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How metal ions control protein structure and dynamics (and biomolecular interactions)

How metal ions control protein structure and dynamics (and biomolecular interactions). A: Calmodulin B: Zinc fingers. Calcium-binding proteins: Calcium regulates protein structure, dynamics and interactions. The EF hand motif. Numerous Ca-binding proteins contain pairs of this motif.

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How metal ions control protein structure and dynamics (and biomolecular interactions)

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  1. How metal ions control protein structure and dynamics(and biomolecular interactions) A: Calmodulin B: Zinc fingers

  2. Calcium-binding proteins:Calcium regulates proteinstructure, dynamics and interactions

  3. The EF hand motif Numerous Ca-binding proteins contain pairs of this motif http://www.agr.nagoya-u.ac.jp/~mcr/Research/EF-hand.html Lewit-Bentley, A. & Réty, S. (2000) Curr. Op. Struct. Biol., 10, 637 - 643 Labels E and F stem from parvalbumin, where this motif was first discovered

  4. Calmodulin • Monomeric, 148 aa, 17 kDa • Binds up to 4 Ca2+ • 2 x 2 EF-hands • Why specific for Ca2+ ? • log K≈ 6-7 (Mg2+: 3-4) • 7 oxygen ligands (hard !): • Not suitable for soft/borderline ions (Cu(I/II), Zn(II)) • Mg2+ or Fe3+ too small

  5. 1 EF hand A pair of EF hands Full-length Ca4-calmodulin Calmodulin X-ray: pdb 1cll; Chattopadhyaya, R.,  Meador, W.E.,  Means, A.R.,  Quiocho, F.A. J. Mol. Biol. 228, 1177-1192 (1992) Two domains connected by long a-helix

  6. Apo CaM: Disruption of central helix • NMR solution structure • No fixed orientation of the two domains w.r. to each other • Key point: Ca2+ binding changes protein structure and dynamics NMR structure: pdb 1dmo; M Zhang, T Tanaka, M Ikura: Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nature structural biology. (1995) 2, pp. 758-67

  7. Cooperativity • Usually, simple binding curves are hyperbolical • Cooperativity is frequent in biological systems • Binding curves are sigmoidal • Means: Binding of the first Ca enhances binding of the second and so on • Will see again when discussing Hb non-cooperative binding cooperative binding in CaM Bound Ca2+ http://ead.univ-angers.fr/~jaspard/Page2/COURS/7RelStructFonction/2Biochimie/5Signalisation/2Calmoduline/1Calmodulin.htm

  8. Ca-loaded CaM interacts with a plethora of proteins • Strong interaction (Kd 10-100 nM) with many different proteins: Strange

  9. How ? • Ca2+ binding induces the formation of hydrophobic patches on calmodulin surface • CaM-binding proteins have a positively charged amphipathic helix (hydrophobic and hydrophilic sides) • Interacts with both hydrophobic patches calmodulin “mitts”

  10. Less schematic: WE Meador, AR Means, FA Quiocho: Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. (1992) 257, 1251-5

  11. Summary • Binding of Ca2+ to calmodulin changes protein structure and dynamics • This change in properties enables CaM to bind to a plethora of other proteins, but only in the presence of Ca2+ (and only Ca2+) • This is how, by regulation of intracellular Ca2+ concentrations, the activity of many other proteins can be regulated

  12. Bio-Inorganic Chemistry Lecture 6b Zinc fingers: Control of protein structure and biomolecular interactions

  13. Zinc fingers and other proteins with structural zinc Zinc proteins Zinc transporting proteins Zinc enzymes Enzymes in which zinc modulates activity others Hydrolases Metallo-b-lactamases others nucleases peptidases

  14. Zinc fingers • Small protein domains • Classical: C2H2 ligand set • Tetrahedral coordination • Unfolded without zinc • DNA binding (only in presence of zinc) C N • Transcription factors • Often occur in multiples

  15. Zif268 binding to DNA 3 fingers in one protein Interaction with the major groove M Elrod-Erickson, TE Benson, CO Pabo

  16. DNA recognition: a code ? Corbi, Nicoletta; Libri, Valentina; Onori, Annalisa; Passananti, Claudio. Biochemistry and Cell Biology (2004), 82(4), 428-436.

  17. Interactions are mediated by H-bonds in major groove G His (+3) G Arg (-1) Asp (+2) C

  18. Artificial zinc fingers • Based on unravelling the details of zinc finger DNA recognition • Re-design zinc finger proteins for recognition of other DNA sequences • Promising for medical applications: e.g. Gene regulation/targeting, antiviral therapy

  19. Today: 116 different zinc finger families Zinc fingers are amongst the most populous domains in the human genome

  20. The zinc fingers • Can have His2Cys2, Cys3His, or Cys4 ligand set • Can have 1 or 2 zinc sites • Can be classified by the pattern of ligands in the sequence • E.g. Pattern for GATA-type zinc fingers: • C - x - [DN] - C - x(4,5) - [ST] - x(2) - W - [HR] - [RK] - x(3) - [GN] - x(3,4) - C - N - [AS] - C

  21. Some principles in DNA-binding zinc fingers • Isolated fingers, but more than one required (can be 2-9, or even more) • 2 zinc working together to structure protein • Directly bridged by 2 Cys sulfurs Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains, BERT L. VALLEE, JOSEPH E. COLEMAN, AND DAVID S. AULD, PNAS 88, 999-1003 (1991).

  22. HIV nucleocapsid zinc fingers • Small, basic, 2 zinc fingers • Nucleic acid chaperone: protects viral RNA and is important for reverse transcription (RNA as template for DNA synthesis) • Drugs targeting the zinc fingers are in development: e.g. 2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA)) http://jlevinlab.nichd.nih.gov/research.html CCHC ligand set TL South, MF Summers:Protein science (1993) 2, pp. 3-19

  23. Summary • Zinc fingers are important for mediation of bio-molecular interactions • Cys2His2, Cys3His, Cys4 coordination modes • Unfolded without zinc

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