Transcript 投影片 1 - Wellesley College

Characterization of the
2+
Ca
Binding Affinity and Coordination Site of the LIN-12/Notch-Repeat
Christina Hao, Didem Vardar-Ulu
Wellesley College Chemistry Department
Wellesley, Massachusetts
Introduction
Results
• Notch receptors are transmembrane glycoproteins that regulate cell fate in multicellular organisms via a
highly conserved signaling pathway (Figure A).
Ca2+ dependency during folding of hN1 LNRA, glucose transferase
and hN4LNRA
Representations of overall structures and calcium binding sites for (A) hN1LNRA (B)
Glucose Transferase LNRA (C) hN4LNRA
(A)NMR Structure. (B) and (C) homology Modeling
• Deregulation of notch signaling pathway in all four identified notch homologues (Notch1 – Notch4) has been
implicated in numerous disease phenotypes.
A. hN1 LNRA
• Three conserved Lin12/Notch Repeat (LNRA, LNRB, and LNRC) modules are located in tandem in the
extracellular region of the notch receptors. They maintain the receptor in a resting conformation prior to ligandinduced activation. (Figure B).
B. Glucose Transferase
LNRA
C. hN4 LNRA
• Each LNR module in the Notch receptor consists of three characteristic disulfide bonds and a Ca2+ ion,
essential for structural integrity (Figure C).
Domain Organization of the
Notch Receptors and the
Notch Signaling Pathway
Crystal Structure of LNR and
HD Domain of human
Notch21
Figure B
NMR Structure of hN1LNRA2
Figure
C
C
4
Key:
N15
C9
C27
Figure A
Nterm
C22
D30
• Calcium binding sites (7 angstroms from the Ca2+): red and green ribbons
Red sticks: Aspartates - Green sticks: other residues besides aspartates - Blue sphere: calcium ion
Ca2+
C34
• Non-binding sites: silver ribbons
D33
S19
C18
C-term
Representative ITC data on the calorimetric titrations of hN1LNRA
with Ca2+,Zn2+ ,Tb3+
Distances and distribution of coordinating residues from Ca2+
Objectives
• Quantify calcium binding affinity and specificity of LNR homologues across different proteins using ITC
• Determine calcium dependency of different LNR homologues for autonomous folding
• Understand the molecular basis of calcium binding in LNR using computer modeling
Tb
Zn2
3+
+
Ca2+
Key: Red residues: residues that coordinate calcium with both side chain and backbone oxygen moiety
Sequence Alignment of LNR homologues investigated in this study:
Distances ranges highlighted yellow: aspartate is present in this distance range
Conclusions
• HN1 LNRA exclusively binds to Ca2+ in an exothermic reaction with a dissociation constant of 22.05 +/3.27 µM and a stoichiometry of 1:1 at pH 7.0.
Representative ITC data on the calorimetric titrations of glucose
transferase and hN4LNRA with Ca2+
Material and Methods
Glucose transferase
LNRA
hN1 LNR A
hN4 LNR A
Protein Acquisition:
• Human N1LNRA recombinantly expressed in Escherichia coli. Human Notch 4 LNRA and glucose
transferase LNRA were synthesized by EZ Biolabs.
• All proteins were folded and purified as follows:
• Folding: 6-8 hours dialysis against refolding buffer: 2.5mM cysteine, 0.5mM cystine,
100mM NaCl, 200mM sucrose, 10mM CaCl2 and 20mM Tris pH 8.
• Purification: Elution through reverse phase high pressure liquid chromatography (RPHPLC) using 0.1% formic acid in acetonitrile based buffer systems. Peaks corresponding
to the folded species were collected and lyophilized.
Folding Experiments:
• Denatured proteins were refolded in redox solution containing 5:1 cysteine/cystine ratio, 100mM NaCl and
20mM Tris at pH 8 and with/without 10mM CaCl2. Proteins were folded under partial nitrogen atmosphere for
six hours and promptly analyzed on RP-HPLC.
Isothermal titration calorimetry (ITC) Experiments:
• Lyophilized protein of appropriate concentration was demetalized with sigma chelex beads and suspended
in 35mM Hepes pH7, 100mM NaCl buffer .
• Stock metal solution of 0.2 – 1mM CaCl2 was used
• 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.
Computer Modeling Software used:
• Clustal W: sequence alignment
• Modeller: homology modeling
• Pymol: visualization
• Glucose transferase LNRA display strong binding to calcium in a non-stoichiometric manner
• HN4 LNRA does not bind to calcium
• Calcium is necessary for the folding of HN1 LNRA and glucose transferase LNRA but not for HN4
LNRA
• Homology modeling suggests differences in distribution of aspartic acids lead to distinct calcium binding
behaviors of the LNR repeats.
Future Directions
• Determine precise roles of disulphide bonds and aspartic acids in calcium binding affinity
mutational studies.
through
• Correlate calcium binding affinity and specificity with structural stability to gain insight into the biological
significance of calcium binding by the LNRs in vivo.
• Design of calcium binding peptides through de novo experiments based on understanding of the LNRs
Summary of thermodynamic parameters associated with the binding
of Ca2+ to Zn2+ and Tb3+ presaturated hN1LNRA
Constructs
hN1LNRA
Metal
Calcium
N
0.9600.005
Kd (µM)
22.053.27
Zinc
Calcium
hN4LNRA
Calcium
-9.140.25
No binding
Terbium
GlucTran
H (kcal/mol)
Undefined mode of binding
0.07
-4.64 E4  1.36E4
9
No binding
References
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.
2) 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.
3) N. Eswar, M.A. Marti-Renom, b. Webb, m.S. Madhusudhan, D. Eramian, M. Shen, U. Pieper, A. Sali, Comparative Protein
Structure Modeling with MODELLER. Current Protocols in Bioinformatics, John Wilery & Sons, Inc., Supplement 15, 5.6. 1-5.6.30,
2000
4) Cheng G, Baker D and Samudrala R. A Novel Small Molecule Crystal Structure Derived Potential Function To Predict Protein
Metal Ion Binding Site, Affinity and Specificity From Structure. xxxx.YYYY,aa-bb,2007
http://protinfo.compbio.washington.edu/soak/
hN1 LNRA
GL
N4