Conformational selection is present in ligand binding to cytochrome P450 19A1 lipoprotein nanodiscs

Zárate‐Pérez, Francisco; Hackett, John C.

doi:10.1016/j.jinorgbio.2020.111120

Cited by 12 publications

(12 citation statements)

References 54 publications

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“…Finally, the differential HDX supports the fact that ASD eliminates detection of bimodal patterns, favoring binding to one state and thereby supporting the possibility that conformational selection, or the process by which a ligand preferentially binds to one of several conformations in equilibrium, occurs in 19A1. This observation is in agreement with recent stopped-flow data supporting the presence of conformational selection in ligand binding to 19A1 …”

supporting

confidence: 93%

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase

et al. 2020

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Aromatase (CYP19A1) catalyzes the synthesis of estrogens from androgens and is an invaluable target of pharmacotherapy for estrogen-dependent cancers. CYP19A1 is also one of the most primordial human CYPs and, to the extent that its fundamental dynamics are conserved, is highly relevant to understanding those of the more recently evolved and promiscuous enzymes. A complementary approach employing molecular dynamics simulations and hydrogen–deuterium exchange mass spectrometry (HDX-MS) was employed to interrogate the changes in CYP19A1 dynamics coupled to binding androstenedione (ASD). Gaussian-accelerated molecular dynamics and HDX-MS agree that ASD globally suppresses CYP19A1 dynamics. Bimodal HDX patterns of the B′–C loop potentially arising from at least two conformations are present in free 19A1 only, supporting the possibility that conformational selection is operative. Random-acceleration molecular dynamics and adaptive biasing force simulations illuminate ASD’s binding pathway, predicting ASD capture in the lipid headgroups and a pathway to the active site shielded from solvent. Intriguingly, the predicted access channel in 19A1 aligns well with the steroid binding sites of other human sterol-oxidizing CYPs.

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supporting

confidence: 93%

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase

et al. 2020

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“…Moreover, the enthalpic component involved in binding measured in the isothermal titration experiment is more pronounced in the case of the substrate free compared to the substrate bound form of the enzyme, indicating that, without the substrate, more interactions between the two partners (aromatase and CPR) are formed suggesting also conformational changes upon substrate binding [ 47 , 54 , 55 , 56 ]. In addition to the effector role of CPR, aromatase was also found to follow a conformational selection process [ 54 , 57 ] for substrate binding that is a well-known substrate binding process for P450s [ 58 , 59 ]. As shown in Figure 5 , in a conformational selection model, we can assume that aromatase is present as a heterogeneous population of different conformational states before binding the substrate.…”

Section: Discussionmentioning

confidence: 99%

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins

Catucci

Ciaramella

Nardo

et al. 2022

IJMS

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The cytochrome P450 superfamily are heme-thiolate enzymes able to carry out monooxygenase reactions. Several studies have demonstrated the feasibility of using a soluble bacterial reductase from Bacillus megaterium, BMR, as an artificial electron transfer partner fused to the human P450 domain in a single polypeptide chain in an approach known as ‘molecular Lego’. The 3A4-BMR chimera has been deeply characterized biochemically for its activity, coupling efficiency, and flexibility by many different biophysical techniques leading to the conclusion that an extension of five glycines in the loop that connects the two domains improves all the catalytic parameters due to improved flexibility of the system. In this work, we extend the characterization of 3A4-BMR chimeras using differential scanning calorimetry to evaluate stabilizing role of BMR. We apply the ‘molecular Lego’ approach also to CYP19A1 (aromatase) and the data show that the activity of the chimeras is very low (<0.003 min−1) for all the constructs tested with a different linker loop length: ARO-BMR, ARO-BMR-3GLY, and ARO-BMR-5GLY. Nevertheless, the fusion to BMR shows a remarkable effect on thermal stability studied by differential scanning calorimetry as indicated by the increase in Tonset by 10 °C and the presence of a cooperative unfolding process driven by the BMR protein domain. Previously characterized 3A4-BMR constructs show the same behavior of ARO-BMR constructs in terms of thermal stabilization but a higher activity as a function of the loop length. A comparison of the ARO-BMR system to 3A4-BMR indicates that the design of each P450-BMR chimera should be carefully evaluated not only in terms of electron transfer, but also for the biophysical constraints that cannot always be overcome by chimerization.

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“…This is the case of cytochromes P450 that display remarkable plasticity in their ability to bind substrates and catalyze a broad array of chemical reactions. 86–89 Strong evidence exists that such proteins bind ligands according to CS. In the case of cytochrome P450 P19A1, a detailed kinetic analysis using stopped flow has revealed the complexity of the recognition mechanism of three distinct ligands, i.e., androstenedione, testosterone, and 7-hydroxyflavone.…”

Section: Distinguishing Between If and Csmentioning

confidence: 99%

“…(42) . 86 The glucokinase regulatory protein plays an essential role in glucose homeostasis by acting as a competitive inhibitor of glucokinase and triggering its localization to the hepatocyte nucleus upon glucose deprivation. A small-molecule inhibitor of this protein-protein interaction utilizes CS by binding to a conformation of the regulatory protein that accounts for only 3% of the total population.…”

Section: Distinguishing Between If and Csmentioning

confidence: 99%

Mechanisms of ligand binding

2020

Biophysics Reviews

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Many processes in chemistry and biology involve interactions of a ligand with its molecular target. Interest in the mechanism governing such interactions has dominated theoretical and experimental analysis for over a century. The interpretation of molecular recognition has evolved from a simple rigid body association of the ligand with its target to appreciation of the key role played by conformational transitions. Two conceptually distinct descriptions have had a profound impact on our understanding of mechanisms of ligand binding. The first description, referred to as induced fit, assumes that conformational changes follow the initial binding step to optimize the complex between the ligand and its target. The second description, referred to as conformational selection, assumes that the free target exists in multiple conformations in equilibrium and that the ligand selects the optimal one for binding. Both descriptions can be merged into more complex reaction schemes that better describe the functional repertoire of macromolecular systems. This review deals with basic mechanisms of ligand binding, with special emphasis on induced fit, conformational selection, and their mathematical foundations to provide rigorous context for the analysis and interpretation of experimental data. We show that conformational selection is a surprisingly versatile mechanism that includes induced fit as a mathematical special case and even captures kinetic properties of more complex reaction schemes. These features make conformational selection a dominant mechanism of molecular recognition in biology, consistent with the rich conformational landscape accessible to biological macromolecules being unraveled by structural biology.

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Conformational selection is present in ligand binding to cytochrome P450 19A1 lipoprotein nanodiscs

Cited by 12 publications

References 54 publications

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase

Dynamics and Mechanism of Binding of Androstenedione to Membrane-Associated Aromatase

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins

Mechanisms of ligand binding

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