Ischemia Oct 2011
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Ischemia Oct 2011



Presentation about work to be published in the journal of biological chemistry

Presentation about work to be published in the journal of biological chemistry



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Ischemia Oct 2011 Ischemia Oct 2011 Presentation Transcript

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