CoA diphosphohydrolase.

Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor.

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here, we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of 'energetic frustration'. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.

Structural insight into the inactivation of Mycobacterium tuberculosis non-classical transpeptidase LdtMt2 by biapenem and tebipenem.

BACKGROUND: The carbapenem subclass of ß-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of ß-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb). RESULTS: Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by ß-lyases. CONCLUSION: The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.