Modulating Enzyme Function via Dynamic Allostery within Biliverdin Reductase B.
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ABSTRACT:
SUBMITTER: Redzic JS
PROVIDER: S-EPMC8173106 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Many protein systems rely on coupled dynamic networks to allosterically regulate function. However, the broad conformational space sampled by non-coherently dynamic systems has precluded detailed analysis of their communication mechanisms. Here, we have developed a methodology that combines the high sensitivity afforded by nuclear magnetic resonance relaxation techniques and single-site multiple mutations, termed RASSMM, to identify two allosterically coupled dynamic networks within the non-coherently dynamic enzyme cyclophilin A. Using this methodology, we discovered two key hotspot residues, Val6 and Val29, that communicate through these networks, the mutation of which altered active-site dynamics, modulating enzymatic turnover of multiple substrates. Finally, we utilized molecular dynamics simulations to identify the mechanism by which one of these hotspots is coupled to the larger dynamic networks. These studies confirm a link between enzyme dynamics and the catalytic cycle of cyclophilin A and demonstrate how dynamic allostery may be engineered to tune enzyme function.
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Project description:Biliverdin Reductase B (BLVRB) is an NADPH-dependent reductase that catalyzes the reduction of multiple substrates and is therefore considered a critical cellular redox regulator. In this study, we sought to address whether both structural and dynamics changes occur between different intermediates of the catalytic cycle and whether these were relegated to just the active site or the entirety of the enzyme. Through X-ray crystallography, we determined the apo BLVRB structure for the first time, revealing subtle global changes compared to the holo structure and identifying the loss of a critical hydrogen bond that "clamps" the R78-loop over the coenzyme. Amide and Cα chemical shift perturbations were used to identify environmental and secondary structural changes between intermediates, with more distant global changes observed upon coenzyme binding compared to substrate interactions. NMR relaxation rate measurements provided insights into the dynamic behavior of BLVRB during the catalytic cycle. Specifically, the inherently dynamic R78-loop that becomes ordered upon coenzyme binding persists through the catalytic cycle while similar regions experience dynamic exchange. However, the dynamic exchange processes were found to differ through the catalytic cycle with several groups of residues exhibiting similar dynamic responses. Finally, both local and distal structural and dynamic changes occur within BLVRB that are dependent solely on the oxidative state of the coenzyme. Thus, through a comprehensive analysis here, this study revealed structural and dynamic alterations in BLVRB through its catalytic cycle that are not simply relegated to the active site, but instead, are allosterically coupled throughout the enzyme.
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Project description:Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and ?-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed ?-adrenergic inotropic responses that were associated with impaired ?-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.
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Project description:Bioenergetic requirements of hematopoietic stem cells and pluripotent stem cells (PSCs) vary with lineage fate, and cellular adaptations rely largely on substrate (glucose/glutamine) availability and mitochondrial function to balance tricarboxylic acid (TCA)-derived anabolic and redox-regulated antioxidant functions. Heme synthesis and degradation converge in a linear pathway that utilizes TCA cycle-derived carbon in cataplerotic reactions of tetrapyrrole biosynthesis, terminated by NAD(P)H-dependent biliverdin reductases (IXα, BLVRA and IXβ, BLVRB) that lead to bilirubin generation and cellular antioxidant functions. We now demonstrate that PSCs with targeted deletion of BLVRB display physiologically defective antioxidant activity and cellular viability, associated with a glutamine-restricted defect in TCA entry that was computationally predicted using gene/metabolite topological network analysis and subsequently validated by bioenergetic and isotopomeric studies. Defective BLVRB-regulated glutamine utilization was accompanied by exaggerated glycolytic accumulation of the rate-limiting hexokinase reaction product glucose-6-phosphate. BLVRB-deficient embryoid body formation (a critical size parameter of early lineage fate potential) demonstrated enhanced sensitivity to the pentose phosphate pathway (PPP) inhibitor 6-aminonicotinamide with no differences in the glycolytic pathway inhibitor 2-deoxyglucose. These collective data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism and suggest that early stages of lineage fate potential require glutamine anaplerotic functions and an intact PPP, which are, in part, regulated by BLVRB activity. In principle, BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization with consequences for cancer and hematopoietic metabolism.
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Project description:Chronic hypoxia in the renal parenchyma is thought to induce epithelial-to-mesenchymal transition (EMT), leading to fibrogenesis and ultimately end-stage renal failure. Biliverdin reductase, recently identified as a serine/threonine/tyrosine kinase that may activate phosphatidylinositol 3-kinase (PI3K) and Akt, is upregulated in response to reactive oxygen species that may accompany hypoxia. We investigated this potential role of biliverdin reductase in hypoxia-induced renal tubular EMT. Expression of biliverdin reductase was upregulated in a human proximal tubule cell line (HK-2) cultured in hypoxic conditions (1% O2), and this was accompanied by reduced expression of E-cadherin and increased expression of the mesenchymal marker vimentin. Inhibiting PI3K reversed these changes, consistent with EMT. In normoxic conditions, overexpression of biliverdin reductase promoted similar characteristics of EMT, which were also reversed by inhibiting PI3K. Furthermore, using small interfering RNA (siRNA) to knockdown biliverdin reductase, we demonstrated that the enzyme associates with phosphorylated Akt and mediates the hypoxia-induced EMT phenotype. In vivo, expression of biliverdin reductase increased in the tubular epithelia of 5/6-nephrectomized rats, and immunohistochemistry of serial sections demonstrated similar localization of phosphorylated Akt and biliverdin reductase. In conclusion, biliverdin reductase mediates hypoxia-induced EMT through a PI3K/Akt-dependent pathway.
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Project description:Tissue absorbance, light scattering, and autofluorescence are significantly lower in the near-infrared (NIR) range than in the visible range. Because of these advantages, NIR fluorescent proteins (FPs) are in high demand for in vivo imaging. Nevertheless, application of NIR FPs such as iRFP is still limited due to their dimness in mammalian cells. In contrast to GFP and its variants, iRFP requires biliverdin (BV) as a chromophore. The dimness of iRFP is at least partly due to rapid reduction of BV by biliverdin reductase-A (BLVRA). Here, we established biliverdin reductase-a knockout (Blvra-/-) mice to increase the intracellular BV concentration and, thereby, to enhance iRFP fluorescence intensity. As anticipated, iRFP fluorescence intensity was significantly increased in all examined tissues of Blvra-/- mice. Similarly, the genetically encoded calcium indicator NIR-GECO1, which is engineered based on another NIR FP, mIFP, exhibited a marked increase in fluorescence intensity in mouse embryonic fibroblasts derived from Blvra-/- mice. We expanded this approach to an NIR light-sensing optogenetic tool, the BphP1-PpsR2 system, which also requires BV as a chromophore. Again, deletion of the Blvra gene markedly enhanced the light response in HeLa cells. These results indicate that the Blvra-/- mouse is a versatile tool for the in vivo application of NIR FPs and NIR light-sensing optogenetic tools.Key words: in vivo imaging, near-infrared fluorescent protein, biliverdin, biliverdin reductase, optogenetic tool.
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Project description:Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the ?2:?2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122? ? [W48??] ? Y356? ? Y731? ? Y730? ? C439?). The mutant R411A(?) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in ?2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active ?2:?2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-?2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns-?s time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 104 s-1 when comparing wild type to R411A-?2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.
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Project description:Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.
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Project description:Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.
| S-EPMC6572854 | biostudies-literature
Project description:Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78?Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.
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