Dataset Information

In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals.

ABSTRACT: Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.

SUBMITTER: Plavsa JJ

PROVIDER: S-EPMC5931143 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals.

Plavša Jovana J JJ Řezáčová Pavlína P Kugler Michael M Pachl Petr P Brynda Jiří J Voburka Zdeněk Z Ćelić Anđelka A Petri Edward T ET Škerlová Jana J

Acta crystallographica. Section F, Structural biology communications 20180424 Pt 5

Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His<sub>6</sub> tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the ...[more]

PMID: 29717998

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Project description:Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.

Dataset Information

In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals.

Publications

In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals.

Similar Datasets

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