Ischemia Oct 2011

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

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

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 HSQC HCBCGCO

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 HSQC HCBCGCO

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 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

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

Ischemia Oct 2011

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Ischemia Oct 2011