CalbindinD9k metal binding properties

1. CalbindinD9k metal binding properties Yanyi Chen

2. Introduction • Ca2+ properties & EF hand Ca2+ binding proteins • Properties of Calbindin D9k • Calcium-binding cooperativity of Calbindin D9k • Calbindin D9k as a model protein

3. Coordination properties of Ca2+ • Ca2+ is a hard ion; it forms complexes with many weak M-Ligand contacts and large coordination numbers (> 6); • typical ligands are O-containing (e.g. water, neutral carbonyl, anionic carboxylates); it has no affinity toward N-containing ligands; • exchange of ligands from coordination sphere of Ca2+ is quick(for water complexes ligand exchange time:10-10-10-7 s).

4. EF hand Ca2+ binding proteins Affinity of these proteins to Ca2+ is in the range 108-106 M-1. • Calmodulin- interaction with kinases • Troponin C- activation of actinmyosin interaction in muscles. • Calbindin D9k- Ca2+ transport in intestine cells • Parvalbumin- buffering Ca2+ muscles http://www.rcsb.org/pdb/Welcome.do

5. Ca2+ uptake in small intestine • TRPV6 is found in intestine and kidney; • PMCA1 – Ca-pump fueled by ATP hydrolysis • Calbindin D9k concentration in cell correlates with rate of Ca2+ transport.

6. Vitamin D Active form of vitamin D is called calcitriol. It is a hormone, which activates transcription of TRPV6 and Calbindin D9k.

7. Basic Chemical and Physical Information of Calbindin D9k • Number of amino acids: 78 • Molecular weight: around 9000 Kd • pI: calculated pI = 4.7 • Number of functional calcium binding sites: 2

8. Biological roles of Calbindin D9k • fetal calcium uptake: Movement of calcium across the placenta increase dramatically in the last trimester, when fetal skeletal mineralization is occurring • uterine contractions: It may help regulate the strength duration, and frequency of contractions by altering the free calcium levels in the cytoplasm • calcification of bone : Calbindin D9k is found in the correct locations to support activity in calcification of bone • calcification of teeth : Calbindin D9k is found in the correct locations to support activity in calcification of teeth • calcium transport and uptake in mammalian intestine: Active transport of calcium across the intestine is directly proportional to levels of calbindin D9k

9. Calcium-binding Cooperativity Macroscopic calcium binding constants (Bos taurus ): low salt: K1 = 1.6 x 108; K2 = 4 x 108 medium salt: K1 = 5 x 106; K2 = 1.6 x 107 high salt: K1 = 2 x 106; K2 = 3.2 x 106

10. Positive Cooperativity • An enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site. • A free energy coupling between binding sites • Occurs in systems with sites having identical ligand affinities or have different affinities • Wild-type Calbindin D9kbinds two calcium ions with similar affinities • N56A, with the substitution Asn 56 to Ala, binds calcium in a sequential manner

11. Initial 1H NMR characterization of the calcium binding properties of N56A • stepwise additions of Ca2+ 0.10 equivalents of calcium. • At additions up to 1.0 equivalent, new resonances appear and increase in intensity • At additions up to 1.0 equivalent, new resonances appear and increase in intensity • the largest effect at Ca2+ 1.5 equivalents of added calcium

12. A: Titration in the absence of Ca2+ B: Titration in the presence of Ca2+ La3+ binding in the absence and presence of Ca2+

13. Titrated with La(ClO4)3 in steps of Ca2+ 0.15 equivalents Between 0 and 1 equivalent, a slow exchange process was observed A maximum linewidth, W, of 30 Hz at Ca2+ 0.5 La3+ equivalents La3+ has the same site preference as observed for wild-type calbindin D9k Aslow exchange process was observed in the La3+ titration at additions between zero and one equivalent of lanthanum, with no significant line-broadening Nofurther La3+ binding was observed at additions above one equivalent. A: Titration in the absence of Ca2+ B: Titration in the presence of Ca2+

14. Calculation Formulation koff= πW - l/T2. • When l/T2≤10Hz, which corresponds to the linewidth of the resonances in the absence of exchange, W from scalar coupling as large as 10 Hz, a lower limit of koff ≥ 54 s-1 is obtained • When a calcium ion is present in site I, the La3+ off-rate in site II, is Ca2+6 s-1 or lower

15. 'H NMR parameters from two Cd2+titrations of the N56Amutant in the absence (o) and presence (•) of Ca2+ in site I

16. Calculation Formula • The Cd2+off-rate is 8,200 ±1,200 s-1 for N56A when Ca2+ is absence • The Cd2+off-rate is 675 ±40 s-1 for N56A with Ca2+bound in site I

17. Conclusions regarding cooperativity • The N56A mutant of calbindin D9k binds metal ions with positive cooperativity • The concepts of sequential and cooperative binding are not mutually exclusive • The cooperative mechanism involves a reduction in the metal-ion dissociation rates.

18. Hydrophobic Core • Substantial effects on Ca2+ affinity and dissociation rates in calbindin D9k due to large volume changes in the central parts of the core. • The hydrophobic core residues promote Ca2+ binding by contributing to preformation of the sites in the apo state.

19. Isolated Ca2+-binding EF-handpeptides have a tendency to dimerize

20. Dimerization Model The 5-state model including all possible dimerization and Ca2+-binding events that can take place with an isolated EF-hand fragment.

21. References • Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding SARA LINSE AND WALTER J. CHAZIN • Site−site communication in the EF-hand Ca2+ -binding protein calbindin D9k Lena Mäler, John Blankenship, Mark Rance & Walter J. Chazin • Hydrophobic Core Substitutions in Calbindin D9k: Effects on Ca2+ Binding and Dissociation Birthe B. Kragelund Malin Jonsson SARA LINSE • Coupling of Ligand Binding and Dimerization of Helix-Loop-Helix Peptides: Spectroscopic and Sedimentation Analyses of Calbindin D9k EF-HandsKarin Julenius, James Robblee, Eva Thulin, Bryan E. Finn, Robert Fairman, and Sara Linse • Ca status and osteoporosis lectureAndriy Mokhir • The CABP Data Library

22. Thank you THE END