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Structural basis for Hif-1 a/ CBP recognition in the cellular hypoxic response

Structural basis for Hif-1 a/ CBP recognition in the cellular hypoxic response. 2010. 3. 10 권 혁 성. Introduction : Hypoxia Inducible Factor-1 a. Regulation of the hypoxia-inducible factor-1 a (HIF-1 a ) pathway. Introduction : Hypoxia Inducible Factor-1 a.

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Structural basis for Hif-1 a/ CBP recognition in the cellular hypoxic response

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  1. Structural basis for Hif-1a/CBP recognition in thecellular hypoxic response 2010. 3. 10 권 혁 성

  2. Introduction : Hypoxia Inducible Factor-1a Regulation of the hypoxia-inducible factor-1a(HIF-1a) pathway

  3. Introduction : Hypoxia Inducible Factor-1a Transcription of hypoxia responsive genes Mechanisms of HIF-1a

  4. Introduction : CBP/P300 • Contain two transcriptional adapter zinc-binding (TAZ) motifs • Cysteine/histidine-rich domain-1(the CH1 domain)->TAZ1 • Cysteine/histidine-rich domain-3(the CH3 domain)->TAZ2 and zz zinc-binding domain • Transcriptional regulation • - the interaction between the HIF-1a C-terminal activation domain (CAD) and • the TAZ1 domain of CBP/P300 • - If in normoxic condition, hydroxylation of an asparagine(803) residue • occurs and inhibits binding to CBP/P300

  5. Overall structure of the complex • Stereo view of the HIF-1a/TAZ1 complex • Residues 372-380 of TAZ1 are the first zinc-binding site • TAZ1 formed by residues P347~A372(a1), H383~H396(a2), A406~C421(a3), C429~N434(a4) packed to enclose a hydrophobic core • The zinc atom binds to H362, C366, C379, C384(Zn1), H393, C397, C403, C408(Zn2), H417, C421, C426, C429(Zn3)

  6. Interaction between the Hif-1a CAD and the TAZ1 domain • The dissociation constant of TAZ1 (345-439)/HIF-1a(776-826) was determined to be 7±1 nM by isothermal titration calorimetry(ITC) • The intermolecular interactions in HIF-1a 784-788(aA) region seem to be primarily hydrophobic, with A779, L783, M787 of the HIF-1a CAD contacting V357, L359 on the TAZ1 a1-helix • Deletion of this region, The dissociation constant of TAZ1 and HIF1a truncated form (790-826) was determined to be 34±5 nM by ITC

  7. Interaction between the Hif-1a CAD and the TAZ1 domain • The side chain of L795 project into a deep hydrophobic cavity • -substitution of L795 by small or polar side chains impairs HIF-1a • transcriptional activity • The stringency of leucine favors HIF1a-CAD transcriptional activity • Replacement of L795 with P, Q, V, A abrogated CAD transcription J. Biol. Chem. 276, 3550–3554, 2001

  8. Interaction between the Hif-1a CAD and the TAZ1 domain • The G791 backbone carbonyl forms a hydrogen bond to the side- • chain amide group of Q355 in TAZ1 a1-helix • L792 makes hydrophobic contacts, which fits into a shallow • depression at the a1/a2 of TAZ1. • P793 makes also hydrophobic contacts, which fits neatly into a groove • between L352 and Q356 on helix a1 of TAZ1.

  9. Interaction between the Hif-1a CAD and the TAZ1 domain J. Biol. Chem. 276, 3550–3554, 2001 • A short a-helix(residues 796-804, aB) of the HIF1a CAD is docked to TAZ1 through its more polar face, with C800 and N803 deeply buried in the molecular interface • This feature is consistent with mutagenesis data assigning C800 and N803 essential roles in CBP/P300 complex formation and hypoxic signal transduction • The Conserved Y798 and V802 side chains make little contact with TAZ1 • The side-chain carboxyl groups of D799 and E801 are in close contact with K 349 and K401 in TAZ1, respectively.

  10. Interaction between the Hif-1a CAD and the TAZ1 domain • L795P, C800R, L818S, L822S didn’t exhibit hypoxia-induced transcriptional activity, even through their protein expression levels were similar to tat of the wild type • L818 and L822 are critical for binding, and mutation of either of these residues impairs or abrogates transcriptional activation • The invariant L818, L819 and L822 side chains from the hydrophobic face of aC and are docked deeply within the groove J. Biol. Chem. 276, 3550–3554, 2001

  11. Interaction between the Hif-1a CAD and the TAZ1 domain • V825 makes hydrophobic contacts with TAZ1 • The E817 and D823 of HIF1a are in close proximity to K419 and R368 in the TAZ1 domain. • Despite the critical contacts provided by helix aC, it binds TAZ1 only weakly(Kd>500 mM) for HIF-1a (808-826) in the absence of stabilizing contacts involving other regions of the CAD

  12. Conclusion • This structure reveals the detail of the molecular interface between HIF-1aCAD and the CBP/P300 TAZ1 domain • That complex provides a basis for design of inhibitors • The structure of the complex of the HIF-1aCAD with the TAZ1 domain of CBP is of interest for the insights it provides into the molecular basis of the hypoxic response • Because tumor growth leads to hypoxia, this structure provides information for design of antitumor agents

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