Transcript Slide 1

Characterization of protein folding determinants for LIN-12/Notch-Repeats (LNRs)
using Human Notch1 LNR-B as a model system
Sharline Madera
Advisor: Dr. Didem Vardar-Ulu
Wellesley College
Human Notch1 is a member of a conserved family of heterodimeric type 1 transmembrane receptors that
control differentiation in multicellular animals. Notch proteins exhibit a highly conserved modular
architecture which includes three contiguous LIN-12/Notch-Repeats (LNRs), LNRA, LNRB and LNRC, in
its extracellular domain that maintain the receptor in its resting conformation in the absence of ligand.
These conserved LNRs are separated by two linkers, linker_AB and linker_BC, 10 amino acids and 5
amino acids long, respectively (Figure 1). The objective of this study is to determine the minimum
requirements for the folding of an LNR module using LNRB as a model system. For this work, we studied
the effects of metal ion specificity, linker residue and redox potential dependence on LNRB folding and
compared it to the prototype human Notch1 LNRA. Metal ion specificity was determined by exposing
unfolded protein to metal ions present in refolding buffer. Redox potential sensitivity was examined by
monitoring LNRB folding under varying reducing environments. The effect of linker_AB and linker_BC
residues on LNRB folding were studied using LNRB constructs varying in length in the two linker regions.
LNRB constructs lacking linker_BC residues displayed no major changes in protein folding. However, key
residues in linker_AB were identified and shown to directly affect proper folding. These findings
demonstrate the importance of additional N-terminal residues to the initial cysteine that define an
autonomously folding LNRB module, introducing a crucial parameter alongside redox potential sensitivity
and metal ion specificity. This work represents the initial step toward defining the minimum requirements
for a correctly folding LNR module using LNRB from human Notch1 as a model system.
C
SLNFNDPWKN
LNRA
LNR Domain
A B C
Table 1. Construct Sequences
15:1
HD Domain
2:1 30:1 5:1
5 Constructs
1:5
LNRB_orig:
LNRB_short:
10:1
3:6
LNFNDPW KNCTQ SLQCWKYFSDGH CDSQ CNSAG CLFDGFDCQRAEG Q
KNCTQ SLQCWKYFSDG HCDSQ CNSAG CLFDG FDCQ
LNRB_delAB:
LNRB_delBC:
2:4
KNCTQ SLQCWKYFSDG HCDSQ CNSAG CLFDG FDCQRAEG Q
LNRB_int:
DPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQ
Table 3. Intrinsic Redox Potentials
Red Residues: Coordinate Ca2+ ions
Orange Residues: Disulfide bonded cysteines
C
C
LNRB
QRAEGQ
C
C
Figure 6. Chromatograms of LNRB_orig after Dialysis 3 folded
under varying redox conditions: 2:1-grey, 5:1-orange, 10:1black, 15:1-red, 30:1-purple (Table 3). Panel A: Note the
abundance of misfolded peaks surrounding the major peak
representing the correctly folded protein. Panel B: Close up of
5:1 redox ratio. This condition minimizes the misfolded species.
LNRC
Figure 1. Human Notch 1 LNRs and linkers.
Notch Proteins are large Ca2+ binding, transmembrane receptors that control differentiation in multicellular
animals. In mammals, there are four Notch homologs: Notch1-4. These proteins function via a highly
conserved mechanism referred to as the Notch signaling pathway, which is important for cell-cell
communication, involving gene regulation mechanisms that control multiple cell differentiation processes
during embryonic and adult life. Deregulation of normal Notch activation has been noted in certain human
leukemias, (1) Alagille (2, 3) and CADASIL (4) syndromes, indicating that perturbations of Notch signaling
underlie several forms of human diseases (5). Notch proteins exhibit a highly conserved modular
architecture (Figure 2), in which distinct repeated structural units are associated with different functional
roles in the intact receptor (6). Ligand binding to the N-terminal EGF-repeats activates these proteins by
facilitating a proteolytic cleavage by a metalloprotease at site S2, which is a necessary prerequisite for the
gamma-secretase cleavage at S3 that permits the translocation of intracellular Notch (ICN) into the
nucleus, and activates transcription of target genes (7, 8, 9, 10). The Negative Regulatory Region (NRR)
of all Notch receptors has three tandem, independently folding LIN-12/Notch Repeats (LNRs) that wrap
around the HD domain containing the regulatory cleavage site S2, and mask the S2 site in the resting
receptor (Figure 3) (11-13). Hence the interactions between the LNRs and the HD are critical in stabilizing
the NRR and preventing activation prior to ligand binding. Each of the LNRs contains six cysteines with a
unique three disulfide bonding pattern and coordinate a single Ca2+ (Figure 3), however the minimum
requirements that would ensure an LNR to fold independently are not known. This work utilizes the 32
amino acid stretch from cysteine 1 to cysteine 6 and the residues that flank these residues in the second
LNR of hN1 (Table 1), to define the minimum length requirement for hN1 LNRB and to investigate the
impact of metal ions and number of disulfide bonds on its autonomous folding.
LNRB_delBC
A
LNRB_int
Redox Potential
(mV)
-51.5
-35.6
-31.6
-4.5
37.2
B
10 mMCaCl2
1 mMCaCl2
Figure 7. Chromatograms of LNRB_orig and LNRB_int after dialysis 3 folded under varying metal ion
conditions. Panel A: Chromatograms of LNRB_orig folded in the presence of 1mM CaCl2-green and 1mM
ZnCl2-blue. These chromatograms demonstrate the selectivity of LNRB folding for Ca2+ which shows one
thermodynamically favored species unlike Zn2+ which shows an array of multiple peaks indicating the lack of
one predominant native fold. Panel B: Chromatograms of LNRB_int folded in the presence of 1mM CaCl2brown and 10mM CaCl2-purple. These chromatograms illustrate the sensitivity of shorter constructs to the
CaCl2 concentration.
Figure 4. Chromatograms of folded constructs after dialysis 3: LNRB_orig- green, LNRB_int- purple, LNRB_delBC- blue.
Major peaks represent the correctly folded species, small neighboring peaks are indicative of misfolded species.
Top left panel: Representative chromatogram detailing the elution gradient used and the pressure during the run.
Top right panel: post DTT incubation chromatogram, note peak collapse and elution shift in Table 2.
LNRB_delAB
LNRB_short
Current results indicate the importance of linker_AB on proper LNR folding. Verification of the length of
each construct via mass spectrometry coupled with the RP-HPLC elution shifts pre and post DTT incubation
identify the predominant peaks as being the correctly folded species. These data show that the minimum
length requirements for folding previously determined for the prototypical LNR module, LNRA, are in fact not
applicable to all other LNR modules. Furthermore, folding conducted under various redox potentials provide
an optimal intrinsic reducing potential of approximately -4.5mV, which is obtained with a 5:1 cysteine:cystine
ratio.
Further optimization of folding conditions provided by metal specificity experiments show that a
proper folding module cannot be obtained at low CaCl2 concentrations of only 1mM for shorter constructs.
Similarly, even the longest construct refolded under 1mM ZnCl2 failed to achieve correct folding,
underscoring the specificity of this module for Ca2+ in order to obtain proper folding.
Figure 3. Crystal structure of Human Notch2 NRR (14).
• All constructs were expressed as inclusion bodies using •On day 3 the constructs were moved into a dialysis buffer that
BL21(DE3) PlysS E. coli cell line.
did not contain any redox reagent (cysteine/cystine).
• LNRB was cleaved from the hydrophobic leader sequence • Day 3 dialysis samples of all constructs under the
by cyanogen bromide cleavage in 70% formic acid and was experimental conditions were run on a reverse phase HPLC
separated from the leader sequence through precipitation of using a C18 column and 0.25%/min gradient elution :
the leader sequence upon pH increase.
Buffer A: 10% Acetonitrile, 90% H2O, 0.1% TFA
Buffer B: 90% Acetonitrile, 10% H2O, 0.1% TFA
• Soluble LNRB constructs (~175 M) were folded for two
days in a refolding buffer with daily buffer changes.
• A sample of each folded construct was also incubated in
100mM NaCl
100mM DTT at room temp for 2 hrs and run on the RP-HPLC.
 20mM Tris pH 8
 10mM CaCl2
• Significant peaks on the HPLC chromatograms were analyzed
 2.5mM cysteine
•5:1 Red:Ox
by Mass Spectrometry.
 0.5mM cystine
Effect of Redox potentials on Folding:
Red:Ox ratios of 30:1, 15:1, 10:1, 5:1, 2:1 were tested
during folding in order to identify a potential redox potential
range at which protein folding was optimal.
CaCl2
ZnCl2
LNRB_orig
Cysteine:Cystine
Ratio
30:1
15:1
10:1
5:1
2:1
Metal Specificity
Post DTT incubation
Post DTT incubation
Figure 2. Domain organization of the Notch Receptor.
5:1 Best
B
LNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQ
linker_BC
linker_AB
C
Redox Potential
Effect of Metal Ions on Folding:
Constructs were folded in the presence of 1mM ZnCl2 and 1mM
CaCl2 under the optimal redox potential conditions and the
results were analyzed by RP-HPLC.
Figure 5. Chromatograms of unfolded constructs after dialysis 3: LNRB_delAB- brown, LNRB_short- orange.
Note no predominant peak is obtained suggesting no preference for correctly folded species for these two constructs. Panel
on top right: post DTT incubation chromatogram, note peak collapse of misfolded peaks to a single individual peak unique to
each construct as reflected by the elution shift in Table 2.
Future directions include altering the cysteine arrangement of hN1 LNRB after that of hN4 LNRA through
various mutations in order to correlate the extent to which the number of disulfide bonds specify proper LNR
folding. Isothermal Calorimetry will also be used to determine the affinity and specificity of different divalent
metals (Ca2+, Mg2+, Mn2+ and Zn2+) and their impact on LNR folding. These experiments will aid in the
definition of the minimum requirements for the proper autonomous folding of an LNR module using LNR_B
as a model from human Notch1.
Table 2. HPLC & Mass Spectrometry Results
Construct
% Buffer B
Elution
% Buffer B Elution
100mM DTT
Correctly Folded
Calculated MW
(Da)
Mass Spec MW
(Da)
LNRB_orig
28
30
Yes
5368.8
5365.65
LNRB_short
21-25
26
No
3940.3
3937.54
LNRB_int
25
27
Yes
4338.7
4291.93
LNRB_delAB
23-25
25
No
4481.8
4480.47
LNRB_delBC
27
30
Yes
4827.2
4823.22
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