Ischemia Oct 2011

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Ischemia Oct 2011

  1. 1. Understanding the Histidine Button: Structural Evidence for the Isoform- Dependent pH Sensitivity of Troponin I Northern Lights Seminar Series Ian Robertson October 21, 2011 Photograph by JT Austin
  2. 2. Troponin Peter Holmes
  3. 3. Ischemic Heart Disease <ul><li>Cardiac ischemia is the restriction of blood flow to the myocardium </li></ul><ul><li>Most commonly caused by the accumulation of cholesterol-rich plaques in the coronary arteries </li></ul><ul><li>Ischemia can result in heart failure (decreased cardiac output) </li></ul><ul><ul><li>partly caused by intracellular acidosis (≤ 6.5) </li></ul></ul><ul><li>Myocardial calcium sensitivity is reduced by this acidic pH </li></ul><ul><ul><li>The intracellular Ca 2+ transient remains the same is even increased </li></ul></ul><ul><ul><li>It has been shown that the affinity of troponin C for Ca 2+ is reduced at acidic pH </li></ul></ul><ul><li>Skeletal and slow skeletal myofilaments are less sensitive to deactivation by low pH </li></ul>2009 Nucleus Medical Art, Inc.
  4. 4. The role of Troponin I <ul><li>Skinned fibers force under acidic conditions </li></ul><ul><ul><li>A. Muscle containing cTnC and sTnI </li></ul></ul><ul><ul><li>B. Muscle containing sTnC and cTnI </li></ul></ul><ul><ul><li>C. Muscle containing sTnC and sTnI </li></ul></ul><ul><li>All compared to cTnC-cTnI (square symbols) </li></ul>Filled Symbols – pH 7 Open Symbols – pH 6.5 <ul><li>The isoform of TnI dictates the muscle fiber’s pH sensitivity </li></ul>
  5. 5. The role of Troponin I <ul><li>the C-terminal domain of sTnI is the region responsible for the difference in myofilament response to pH </li></ul>
  6. 6. The role of Troponin I <ul><li>the isoform of TnI is responsible for the difference in myofilament response to pH </li></ul><ul><ul><li>consistent with Solaro’s findings </li></ul></ul>
  7. 7. The role of histidine <ul><li>A162 in the switch region of cTnI was replaced by a histidine </li></ul><ul><ul><li>The deleterious effects of acidic pH were reduced </li></ul></ul><ul><li>H130 in the switch region of sTnI was substituted with an alanine </li></ul><ul><ul><li>the pH sensitivity resembled cardiac </li></ul></ul>
  8. 8. The role of histidine
  9. 9. Methods <ul><li>Both (slow skeletal TnI) ssTnI and (fast skeletal TnI) sTnI have been shown to reduce pH sensitivity </li></ul><ul><ul><li>Sequences are almost identical in the switch region of sTnI and ssTnI </li></ul></ul><ul><li>NMR spectroscopy used to probe the molecular mechanism by which H130 of sTnI decreases muscle deactivation at low pH </li></ul><ul><ul><li>Electrostatic interactions assessed by measuring pK a values </li></ul></ul><ul><ul><li>PRE and intermolecular NOEs employed to propose a model </li></ul></ul><ul><ul><li>Measured affinity of sTnI as a function of pH to compile a mechanism </li></ul></ul>
  10. 10. HSQC NMR experiment <ul><li>pH titrations of four states of cNTnC </li></ul><ul><li>Concerns: </li></ul><ul><li>The HN is far from its side chain and chemical shifts may not represent titration of intraresidue side chain </li></ul><ul><li>amide signals disappeared at pH 7.0-7.5 because of increased exchange </li></ul><ul><ul><li>At around pH 4.25, sample began to precipitate and HSQC spectra resembled apo </li></ul></ul>
  11. 11. HCBCGCO NMR experiment Peter Holmes
  12. 12. Assignment of carboxyl groups 1SPY
  13. 13. HCBCGCO NMR experiment <ul><li>E15 and E19 are near H130 in 1YTZ and may interact with H130 when cNTnC is bound to sTnI </li></ul><ul><li>E55 is far from TnI and Ca 2+ so its pK a should be constant in all four states </li></ul>
  14. 14. Glutamate pK a cNTnC-cTnI <ul><li>pK a values </li></ul><ul><li>E55: 4.6 ± 0.08 </li></ul><ul><li>E15: 4.66 ± 0.11 </li></ul><ul><li>E19: 5.10 ± 0.03 </li></ul>HSQC HCBCGCO
  15. 15. Glutamate pK a cNTnC-sTnI <ul><li>pK a values </li></ul><ul><li>E55: 4.6 ± 0.05 </li></ul><ul><li>E15: 4.85 ± 0.06 </li></ul><ul><li>E19: 4.84 ± 0.05; 6.73 ± 0.11 </li></ul>HSQC HCBCGCO
  16. 16. Glutamate pK a summary <ul><li>The lowered pK a of E19 suggests it is involved in an electrostatic interaction </li></ul><ul><li>E19 is proximal to a residue with a pKa of ~6.7 </li></ul><ul><ul><li>E15 and E55 do not seem to be involved in making electrostatic interactions with sTnI (or cTnI) </li></ul></ul>cNTnC E15 E19 E55 cNTnC(Apo) 4.92 ± 0.05 5.09 ± 0.04 4.53 ± 0.09 cNTnC-Ca 2+ 4.85 ± 0.06 5.07 ± 0.03 4.63 ± 0.04 cNTnC-Ca 2+ -cTnI 4.66 ± 0.11 5.10 ± 0.03 4.60 ± 0.08 cNTnC-Ca 2+ -sTnI 4.85 ± 0.06 4.84 ± 0.05 6.73 ± 0.11 4.61 ± 0.05
  17. 17. H130 pK a of free sTnI
  18. 18. H130 pK a of bound sTnI <ul><li>4:1 excess sTnI </li></ul><ul><li>Did not fit to the simple model, so used the modified hill equation </li></ul><ul><li>*Secondary peak: 6.07 ± 0.07 (n=1) </li></ul>
  19. 19. Histidine pK a Summary <ul><li>Low Hill coefficients fit for H2 (n=0.8) and H5 (n=0.66) indicated that H130 is most likely interacting with other ionizable group(s) </li></ul><ul><li>Fit H2 to two pK a : </li></ul><ul><ul><li>pK a 1 = 5.79 ± 0.07 and pK a 2 = 6.66 ± 0.07 </li></ul></ul><ul><li>Fit H5 to two pK a : </li></ul><ul><ul><li>pK a 1 = 5.16 ± 0.07 and pK a 2 = 6.46 ± 0.03 </li></ul></ul><ul><li>Elevated pK a of H130 when in complex with cNTnC is consistent with the findings for E19 </li></ul>
  20. 20. Histidine pK a Summary <ul><li>Do the two His peaks represent free and bound or two bound conformations? </li></ul><ul><ul><li>Impurity ruled out by Mass Spectrometry </li></ul></ul><ul><li>McKay et al. showed that sTnI 96-148 binds to sNTnC in several conformations </li></ul>
  21. 21. Paramagnetic relaxation enhancement (PRE) <ul><li>Gd 3+ has seven unpaired f-electrons in each of its seven f-orbitals </li></ul><ul><li>The unpaired electrons of Gd 3+ strongly enhance the relaxation rate of NMR signals in a distance dependent manner </li></ul><ul><li>Assume that Gd 3+ binds to cNTnC in a similar manner as Ca 2+ </li></ul>
  22. 22. PRE <ul><li>Titrated Gd 3+ into cNTnC-Ca 2+ -sTnI </li></ul><ul><li>Measured line widths of H2 and H5 as a function of [Gd 3+ ] </li></ul>
  23. 23. PRE conclusions <ul><li>H2* and H2 represent two bound conformations of sTnI </li></ul><ul><ul><li>H2 may be slightly closer than H2* to the Gd 3+ metal ion </li></ul></ul><ul><li>Distance measurements place the aromatic protons ~25-28 Angstroms away from Gd 3+ </li></ul><ul><ul><li>Consistent with H130 forming an interaction with E19 as in 1YTZ </li></ul></ul><ul><ul><li>Alanine β-protons are ~32-34 Å away in the NMR structure of cNTnC-cTnI and 31 Å away in the X-ray structure of core cardiac troponin complex </li></ul></ul>1MXL 1YTZ
  24. 24. NOEs used in docking
  25. 25. Docked Structure <ul><li>Kept backbone atoms of cNTnC rigid and residues 118-126 (helical region) rigid; allowed side chains to be flexible </li></ul>
  26. 26. Docked Structure <ul><li>The C-terminus of sTnI is more similar to sNTnC-sTnI </li></ul>1MXL 1YTZ
  27. 27. pH dependence of sTnI binding <ul><li>Since the pK a of H130 is perturbed when bound to cNTnC, it follows that the protonation state of H130 should influence the binding equilibrium </li></ul><ul><ul><li>Free H130: K a = 7.6 x 10 -7 M (pK a = 6.12) </li></ul></ul><ul><ul><li>Bound H130: K a *= 1.9 x 10 -7 M (pKa = 6.73) </li></ul></ul><ul><ul><li>K D = 3.90 x 10 -4 M (pH 7.5) </li></ul></ul><ul><ul><li>K D = 1.00 x 10 -4 M (pH 6.1) </li></ul></ul><ul><li>K a /K a * (4.1) ~ K D /K D-H (3.9) </li></ul>
  28. 28. Conclusions <ul><li>Second ionization monitored by E19 suggests its carboxylate is in close proximity to another ionizable group with a pK a of ~6.7 </li></ul><ul><ul><li>Elevated pK a of H130 and lowered pK a of E19 in cNTnC-Ca 2+ -sTnI complex suggest they are involved in an electrostatic interaction </li></ul></ul><ul><li>PRE studies indicate that the minor peak in one-dimensional spectrum represents a second bound conformation of H130 </li></ul><ul><li>PRE and NOE driven docking orients sTnI on cNTnC in a similar conformation as when it is bound to sNTnC </li></ul><ul><li>Acidic pH increases the affinity of sTnI for cNTnC </li></ul><ul><ul><li>consistent with the decreased acid dissociation constant of H130 in the presence of cNTnC </li></ul></ul>
  29. 29. Conclusions <ul><li>Acidic pH increases the affinity of sTnI for cNTnC </li></ul><ul><li>At first glance is not consistent with the functional data </li></ul><ul><ul><li>cTnC-cTnI (squares) </li></ul></ul><ul><ul><li>cTnC-sTnI (circles) </li></ul></ul><ul><li>We are working in Ca 2+ excess </li></ul><ul><ul><li>Altered calcium affinity is not being probed </li></ul></ul><ul><li>Protonation of H130 partly compensates for the reduced Ca 2+ affinity at low pH </li></ul>Filled Symbols – pH 7 Open Symbols – pH 6.5 Li, et al. (2001) JMCC
  30. 30. Future directions <ul><li>Peter Holmes is solving the NMR structure of the complex </li></ul><ul><li>PRE experiments with the cardiac complex to measure the PRE rate of A162 </li></ul><ul><li>Sandra Pineda Sanabria is working on the cNTnC-cTnI(A162H) complex </li></ul><ul><ul><li>pK a measurements suggest a similar molecular mechanism </li></ul></ul><ul><ul><li>In contrast to sTnI, E15 as well as E19 seem to interact with H162 </li></ul></ul><ul><ul><li>NMR structure is underway </li></ul></ul>
  31. 31. Acknowledgments Dr. Brian Sykes Dr. Monica Li Dr. Olga Baryshnikova Peter Holmes Sandra Pineda Sanabria Dave Corson Robert Boyko And the rest of the Sykes lab

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