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Topological Specificity in Inhibitor Recognition by Proteolytic Enzymes

Topological Specificity in Inhibitor Recognition by Proteolytic Enzymes. Jeni Lauer-Fields. Matrix Metalloproteinases. Seiki, Current Opinion Cell Biology ( 2002) 14, 624-632. MMP Structures Superimposed. Bode, Cellular Molecular Life Sci. ( 1999) 55, 639-652.

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Topological Specificity in Inhibitor Recognition by Proteolytic Enzymes

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  1. Topological Specificity in Inhibitor Recognition by Proteolytic Enzymes Jeni Lauer-Fields

  2. Matrix Metalloproteinases Seiki, Current Opinion Cell Biology ( 2002) 14, 624-632.

  3. MMP Structures Superimposed Bode, Cellular Molecular Life Sci. ( 1999) 55, 639-652.

  4. Tissue Inhibitors of Metalloproteinases • Regulate activity of MMPs • Form tight non-covalent 1:1 complex with MMPs • Regulate ECM turnover and other cellular processes • Two domains, with N-terminal domain retaining most of the inhibitory activity • Four human TIMPs (1-4), constitutive and regulated expression patterns Abraham et al. Current Vascular Pharmacology ( 2005) 3, 369-379.

  5. Endothelin-Fold as a Scaffold for TIMP-Based Inhibitors Endothelins are 21 residue proteins with vasoactive properties Family members; endothelins and sarafotoxins Contain 2 disulfide bonds and moderate -helical content Comparison with TIMP three-dimensional structure, obvious similarities PDB file: 1SRB

  6. Comparison of Sarafotoxin and TIMP Structures TIMP-1 SRT-6b

  7. Determinants of MMP Binding • ~75% of contacts are from region including residues 1-4 and residues 66-69 • -amino and carbonyl groups of Cys1 coordinate Zn++ • -OH of residue 2 displaces MMP-bound H2O necessary for hydrolysis TIMP-1 Brew et al. Biochim. Biophys. Acta, 1477 (2000) 267-283.

  8. Sarafotoxin Variants H-CSCKDMTDKECLYFCHQDVIW-OH H-CSCKDMTDKECLYFCHQD-OH H-CSCKDMTDKECLYFCVQD-OH H-CSCADMTDKECLYFCHQD-OH H-CSCSDMTDKECLYFCHQD-OH H-CSCKDMTDKECLYFCMSEMS-NH2 H-CSCSDMTDKECLYFCMSEMS-NH2 Ac-CSCSDMTDKECLYFCMSEMS-NH2 H-XSXSDMTDKEXLYFXMSEMS-NH2 Ac-XSXSDMTDKEXLYFXMSEMS-NH2

  9. Inhibition of MMPs by Sarafotoxin Variant STX-S4-CT

  10. Apparent Ki Values Sarafotoxin Model Peptides NI: No inhibition detected

  11. Circular Dichroism Spectra

  12. CD Spectra Sarafotoxin Model Peptides

  13. Molecular Docking with MMP-1 PDB file generated by PatchDock

  14. Apparent Ki Values and CD Spectra

  15. Current Work • 2D NMR Spectra for STX-S4 and STX-S4-CT • Make a new model based on NMR data • Repeat docking and analysis with NMR-based model

  16. Biomolecular Proton NMR http://www.embl.de/nmr/sattler/lab/

  17. Biomolecular Proton NMR http://www.embl.de/nmr/sattler/lab/

  18. Summary • Endothelin-fold is a good template for peptide-based inhibitors of MMPs • Enhance selectivity between MMPs and other metalloproteinases (ADAM/ADAMTS) • Inhibition is related to endothelin-fold as well as N-terminal charge • Mechanism may be similar to TIMPs

  19. Acknowledgements Biomedical Sciences Keith Brew Vijaya Iragavarapu Shuo Wei Tyrone Ferns Gina Spruill Chemistry and Biochemistry Gregg Fields Frank Mari Mare Cudic Imperial College Hideaki Nagase Rob Visse

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