Metal Ion Selectivity and Affinity of the LIN-12/Notch-Repeat

1
Metal Ion Selectivity and Affinity of the
LIN-12/Notch-Repeat Christina Hao, Advisor Didem
Vardar-Ulu Wellesley College, Chemistry Department

• Introduction
• Notch receptors are large transmembrane
  glycoproteins that regulate cell growth,
  differentiation and death in multicellular
  organisms via a highly conserved signaling
  pathway (Figure A).
• Dysregulation of notch signaling pathway in all
  four identified notch homologs (Notch1 Notch4)
  has been implicated in numerous disease
  phenotypes.
• 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).
• Each LNR module exhibit highly conserved
  architecture consisting of three characteristic
  disulfide bonds and a group of aspartate/asparagin
  e residue that coordate a Ca2 ion, which are
  essential for the correct folding of the module.
  (Figure C)

Results

Representative ITC data on the calorimetric
titrations of hN1LNRA with Ca2,Zn2 ,Tb3

• Conclusions
• Ca2 exhibits exothermic binding to wildtype
  Human Notch1 LNRA (hN1LNRA) with a dissociation
  constant of 22.05 /- 3.27 µM and a stoichiometry
  of 11 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.
• 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.
• 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

Representative ITC data on the calorimetric
titrations of hN1LNRA_mt with Ca2,Zn2 ,Tb3

• Objectives
• 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).
• 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).

Sequence Alignment of LNRAs from human Notch
homologs
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
Summary of thermodynamic parameters associated
with the binding of Ca2 to hN1LNRA and
hN1LNRA_mt
N Kd (µM) ?H(kcal/mol) ?S (kcal/mol)
hN1LNRA 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

• 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.
• 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 (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.
• 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.
• Final protein concentration of the sample was
  determined based on UV absorbance of the sample
  at 280 nm using a corrected extinction
  coefficient.
• 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)2H2O, and TbCl3.
• 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 20C.
• Control experiments of metal solutions titrated
  into protein free buffer solutions were performed
  to correct for the heat of dilution.

• Future Directions
• Repeat the preliminary results on hNLNRA_mt to
  determine reproducible thermodynamic profile for
  Ca2 binding.
• Determine a reliable model to quantify Tb3
  binding to hN1LNRA.
• Test additional metals on both wild-type and
  mutant hN1LNRA.
• Design other mutants that alter binding
  specificity of hN1LNRA.
• 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.

Representative ITC data on the calorimetric
titrations of hN1LNRA pre-saturated with Zn2 or
Tb3 with Ca2

• References
• 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, 295300.2.
• 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, 70617067.

Summary of thermodynamic parameters associated
with the binding of Ca2 to Zn2 and Tb3
presaturated hN1LNRA
N Kd (µM) ?H(kcal/mol) ?S (kcal/mol)
hN1LNRA presat with Zn2 0.66?0.03 73.53?2.24 -12.82?0.70 -24.8
hN1LNRA presat with Tb3 Too weak to quantify Too weak to quantify Too weak to quantify Too weak to quantify