Adaptation of organisms to environmental niches is a hallmark of evolution. One prevalent example is that of thermal adaptation, wherein two descendants evolve at different temperature extremes1,2. Underlying the physiological differences between such organisms are changes in enzymes catalyzing essential reactions3, with orthologues from each organism undergoing adaptive mutations that preserve similar catalytic rates at their respective physiological temperatures 4,5. 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 (AK) 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 (i.e. the LID) control substrate affinity, while dynamic attenuation in the other (i.e. the AMPbd) affects rate-limiting conformational changes governing 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 paradigm for understanding the relationship between dynamics and turnover in AK.

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.

No abstract

CoA is the major acyl carrier in mammals and a key cofactor in energy metabolism.

BackgroundThe carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-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).ResultsHere, 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 β-lyases.ConclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-017-0082-4) contains supplementary material, which is available to authorized users.

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.

No abstract

Endogenous D-stereoisomers are being recognized as functionally important molecules in mammals. Here we report the first identification of endogenous D-cysteine in mammalian pancreas. Serine Racemase (SR) is the biosynthetic enzyme for D- cysteine. D-cysteine is present in substantial amounts in the eyes, brain and pancreas of mice. To characterize endogenous D-cysteine, we used SR deficient mice lacking racemizing ability and show 3.5 fold reduction in pancreatic D-cysteine. SR-/- mice produce 6-10 fold higher levels of insulin in the pancreas and plasma and are stored as amyloid aggregates in secretory vesicles and exosomes respectively. Lack of SR and endogenous D-cysteine globally decreased levels of nucleotides and cAMP, followed by reduced phosphorylation of CREB (S133) including lower expression of DNA methyltransferase (DNMT) 1, 3A and 3B, and reduced DNMT enzymatic and promoter activities in the pancreas. This results in decreased DNA methylation globally and specifically of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1 transporters in cells. Dietary supplementation with methyl donors rescues the high insulin levels and low DNMT activity in SR-/- mice. Our data show that SR racemizes cysteine in the pancreas and is a physiologic down regulator of insulin promoter methylation.