Transcript 投影片 1 - Wellesley College

Metal Ion Selectivity and Affinity of the LIN-12/Notch-Repeat
Christina Hao, Advisor: Didem Vardar-Ulu
Wellesley College, Chemistry Department
Introduction
Results
• Notch receptors are large transmembrane glycoproteins that regulate cell growth, differentiation and death in
multicellular organisms via a highly conserved signaling pathway (Figure A).
Representative ITC data on the calorimetric titrations of hN1LNRA
with Ca2+,Zn2+ ,Tb3+
• Dysregulation of notch signaling pathway in all four identified notch homologs (Notch1 – Notch4) has been implicated
in numerous disease phenotypes.
Tb3
Zn2+
Ca2+
C-term
+
Figure B
C-term
D30
D30
Ca2+
•Each LNR module exhibit highly conserved architecture consisting of three characteristic disulfide bonds and a group
of aspartate/asparagine residue that coordate a Ca2+ ion, which are essential for the correct folding of the module.
(Figure C)
Crystal Structure of LNR and
HD Domain of human Notch21
D19
S19
•Three conserved Lin12/Notch Repeat (LNRA, LNRB, and LNRC) modules of about 35 residues each are located in
tandem in the extracellular region of the notch receptors. They decorate the heterodimerization (HD) domain of the
receptor and conceal the activating cleavage site in the absence of a ligand. Therefore, they are responsible for
maintaining the receptor in a resting conformation prior to ligand-induced activation. (Figure B).
Domain Organization of the
Notch Receptors and the
Notch Signaling Pathway
N-term
N-term
Ca2+
D33
D33
N15
N15
NMR Structure of hN1LNRA2
Ca2+ coordination in hN1LNRA
based on the NMR structure2
Figure C
Ca2+ coordination in hN1LNRA_mt
modeled on the hN1LNRA structure
N15
C4
C9
C27
N-term
Figure A
C22
D30
Representative ITC data on the calorimetric titrations of hN1LNRA_mt
with Ca2+,Zn2+ ,Tb3+
Ca2+
C34
D33
S19
C18
0
C-term
Objectives
125
250
Time (min)
Time (min)
Time (min)
375
500
0
625
40 80 120 160 200 240 280 320 360 400
0
Zn2+
0.0
Ca2+
Conclusions
0
40
80
120
160
200
240
1.2
Tb3
1.0
+
-0.5
280
-1.0
-1.5
µcal/sec
µcal/sec
• To obtain a molecular understanding for the metal binding affinity and specificity of the LNRs through the
determination of thermodynamic parameters associated with the binding of different metals to human Notch1 LNRA
(hN1LNRA). For this aim we performed calorimetric titrations of Ca2+, Zn2+, Tb3+ into hN1LNRA with using isothermal
titration calorimetry (ITC).
甥al/sec
0.8
-1
0.6
0.4
0.2
-2.0
0.0
2
-2
2
• Ca2+ exhibits exothermic binding to wildtype Human Notch1 LNRA (hN1LNRA) with a
dissociation constant of 22.05 +/- 3.27 µM and a stoichiometry of 1:1 at pH 7.0.
• No quantifiable binding is observed for hN1LNRA with zinc at pH 7.0. However, preliminary
displacement experiments indicate that Ca2+ binding affinity of hN1LNRA is slightly decreased
after it has been pre-saturated with Zn2+. This finding may imply some very weak binding of
Zn2+ to the Ca2+ site or an indirect effect of nonspecific binding of Zn2+ on the native
conformation of hN1LNRA altering the molecular details of the Ca2+ binding site.
1
hN1LNRA
:
hN1LNRA_mt:
hN2LNRA
:
hN3LNRA
:
hN4LNRA
:
19
35
EEAC--ELPECQ-EDAGNKVCSLQCNNHACGWDGGDC
EEAC--ELPECQ-EDAGNKVCDLQCNNHACGWDGGDC
PATC--LSQYCA-DKARDGVCDEACNSHACQWDGGDC
P-RC--PRAACQ-AKRGDQRCDRECNSPGCGWDGGDC
P-----GAKGCE-GRSGDGACDAGCSGPGGNWDGGDC
Green: wildtype human Notch1 LNRA
Blue: mutant human Notch 1 LNR A
Brown: LNRA from other human Notch homologs
Orange: Cysteines (disulfide bonding pattern
indicated on the top)
Highlighted in yellow: Ca 2+-coordinating residues
Material and Methods
Protein Expression and Purification:
•Wildtype hN1LNRA was expressed in E.coli. as a fusion protein with a modified form of the trpLE sequence in which
the methionine and cysteine residues have been replaced by leucine and alanine ,respectively using the pMML vector
(kind gift of S. Blacklow, BWH). The plasmid for hN1LNRA_mt was obtained from the pMML vector using the
QuikChange Site-Directed Mutagenesis protocol (Stratagene) The expressed fusion proteins were purified from
inclusion bodies and cleaved by cyanogen bromide to obtain hN1LNRA or hN1LNRA_mt.
-4
-6
-8
-2
-4
-6
-8
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Molar Ratio
0
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Molar Ratio
N
Molar Ratio
H(kcal/mol)
Kd (µM)
0.9600.005
22.053.27
-9.140.25
-9.870.73
hN1LNRA_mt
0.0940.039
9.374.99
-36.446.83
-100.823.96
Representative ITC data on the calorimetric titrations of hN1LNRA presaturated with Zn2+ or Tb3+ with Ca2+
Time (min)
0
40
80
120
160
Time (min)
200
240
280
0
0.2
0.1
0.0
0.0
-0.2
40
80
120
160
µcal/sec
-0.8
-1.0
-0.4
-0.5
-1.4
-0.6
• Conduct additional ITC experiments to test the effect of pH and temperature on the binding
affinity of the different metals to both the wild-type and mutant hN1LNRA.
-0.7
-0.6
kcal/mole of injectant
kcal/mole of injectant
• Design other mutants that alter binding specificity of hN1LNRA.
-0.3
-1.2
-2
Data: wtlnCAintoZ_NDH
Model: OneSites
Chi^2/DoF = 1071
N
0.659 ±0.0316
K
1.36E4 ±446
H
-1.282E4
±695.1
S
-24.8
-4
-6
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
-0.8
References
-1.0
-1.2
-1.4
1) Gordon, W. R.;* Vardar-Ulu, D.;* Histen, G.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S.
C. “Structural basis for autoinhibition of Notch” Nat Struct Mol Biol. 2007, 14, 295–300.2.
-1.6
-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5
Molar Ratio
Molar Ratio
2)
Summary of thermodynamic parameters associated with the binding
of Ca2+ to Zn2+ and Tb3+ presaturated hN1LNRA
N
•Experiments were repeated to ensure reproducibility.
hN1LNRA presat
with Zn2+
•The dissociation constant, the binding stoichiometry, the enthalpy of binding, and the calculated entropy were
determined based on a one-site fitting model using the Origin software, version 7.0. after correcting for heat of dilution.
hN1LNRA presat
with Tb3+
• Determine a reliable model to quantify Tb3+ binding to hN1LNRA.
• Test additional metals on both wild-type and mutant hN1LNRA.
-0.2
-0.6
0
•Small aliquot of buffer used to dialyze the protein sample was also chelexed and used to prepare stock metal
solution of 1 or 2mM CaCl2, Zn(CH3COO)2*H2O, and TbCl3.
• Repeat the preliminary results on hNLNRA_mt to determine reproducible thermodynamic
profile for Ca2+ binding.
-0.1
-1.6
• Final protein concentration of the sample was determined based on UV absorbance of the sample at 280 nm using
a corrected extinction coefficient.
Future Directions
200
-0.4
• To ensure protein samples were completely metal-free, prewashed chelex beads (Sigma chelex 100, were
incubated with the sample after dialysis for to remove residual metals.
• Preliminary data suggest mutant human Notch 1 LNR_A where a serine in position 19 is
replaced by an aspartate (nN1LNRA_mt) binds to calcium approximated 2.5 fold more tightly
than the wildtype counterpart. However, the stoichiometry of binding is drastically altered.
Zn2+ and Tb3+ binding to hN1LNRA_mt follows the same trends as the wild-type protein
S (kcal/mol)
hN1LNRA
Isothermal titration calorimetry (ITC) Experiments:
• Lyophilized protein was solubilized in water at concentration of approximately 0.1mM and then extensively dialyzed
against, 35 mM HEPES pH 7, 100 mM NaCl buffer.
•Control experiments of metal solutions titrated into protein free buffer solutions were performed to correct for the heat
of dilution.
• Although Tb3+ clearly exhibits endothermic binding to (hN1LNRA), preliminary results indicate
that a single binding site model does not fit the experimental data. However, displacement
experiments indicate that Ca2+ binding affinity of hN1LNRA is decreased dramatically after it
has been pre-saturated with Tb3+. Taken together, these data suggest strongly that Tb3+ most
likely binds to the Ca2+ coordination site as well as to other specific or non-specific sites on the
protein.
Summary of thermodynamic parameters associated with the binding
of Ca2+ to hN1LNRA and hN1LNRA_mt
•The protein was refolded in vitro through successive dialysis against a redox buffer (50 mM Tris pH 8.0, 150 mM
NaCl, 10 mM CaCl2, 2 mM cysteine, 0.5 mM cystine), purified via reversed-phase HPLC, and lyophilized. The
identity of the constructs were confirmed using MALDI-TOF mass spectrometry.
•Isothermal titration calorimetry experiments (ITC), were carried out using a high-precision VP-ITC titration calorimetry
instrument (Microcal Inc., Northampton, MA) where the metal solution was titrated in 5µL increments into the protein
solution at 20°C.
kcal/mole of injectant
-2
0
甥al/sec
Sequence Alignment of LNRAs from human Notch homologs:
kcal/mole of injectant
• To test if the binding affinity and specificity of these repeats can be altered through a single amino acid replacement
in the Ca 2+ binding pocket of the wild-type hN1LNRA. For this aim we designed a mutant form of hN1LNRA where
serine in position 19 is replaced by an aspartate that is part of the Ca2+ coordination site in most other LNRs (Figure D).
kcal/mole of injectant
0
0.660.03
Kd (µM)
73.532.24
H(kcal/mol)
-12.820.70
Too weak to quantify
S (kcal/mol)
-24.8
Vardar, D.; North, C. L.; Sanchez-Irizarry, C.; Aster, J. C.; Blacklow, S. C. “NMR Structure
of a Prototype LNR Module from Human Notch1” Biochemistry 2003, 42, 7061–7067.