Transcript Poster Presentation

LIN12/NOTCH REPEATS (LNRs):
IMPARTING TUNABLE REDOX RESPONSIVENESS IN LESS THAN 35 AMINO ACIDS THROUGH
2+
AN INTERPLAY BETWEEN Ca BINDING AND DISULFIDE BOND FORMATION
Janelle L. Jackson, Angie Seo, Didem Vardar Ulu, Wellesley College, Chemistry Department, Wellesley, MA, USA.
A.
B.
N15
C4
hN1LNRA : EEAC-ELPECQEDA-GNKVCSLQCNNHACGWDGGDC
GS LNRA :
EC—-AEGCPGSWIKDGYCDKACNNSACDWDGGDC
hN4LNRA :
GAKGCEGRS-GDGACDAGCSGPGGNWDGGDC
C9
C22
Ca2+
C27
D30
D33
N-term
S19
Figure 1. (A) Sequence alignments of the first LNRs from three
C34
different proteins: Human Notch 1 (hN1LNRA), NC18
acetylglucosamine-1-phosphate transferase (GS LNRA), and
C-term
Human Notch 4 (hN4LNRA). Cysteine residues are in orange, and
(B) NMR structure of hN1LNRA with disulfide bonds
2+
the Ca coordinating D/N residues in yellow. The characteristic
shown as orange sticks and Ca2+ coordinating
disulfide bonding-pattern is indicated above the sequence.
residues represented as yellow sticks.1
In this work we investigated the impact of free [Ca2+] and the total number of disulfides on the reduction of
LNRs under various redox potentials through a comparative study of multiple LNRs. Our results indicate
that while bound Ca2+ provides significant protection and prolonged chemical stability against reduction
under even strong reducing conditions for the canonical LNRs, this Ca2+ based tunable stability is
eliminated for LNRs missing the first pair of disulfide bonds, despite the presence of all the conserved
Ca2+ binding residues. Taken together with our earlier findings we propose that LNRs are small protein
modules that have evolved to provide varying amounts of redox sensitivity to the multi-domain protein
they are incorporated in through a protein specific arrangement of multiple LNR modules with subtle, yet
critical sequence variations.
Materials and Methods:
Sample Preparation: Bacterially expressed hN1LNRA and synthesized GS and hN4LNRA were folded in a
refolding buffer that allowed the formation of native disulfide bonds and were purified via reverse phase High
Performance Liquid Chromatography (RP-HPLC). Folded proteins were then dialyzed against 20 mM HEPES, pH
8.0, 150 mM NaCl, containing predetermined concentrations of free Ca2+ as calculated by Visual Minteq.2
Reduction Reactions: Dialyzed proteins were aliquoted into reaction tubes to have a final concentration of
15 μM and were placed in an AtmosBag (Sigma-Aldrich) purged with N2. Dialysis buffers supplemented with
250 μM or 2.5 mM DTT were added under these anaerobic conditions to start the reduction reaction. At
predetermined time points, reactions were quenched via acidification and assayed via analytical RP-HPLC, using a
C18 column running a 0.1% / min. acetonitrile gradient.
Data Analysis: The areas under each corresponding peak on the 280 nm chromatograms were integrated to
quantitate the amounts of oxidized, reduced, and misfolded proteins (Figure 2). Percent oxidized, reduced, and
misfolded proteins were calculated by dividing the integrated peak area of the corresponding peaks by the total
area of all protein peaks from individual chromatograms (Table 1).
Figure 2. Overlay of
chromatograms recorded at
280 nm at 0, 15, 30, 60, 180
min. for the series of
hN1LNRA reduction
experiments. The four sets of
quantified peaks are
annotated on the figure.
Table 1. Integrated peak
areas (IPA) of the
corresponding peaks on the
chromatogram, in addition to
the calculated oxidized and
reduced hN1LNRA
percentages and redox
potentials for 250 μM DTT in
0 μM Ca2+ (50 μM EDTA) at
0, 15, 30, 60, 180 min.
Correctly folded
hN1 LNRA peak
(13.0 min.)
0.030
0.025
Reduced
hN1 LNRA peak
(17.0 min.)
0.020
AU
Oxidized
DTT peak
(9.50 min.)
0.015
0.010
Misfolded
hN1LNRA Peaks
Below the fully saturating Ca2+ concentration, the rate of
hN1LNRA reduction is redox potential dependent
Ca2+ protects the disulfide bonds against reduction and
prolongs the chemical stability of folded hN1LNRA
0% LNRA-Ca2+
complex
90
80
42.7% LNRA-Ca2+
complex
70
60
77.9% LNRA-Ca2+
complex
50
40
97.5% LNRA-Ca2+
complex
30
20
10
99.8% LNRA-Ca2+
complex
0
0
15
30
45
60
Time (min.)
Figure 3. Reduction of folded hN1LNRA as a function of time in varying [Ca2+] and 2.5 mM DTT.
Percent values of reduced hN1LNRA in 0 μM, 25 μM, 100 μM, 1 mM and 10 mM free [Ca2+] were
plotted as a function of time in 15 minute intervals up to 1 hr.
2.5 mM DTT
[Ca2+]
0 μM Ca2+ (500 μM EDTA)
0% LNRA-Ca2+ complex
25 μM Ca2+
42.7% LNRA-Ca2+ complex
100 μM Ca2+
77.9% LNRA-Ca2+ complex
1 mM Ca2+
97.5% LNRA-Ca2+ complex
10 mM Ca2+
99.8% LNRA-Ca2+ complex
Redox Potential (mV) in EDTA
Mean Redox Potential (± SD) (mV) in Ca2+
0 min.
3.16
0.58
% hN1LNRA Reduced
15 min.
30 min.
45 min.
85.33
12.71
92.15
32.71
91.57
38.73
60 min.
88.5
49.64
0
4.93
13.46
14.99
19.9
0
0
0.43
1.55
1.87
0
0
0
0
0
-373
-346 ± 7
-365
-341 ± 8
-364
-343 ± 6
-364
-341 ± 7
-367
-343 ± 7
Table 2. Quantification of hN1LNRA reduction over time in 20 mM HEPES, pH 8.0, 150 mM NaCl,
2.5 mM DTT, and varying free [Ca2+]. The percent of reduced hN1LNRA and the redox potentials for
each experiment were determined as described in the Materials and Methods section. Since the
presence of metal ions impacts the stability of DTT5, the redox potentials calculated in the presence of
different [Ca2+] were averaged for each time point, excluding the EDTA conditions, which are reported
separately. Based on the calculated redox potentials, oxidized hN1LNRA experienced very similar
reducing environments (redox potential = -343±6) in the presence of any amount of Ca2+ during the
course of the experiment. Under these redox conditions, essentially full protection against reduction was
achieved when there was enough free Ca2+ to ensure >97.5% LNRA-Ca2+ complex.
Folded hN1LNRA and GS LNRA are more protected
against reduction by Ca2+ than hN4LNRA
0.005
79.27
80
100
% Reduced hN1 LNRA
Many multi-domain proteins contain small protein modules whose global folds are stabilized by metal
binding or disulfide bonds rather than an extensive hydrophobic core or secondary structures. Lin12/Notch Repeat (LNR) is such a module, first identified in Notch receptors and more recently, within
functionally unrelated multi-domain proteins, such as pregnancy associated plasma proteins and stealth
proteins. Prosite database defines LNR (PDOC50258) as a 35 amino acid module with three conserved
Asp/Asn residues and six Cys residues engaged in a particular disulfide pattern favored by the presence
of Ca2+. However, homology searches reveal naturally occurring LNRs with only four of the conserved
Cys residues, as well as deviations in the proposed Ca2+ binding residues (Figure 1A).
Results:
% Reduced hN1LNRA
Introduction:
60
40
19.90
20
23.86
1.87
0
77.9% LNRACa2+ complex
97.5% LNRACa2+ complex
0
0
-338 ± 6 mV
-361 ± 1 mV
99.8% LNRACa2+ complex
Figure 4. Percent of reduced hN1LNRA, complexed with Ca2+ to varying degrees, after 60 min. of
reaction with 250 μM (E=-338±6 mV) and 2.5 mM DTT (E=-361±1 mV). At Ca2+ concentrations
below what is needed to form essentially 100% LNRA-Ca2+ complex, the amount of reduction at a given
time was directly proportional to the reducing power of the environment. When hN1LNRA was fully
saturated with Ca2+, however, it was fully protected against reduction at both redox potentials.
Conclusions:
 Ca2+ binding LNRs, like hN1LNRA and GS LNRA, are protected against reduction by free Ca2+ in the
environment. The rate of reduction is inversely proportional to the free [Ca2+ ].
 The rate of reduction of an LNR is dependent on both the free [Ca2+ ] and the redox potential of the
environment, unless the free [Ca2+ ] is fully saturating. At this point, there is complete protection against
reduction for the range of tested redox potentials.
 Ca2+ does not does not have an impact on the reduction of hN4 LNRA because hN4 LNRA does not
bind Ca2+ . However, even in the absence of any Ca2+, hN4LNRA, with one fewer disulfide bond is still
more susceptible to reduction at a given redox potential than its three disulfide-bonded homologs.
Discussion:
 Ca2+ is an integral
part of the LNR structure possessing three disulfide bonds1. Fluorescence
experiments have shown that binding of Ca2+ to the LNR alters the surface exposure of residues
around the binding pocket. Hence the [Ca2+] dependent protection against reduction observed for
these LNRs can be attributed to the increased chemical stability of the LNR-Ca2+ complex compared
to its apo form due to a decrease in the solvent accessibility of the disulfide bonds upon Ca2+ binding.
 In comparison to the cytoplasm, which has a resting redox potential of approximately -230 mV that
can be significantly altered by cellular status (-240, -200, and -170 mV during cell proliferation,
differentiation, and apoptosis, respectively), the ER lumen provides a relatively constant and more
oxidative environment (-180 mV)4 critical for the proper folding of extracellular proteins. Unlike redox
potential, though, the free [Ca2+ ] in the ER can change significantly based on cellular demands.6
 LNRs
that are the focus of this study are found as repeated units within different multi-domain
proteins targeted to the cell membrane. During folding in the ER, their disulfide bonds are formed and
broken until the correct bonding pattern is achieved. The ability of free Ca2+ in the environment to
selectively fine tune the chemical stability of the LNRs by altering their redox sensitivity offers a novel
mechanism for the cells to regulate this process and preserve any correctly folded regions of the
protein over misfolded regions, which can continue to shuffle their disulfide bonds in search of the
most stable conformation.
0.000
9.00
Time
(min.)
0
15
30
60
180
10.00
Oxidized
DTT
IPA
N/A
17934
27861
48648
82116
11.00
12.00
Oxidized
hN1 LNRA
IPA
409529
318971
250626
151864
24604
13.00
14.00
Minutes
15.00
16.00
17.00
18.00
19.00
Reduced
Calculated
Mis% Oxidized % Reduced
hN1 LNRA
redox potential
Folded PA hN1LNRA hN1LNRA
IPA
(mV)
N/A
0
8333
98.01
0
18902
28092
87.16
5.16
-325
44043
39291
75.05
13.19
-319
96588
34173
53.73
34.18
-311
216413
68202
7.96
69.99
-303
Calculation of protein-Ca2+ complex percentage: The percent of protein complexed with Ca2+ at each free
[Ca2+] was calculated using the formula3 ML = {Lo+Mo+KD) – ((Lo+Mo+KD)2 – 4MoLo)1/2} / 2, describing reversible
binding between a receptor and a ligand (ML: % bound complex, Mo: initial [protein] = 15 μM, Lo: initial [Ca2+] of the
experiments included in the tables, and Kd: dissociation constant, previously determined to be 25 μM).
Calculation of Redox Potentials: To determine the amount of reduced and oxidized DTT present at each
experimental time point, a set of experiments with known concentrations of oxidized and total DTT were performed
in the absence of protein. Peaks on the chromatograms corresponding to the oxidized (only at 280 nm) and
reduced (at 229 and 280 nms) were integrated to determine the corresponding absorbances. These values were
substituted into Beer’s Law (A = εlc) to calculate the extinction coefficients of the oxidized and reduced forms of
DTT. These extinction coefficients were used to determine the actual concentration values at the assayed time
points of the reduction experiments. The redox potentials for each experiment and time point were calculated using
the Nernst equation Eh (in mV) = Eo – (RT/nF) ln([DTTred]2/[DTToxid]); Eo = -323 mV at pH 7.0 with an adjustment of
-5.9 mM / 0.1 increase in pH.4
250 μM DTT
% Reduced
Time
Redox Potential
hN1LNRA
GS LNRA
hN4LNRA
(min.)
(mV)
0
0
0.74
0
-333
15
5.16
2.63
9.11
-320
0 μM Ca2+ (50 μM EDTA)
30
13.19
15.99
25.61
-313
0% LNRA-Ca2+ complex
60
34.18
43.18
54.05
-305
0
0
0
1.95
-324
15
0
0
9.92
-304
10 mM Ca2+
30
0.65
0
28.41
-309
99.8% LNRA-Ca2+ complex
60
1.02
0
54.72
-305
Table 3. Quantification of the three homologous LNRs over time in Ca2+-free and Ca2+-saturated
environments. The percent of reduced hN1LNRA, GS LNRA, and hN4LNRA in 0 μM and 10 mM free
Ca2+ and 250 μM DTT at 0, 15, 30, and 60 minutes, as well as the redox potentials calculated as
described in the Materials and Methods section. hN4LNRA, with one fewer disulfide bond, has a faster
rate of reduction in comparison to hN1LNRA and GS LNRA. The varying concentrations of free Ca2+, in
fact, had no effect in the reduction of hN4LNRA, while the saturation of Ca2+ significantly protected and
prolonged the chemical stability of hN1LNRA and GS LNRA against reduction.
[Ca2+]
References:
1. Vardar, D., North, C.L., Sanchez-Irizarry, C., Aster, J. C., Blacklow, S. C. (2003) Nuclear Magnetic Resonance
Structure of a Prototype Lin12-Notch Repeate Module from Human Notch1. Biochemistry, 42, 7061-7067.
2. Visual MINTEQ: http://www2.lwr.kth.se/English/OurSoftware/vminteq/
3. Jakubowski. “Chapter 5 – Binding. A: Reversible Binding 1 Equations and Curves.” (2010)
http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindderveq.html.
4. Shafer, F.Q., & Buettner, G.R. (2001) Redox environment of the cell as viewed through the redox state of the
glutathione disulfide/glutathione couple. Free Radical Biology & Medicine, 30(11), 1191-1212.
5. Burmeister Getz, E., Xiao, M., Chakrabarty, T., Cooke, R., & Slevin, P.R. (1999) A comparison between the
sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry,
Analytical Biochemistry, 273, 73-80.
6. Bygrave, F.L, & Benedetti, A. (1996) What is the concentration of calcium ions in the endoplasmic reticulum?
Cell Calcium, 19 (6), 547-551.
Funding:
•
•
•
•
Camille and Henry Dreyfus Faculty Start-up Award (DVU)
Wellesley College Sophomore Early Research Program (JJ, AS)
Protein Science Young Investigator Travel Grant (JJ)
Wellesley College Science Center Travel Award (JJ, AS)