Understanding the Histidine Button: Structural Evidence for the Isoform-Dependent pH Sensitivity of Troponin I

Northern Lights Seminar SeriesIan RobertsonOctober 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 Ca2+ transient remains the

same is even increased It has been shown that the affinity of

troponin C for Ca2+ 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 histidineA. A162 in the switch region of

cTnI was replaced by a histidine

The deleterious effects of acidic pH were reduced

B. 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 pKa 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 experimentpH 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 chainamide 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 Ca2+ so its pKa should be constant in all four states

Glutamate pKa cNTnC-cTnI

)1(1

101 pHpKaHAobs

pKa valuesE55: 4.6 ± 0.08 E15: 4.66 ± 0.11E19: 5.10 ± 0.03

HSQC HCBCGCO

Glutamate pKa cNTnC-sTnI

pKa valuesE55: 4.6 ± 0.05 E15: 4.85 ± 0.06E19: 4.84 ± 0.05; 6.73 ± 0.11

)2(2

)1(1

101101 pHpKapHpKaHAobs

HSQC HCBCGCO

Glutamate pKa summary

The lowered pKa 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 E55cNTnC(Apo) 4.92 ±

0.05 5.09 ± 0.04 4.53 ± 0.09

cNTnC-Ca2+ 4.85 ± 0.06

5.07 ± 0.03 4.63 ± 0.04

cNTnC-Ca2+-cTnI 4.66 ± 0.11

5.10 ± 0.03 4.60 ± 0.08

cNTnC-Ca2+-sTnI 4.85 ± 0.06

4.84 ± 0.056.73 ± 0.11

4.61 ± 0.05

H130 pKa of free sTnI

)1(1

101 pHpKaHAobs

H130 pKa 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 pKa 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 pKa: pKa

1= 5.79 ± 0.07 and pKa2 = 6.66 ± 0.07

Fit H5 to two pKa: pKa

1 = 5.16 ± 0.07 and pKa2 = 6.46 ± 0.03

Elevated pKa of H130 when in complex with cNTnC is consistent with the findings for E19

Histidine pKa 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 sTnI96-148 binds to sNTnC in several conformations

Paramagnetic relaxation enhancement (PRE)

Gd3+ has seven unpaired f-electrons in each of its seven f-orbitals

The unpaired electrons of Gd3+ strongly enhance the relaxation rate of NMR signals in a distance dependent manner

Assume that Gd3+ binds to cNTnC in a similar manner as Ca2+ 6

1r

PRE

PRE Titrated Gd3+ into

cNTnC-Ca2+-sTnI Measured line

widths of H2 and H5 as a function of [Gd3+]

2R

PRE conclusionsH2* and H2 represent two bound

conformations of sTnI H2 may be slightly closer than H2*

to the Gd3+ metal ionDistance measurements place the

aromatic protons ~25-28 Angstroms away from Gd3+

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 pKa of H130 is perturbed when bound to cNTnC, it follows that the protonation state of H130 should influence the binding equilibrium Free H130: Ka = 7.6 x 10-7 M

(pKa = 6.12) Bound H130: Ka*= 1.9 x 10-7

M (pKa = 6.73) KD = 3.90 x 10-4 M (pH 7.5) KD = 1.00 x 10-4 M (pH 6.1)

Ka/Ka* (4.1) ~ KD/KD-H (3.9)

Conclusions Second ionization monitored by E19 suggests its

carboxylate is in close proximity to another ionizable group with a pKa of ~6.7 Elevated pKa of H130 and lowered pKa of E19 in cNTnC-Ca2+-

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 Ca2+ excess Altered calcium affinity is not

being probed

Protonation of H130 partly compensates for the reduced Ca2+ affinity at low pH

Filled Symbols – pH 7 Open Symbols – pH 6.5

Li, et al. (2001) JMCC

Future directionsPeter Holmes is solving the NMR structure of the

complexPRE experiments with the cardiac complex to

measure the PRE rate of A162Sandra Pineda Sanabria is working on the cNTnC-

cTnI(A162H) complexpKa measurements suggest a similar molecular

mechanism In contrast to sTnI, E15 as well as E19 seem to

interact with H162NMR structure is underway

AcknowledgmentsDr. Brian Sykes

Dr. Monica LiDr. Olga BaryshnikovaPeter HolmesSandra Pineda SanabriaDave CorsonRobert Boyko

And the rest of the Sykes lab