scholarly journals Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

2013 ◽  
Vol 288 (23) ◽  
pp. 17008-17018 ◽  
Author(s):  
D. Fernando Estrada ◽  
Jennifer S. Laurence ◽  
Emily E. Scott
Keyword(s):  
Cytochrome P450 ◽  
Binding Site ◽  
Active Site ◽  
Catalytic Domain ◽  
Cytochrome B5 ◽  
Structural Basis ◽  
Reaction Conditions ◽  
Reversible Binding ◽  
Important Interaction

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

scholarly journals Molecular determinant of substrate binding and specificity of cytochrome P450 2J2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liang Xu ◽  
Liao Y. Chen
Keyword(s):  
Arachidonic Acid ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Model ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Structural Basis ◽  
Molecular Determinant ◽  
Dynamics Simulations

AbstractCytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein

1994 ◽  
Vol 14 (11) ◽  
pp. 7256-7264
Author(s):  
Y W Kim ◽  
G A Otterson ◽  
R A Kratzke ◽  
A B Coxon ◽  
F J Kaye
Keyword(s):  
Binding Site ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Binding Activity ◽  
Reversible Binding ◽  
Complex Interactions ◽  
Differential Binding ◽  
Cellular Transcription

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

Peroxovanadates and Its Bio-Mimicking Relation with Vanadium Haloperoxidases

2016 ◽  
pp. 197-219
Author(s):  
Pranjal Saikia ◽  
Saitanya Kumar Bharadwaj ◽  
Abu Taleb Miah
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
High Efficiency ◽  
Recombinant Enzymes ◽  
Vanadium Compounds ◽  
Reaction Conditions ◽  
The Cost ◽  
Major Emphasis ◽  
Protein Active Site

Vanadium Haloperoxidases (VHPOs) have been used in a variety of biotransformations showing remarkable stereoselectivity and regiospecificity. The high efficiency of the enzyme is influenced by the protein active site and the role of certain amino acids in activation of vanadium(V)-bound peroxide for halide oxidation. The use of natural or recombinant enzymes, or biomimetic vanadium compounds brings up issues regarding the cost of production and reaction conditions. In this chapter, the primary intent is to provide a simple and clear picture of functional mimicking nature of peroxovanadium compounds with haloperoxidases enzymes to the readers. Major emphasis would be given to examine the reactivity of the vanadium haloperoxidases with mechanism.

Structural Basis for the Inhibition Mechanism of Ecotin against Neutrophil Elastase by Targeting the Active Site and Secondary Binding Site

Biochemistry ◽  
2020 ◽  
Vol 59 (30) ◽  
pp. 2788-2795
Author(s):  
Chacko Jobichen ◽  
Mahalakshmi Tirumuru Prabhakar ◽  
Su Ning Loh ◽  
J. Sivaraman
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Neutrophil Elastase ◽  
Structural Basis ◽  
Inhibition Mechanism

Topological role of cytochrome P450 2D6 active site residues

2006 ◽  
Vol 447 (1) ◽  
pp. 53-58 ◽  
Author(s):  
Robert A.B. van Waterschoot ◽  
Peter H.J. Keizers ◽  
Chris de Graaf ◽  
Nico P.E. Vermeulen ◽  
Richard A. Tschirret-Guth
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Cytochrome P450 2D6 ◽  
Active Site Residues ◽  
P450 2D6

scholarly journals Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein.

1994 ◽  
Vol 14 (11) ◽  
pp. 7256-7264 ◽  
Author(s):  
Y W Kim ◽  
G A Otterson ◽  
R A Kratzke ◽  
A B Coxon ◽  
F J Kaye
Keyword(s):  
Binding Site ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Binding Activity ◽  
Reversible Binding ◽  
Complex Interactions ◽  
Differential Binding ◽  
Cellular Transcription

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

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