Protein-Metal-Ion Interactions Studied by Mass Spectrometry-Based Footprinting with Isotope-Encoded Benzhydrazide

Article abstract of DOI:10.1021/acs.analchem.8b04088

Metal ions, usually bound by various amino-acid side chains in proteins, play multiple roles in protein folding, conformational change, cellular communication, and catalysis. Ca(II) and Mg(II), abundant among biologically relevant cations, execute their c

Full text of DOI:10.1021/acs.analchem.8b04088

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Article
Protein-Metal-Ion Interactions Studied by Mass Spectrometry-
Based Footprinting with Isotope-Encoded Benzhydrazide
Chunyang guo, Ming Cheng, and Michael L. Gross
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04088 • Publication Date (Web): 29 Nov 2018
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Protein-Metal-Ion Interactions Studied by Mass Spectrometry-
Based Footprinting with Isotope-Encoded Benzhydrazide
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Chunyang Guo, Ming Cheng, Michael L. Gross*
Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States
ABSTRACT: Metal ions, usually bound by various amino-acid side chains in proteins, play multiple roles in protein folding, conformational
change, cellular communication, and catalysis. Ca(II) and Mg(II), abundant among biologically relevant cations, execute their cellular
functions associated with the conformational change of bound proteins. They bind with proteins where carboxylic acid residues are dominant
ligands. To develop mass spectrometry for mapping protein-binding sites, we implemented a new carboxyl group footprinter, benzhydrazide,
and refined it with isotope encoding. The method uses carbodiimide chemistry to footprint carboxylic residues, whereby 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide activates a carboxyl group followed by nucleophilic attack by benzhydrazide forming a stable labeled
2+
2+
product. We tested the effectiveness of isotope-encoded benzhydrazide by studying Ca and Mg binding of calmodulin, an EF-hand protein.
The footprinting results indicate that the four active sites for metal-ion binding (EF hands I, II, III, and IV) and the linker region (peptide 78-
86) undergo conformational changes upon Ca(II) and Mg(II) binding, respectively. The outcome is consistent with previously reported results
and 3-D structures, thereby validating a new reagent that is more reactive and discriminating for specific amino-acid protein footprinting. This
reagent should be important for locating metal-binding sites of other metalloproteins.
1
Metal ions are essential for many cellular functions. In the range of
5 −50% of all proteins found within an organism contain metal
10
3
Although nuclear resonance (NMR), X-ray crystallography, and
2
11
Cryo-EM are commonly used to determine metal-binding sites,
2,3
ions. Metal ions bind via functional groups termed ligands that
donate lone pairs to the metal ion. In the bound state, the metal ions
in proteins can catalyze a variety of biochemical reactions or stabilize
the protein structures.
they are limited by size, conformational flexibility, aggregation
propensity, or limited sample amount, rendering them frequently
inapplicable. Our goal is to develop mass spectrometry (MS)-based
methods that complement these high-resolution techniques because
MS is general, fast, sensitive, has high-throughout, and provides
moderate resolution. One MS-based method, hydrogen deuterium
exchange (HDX), has been widely applied in protein structure
characterization.1 HDX can be extended to provide an approach
termed protein-ligand interactions by MS, titration, and HD
exchange (PLIMSTEX) to monitor the conformational changes of
Based on the properties of coordinated metals, we can classify two
types of metals that interact with proteins: 1) borderline/soft ions
e.g., Zn(II), Fe(II) and Cu(II)) that coordinate with nitrogen- and
sulfur-containing groups found in His and Cys, and 2) hard cations
e.g., Ca(II) and Mg(II)) that use oxygen ligands as the preferred
(
2-15
(
4
coordination sites (e.g., Asp and Glu are preferred). Among all
metalloproteins, those containing Mg(II), Ca(II), are relatively
2+
proteins upon Ca binding and even measure the binding
constants. HDX, however, cannot directly monitor the binding of
side chains owing to their fast hydrogen deuterium exchange.
16
1
-5
abundant. In many cases, Mg(II) and Ca(II) binding stabilizes the
structure of folded proteins, and in some cases, locks in a
physiologically active conformation. Specifically, Ca(II) plays a role
in signal transduction, muscle contraction, nerve-impulse
transmission, and calcium homeostasis. To maintain the correct Ca
ion concentration in the intra- and extracellular space, the body uses
Ca pumps. Most Ca-binding proteins possess a highly conserved Ca-
binding motif, the EF hand, that selectively binds Ca in a background
An alternative method for solving this challenge is irreversible
covalent labeling whereby a variety of reactions can be invoked to
modify amino-acid side chains. In this approach, reactive side chains
are stably modified such that the label can be conveniently located
by MS in peptides form from proteolysis. One approach is to use
reactive species such as OH radicals (provided through electron
beam or X-ray radiolysis, Fenton chemistry, or photolysis of
iodide radicals, and trifluoromethyl
radicals. Slower but still effective footprinting can use
carbodiimide , ICAT, or diethylpyrocarbonate (DEPC)
5
of up to 10 -fold higher concentrations of Na(I), K(I), and
6
,7
Mg(II). Although divalent Mg has higher charge density and exists
at higher concentration in the cytosol than Ca(II), it shares physical
and chemical properties with Ca(II) to bind proteins and enable
signaling, enzymatic activation and catalysis, and muscle
relaxation. A clear knowledge of metal/protein interactions,
including the location of the metal binding site and its binding
characteristics, is essential to understand the functional mechanisms
of metal-binding proteins.
17
18,19
20
peroxide), carbenes,
21
22
23
24
chemistries. Because the reactions are slower than those of free
radicals, there is always an issue that the labeling itself doesn’t
perturb the native structures.
8
,9
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Carbodiimide chemistry provides the most versatile method for
labeling or crosslinking carboxylic acids. Earlier, we employed 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC), a crosslinking facilitator, to enable glycine ethyl ester (GEE)
to footprint side chains of Asp and Glu. This approach was
successfully applied to determine the orientation of the FMO
was added dropwise to the hydrazine hydrate solution (78-82%, 1.23
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g, 24.6 mmol) in 6 mL THF over 45 min. The reaction was allowed
o
to go to completion overnight at 25 C, as monitored by TLC.
Solvents were evaporated in vacuum to give a crude white product
that was isolated and purified by silica gel column chromatography
(hexane:ethyl acetate = 1:1) to give the benzhydrazide as a white
2
5
26
antenna protein, to map the binding of Fab-1 and VEGF, to
solid (1.03 g, 93% yield). d
an identical procedure, giving the d
5
-Benzhydrazide was synthesized by using
-benzhydrazide as a white solid
27
locate a membrane-associated tyrosine kinase binding site, to map
the epitope of human interleukin-6 receptor, to determine the
binding interface for the oligomeric apolipoprotein E, to
characterize monoclonal antibody structure, and ELISA antibody-
antigen interactions. Nevertheless, GEE footprinting has
limitations, including the requirement for a large excess of the GEE
reagent (20,000 equivalents with respect to the protein), some
hydrolysis of the product during work-up, and a limited opportunity
to incorporate isotopes for encoding.
5
2
8
(1.00 g, 87% yield). High-resolution mass spectra (HRMS) were
2
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recorded with a Bruker MaXis QToF (ESI). HRMS (ESI) m/z
3
0
+
calcd. for protonated BHD (C
7
H
8
N
2
OH ): 137.0715. Found:
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+
5 7 3 5 2
137.0714. HRMS (ESI) m/z calcd. for d -BHD (C H D N OH ):
142.1029. Found: 142.1028. (Figure S2)
Preparation of Protected amino acids/Peptide/Protein Stock
Solution. All protected amino acids (Z-Glu-OMe and N-
Benzyloxycarbonyl-L-aspartic acid 1-methyl ester) and cyclic
peptide stock solutions of 100 uM concentration were prepared in
Herein, we explore application of carbodiimide compound (EDC),
in the presence of new labeling reagent benzhydrazide (BHD),
2+
10 mM HEPES buffer. Ca -free CaM (50 µM) was prepared by
3
2
inspired by Leitner. To be specific, carboxylic acid residues are
activated by EDC, then BHD, a nucleophilic modifying reagent,
attacks the activated carboxyl group. Our goal is to avoid the
hydrolysis of the modified product while still characterizing metal-
binding sites. We focus on two hard metal ions Ca(II) and Mg(II)
that chelate with carboxylic residues because they are ubiquitous in
metalloproteins. We chose carbodiimide specifically because it
reacts with carboxylic groups whose reactivity will be reduced when
chelated with Ca(II) or Mg(II) ions. Furthermore, its size may allow
high discrimination for metal-bound vs. unbound carboxylates. To
improve the MS quantification, we incorporated five deuteriums in
washing three times with HEPES buffer at 15,000 (×g) speed using
TM
2+
Vivaspin centrifugal concentrators, whereas Ca -bound CaM
was prepared by incubating 50 µM CaM with 500 µM CaCl
2
o
2+
overnight at 25 C. Similarly, Mg -bound CaM was prepared by
incubating 50 µM CaM with 500 µM MgCl overnight at 25 C. The
2
o
concentration of CaM in 10 mM HEPES buffer (pH 7.5) was
determined by using UV absorbance (molar absorptivity of 2980
-
1
-1
Lmol cm at 280 nm) with a NanoDrop spectrophotometer
(Thermo Scientific).
Carboxyl Group Modification. The final concentrations of
protected amino acids, peptide, or protein for all reactions were
adjusted to 10 µM. For protected amino acids, the labeling was
initiated by adding BHD and EDC to give final concentrations of 20
mM and 500 M, respectively. The protected amino acid
modification was quenched after 20 min by adding 20 μL
ammonium acetate (1 M). For cyclic peptides, the labeling follows
the same procedure as protected amino acids, except the labeling
was quenched after 24 h. Experiments with CaM were performed in
triplicate by using 20 mM BDH and 500 M EDC, identical to the
concentration used for cyclic peptide labeling. After 5 min of
reaction at 37 ℃, the solution was quenched by removing the
reagents by using a Zeba spin desalting column according to the
manufacturer’s protocol. (Figure S3)
3
3
the structure to permit encoding. Results show that the method can
accurately measure the binding site of Ca(II) and Mg(II) in a test
protein, calmodulin (CaM). By assisting in the elucidation of metal-
binding sites in proteins, this method should enrich the output of
structural proteomics studies.
EXPERIMENTAL SECTION
Materials. Tris base, HEPES buffer (pH 7.5), urea, water, formic
acid, glycine ethyl ester hydrochloride (GEE), calcium chloride,
magnesium chloride, ammonium acetate, 1,1′-carbonyldiimidazole,
5
benzoic acid, benzoic acid-2,3,4,5,6-d , tetrahydrofuran (THF),
hydrazine hydrate solution (78-82%), Z-Glu-OMe, and thin-layer
chromatography supplies (TLC, silica gel 60 F254) were obtained
from Sigma-Aldrich (St. Louis, MO). N-Benzyloxycarbonyl-L-
aspartic acid 1-methyl ester was obtained from Alfa Aesar
Trypsin Proteolysis Protocol. After desalting, the filtrate was
evaporated to dryness in a vacuum centrifuge. The pellets were
dissolved with 50 μL water to make an equal volume of protein.
TM
TM
(Lancashire, UK) Sartorius
Vivaspin
500 μL centrifugal
concentrators were purchased from Sartorius Corporation
(Goettingen, Germany). The protein bovine calmodulin (CaM)
was from Ocean Biologics (Seattle, WA). The 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDC), trypsin
5
Aliquots of 10 μL filtrate of BHD and d -BHD were combined at 1:1
ratio (v/v) and dried in vacuum (the dry sample can be stored in a
freezer at -80 ℃ for months). Prior to analysis, the protein mixture
pellet was denatured with urea (8 M, 5 μL) at 37 ℃ for 30 min,
followed by adding 45 μL Tris buffer (pH 8.5). Protein samples were
then digested with trypsin at a protease:protein ratio of 1:20 (w/w)
at 37 ℃ overnight. Pure formic acid (1 μL) was added to stop the
digestion.
TM
and Zeba spin desalting columns (7K MWCO) were from
Thermo Scientific (Rockford, IL). Cyclo(Arg-Gly-Asp-D-Phe-Glu)
was from Peptides International, Inc. (Louisville, KY).
Synthesis and Characterization of Benzhydrazide and d
5
-
Benzhydrazide. Reagents (no known hazards) were synthesized
3
4
Solvent Accessible Surface Area (SASA) calculation. We used PDB
based on protocol of Li, (Figure S1) except for the purification
2+
2+
files of Ca -free CaM (PDB ID: 1CFC) and Ca -bound CaM
PDB ID: 1CLL) to GETAREA
process. In 100 mL reaction flask, 1,1′-carbonyldiimidazole (1.73 g,
(
1
0.6 mmol) was added to a solution of benzoic acid (1.00 g, 8.19
o
(http://curie.utmb.edu/getarea.html) to calculate individual side-
chain SASA.
mmol) in THF (15 mL). After stirring at 25 C for 3 h, the mixture
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Analytical Chemistry
Mass Spectrometry. All mass spectra for intact proteins were
acquired on a Bruker MaXis Q-TOF (Bremen, Germany) in the
positive-ion mode, as was reported previously.
accurate quantification. 3) A sufficient mass difference between two
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isotopically encoded adducts is needed to separate the peaks of the
“light and heavy” analogs over a range of from m/z 400 to 2000.
Unfortunately, only a limited number of isotopic footprinters are
commercially available. Therefore, we prepared both the “light” and
“heavy” reagents (see Experimental) that we used in this
footprinting.
1
8
For proteolytic peptides, digests were analyzed on a Q Exactive Plus
hybrid quadrupole orbitrap mass spectrometer coupled with a
Nanospray Flex ion source (Thermo Fisher, Santa Clara, CA). A
C18 column was custom fabricated with an integrated emitter (75
μm × 150 mm, 1.8 μm, 100 Å). The gradient was from 2.0% solvent
B (80% acetonitrile, 0.1% formic acid) to 20% solvent B over 55 min,
then to 50% solvent B over 25 min, and finally to 90% solvent B for
We first measured the relative labeling efficiency of D and E on
protected-aspartic acid and glutamic acid. They show very similar
modification extent (25.1% ± 0.4% for protected D v.s. 25.0% ± 0.1%
for protected E). (Figure. S4)
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10 min followed by a 15 min re-equilibration step. The 15 most
abundant molecular ions were automatically chosen for
fragmentation (DDA) by using the mass spectra scanned from m/z
To test whether the “intrinsic” chemical reactivity is the same for
“light” vs. “heavy”, we chose a cyclic peptide cyclo(RGDFE), which
has no secondary structure and no complicating N- or C-termini.
The EICs (Figure 1a) for unmodified (black), BHD- (light blue),
380−2200 at a resolving power (RP) of 70 K (at m/z 200)
throughout the chromatography. Precursor ions were isolated in the
quadrupole with an isolation window of 2.0 m/z and fragmented
with higher-energy collisional dissociation (HCD) with a
normalized collision energy (NCE) of 32%. The automatic gain
5
and d -BHD (dark blue) labeled cyclic peptides were integrated,
demonstrating that ratio of modified vs. unmodified was nearly the
same, 43.2 ± 1.6% “light” and 44.6 ± 1.9% “heavy” labeling. Thus,
the presence of isotopes in BHD did not affect the reactivity of the
carboxylic residues in cyclo(RGDFE).
5
4
control (AGC) targets were 5 × 10 for MS and 5 × 10 for MS/MS
acquisitions. Maximum injection times (maxIT) were 200 ms for
MS and 100 ms for MS/MS.
LC-MS/MS Data Analysis The LC-MS/MS data were searched for
This cyclic peptide also helps determine whether isotopomer
mixture of BHD can label both Asp and Glu if the sibling residues
are adjoining. Metal ions (e.g., Ca, Mg) generally have multiple
coordinated sites, and usually include several Asp and Glu in a single
binding pocket. It is important that the footprinter can modify all
carboxylic residues in a pocket-like site rather than react with one
and be “silent” with the other. BHD labels both Asp and Glu even
when they are adjoining in the cyclic peptide (cRGDFE). To
validate the peptide assignments, we manually assigned peaks in the
TM
unmodified and modified CaM tryptic peptides by using Byonic
Software (Protein Metrics, San Carlos, CA). All BHD and d -BHD
5
modifications on Asp and Glu were added as modifications to the
database. The parameters were: 10 ppm precursor mass tolerance,
60 ppm fragment mass tolerance, and CID/HCD fragmentation.
Modification-sites were validated by manually checking product-ion
spectra and were double-checked with Thermo Xcalibur (a custom
program from Thermo Fisher).
5
product-ion spectra (MS/MS) for the BHD- and d -BHD modified
Results and Discussion
cyclo(RGDFE), confirming that the modification occurs at both
35
Asp and Glu (Figure 1b and 1c) . In addition, we did not find di-
labeled peptide.
Evaluation of Isotope Effects. A reliable approach in quantitative
methods is to use stable isotope labeling for ratioing. The use of an
isotope-encoded footprinter normalizes the MS intensity of analytes
to their isotopic analogs and, therefore, effectively compensates for
solvent effects, variable ion suppression from other coeluting
analytes, and experimental variations accompanying sample
preparation, injection, and instrument setup. To develop an isotope-
encoded footprinter, certain criteria are required: 1) isotope effects
should not significantly affect the kinetics of footprinting (i.e., the
reaction rates of a footprinting reaction are nearly constant when
one or more atoms of the footprinter are replaced by isotopes. 2)
Analytes and their isotopic analogs (nearly) coelute during the LC;
in this way, a pair of isotope-labeled analytes would experience the
same ionization efficacy during MS analysis, which is important for
We next determined whether there are any isotope effects in the
separation of BHD- and d -BHD modified peptides. If the light and
5
heavy species coelute, matrix effects should not affect the relative
efficiency of ionization of the analytes. Because there is a difference
in the size of the atoms3 , substitution of hydrogen with deuterium
could lead to differences in the binding interactions (van der Waals
interactions) with the stationary phase. Protiated compounds bind
to hydrophobic stationary phase more strongly than their deuterated
6,37
36
counterparts. When comparing the retention time of BHD- and d
BHD modified peptide (Figure 1a), we found a small isotope effect
that d -BHD modified peptides eluted approximately 11 s after the
BHD-modified counterpart.
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Figure 1. a) Extracted ion chromatograms (EIC) for unmodified (black), BHD-modified (light blue) and d
5
-BHD modified (dark blue)
, blue circle:
O, yellow square: addition 118.0524 or 123.0845 mass unit, green square: addition of 118.0524 or 123.0845 mass unit and loss of H O, pink
cyclo(RGDFE), product-ion (MS/MS) spectrum for BHD- b) and d
loss of H
5 3
-BHD- c) modified cyclo(RGDFE). (Orange circle: loss of NH
2
2
∑
퐼
푚표푑푖푓푖푒푑
square: addition of 118.0524 or 123.0845 mass unit and loss of NH
3
.) 푀표푑푖푓푖푐푎푡푖표푛 푒푥푡푒푛푡 = ∑퐼푚표푑푖푓푖푒푑 + ∑퐼푢푛푚표푑푖푓푖푒푑 (The nomenclature for cyclic
3
9
peptides is that by Ngoka and Gross , figure itself is original. Details are shown in Scheme S1.)
Calmodulin Protein Footprinting. Calmodulin (CaM) is a highly
conserved, secondary messenger protein, which is ubiquitous in
eukaryotic cells. More than thirty proteins and enzymes with various
cellular functions are activated by CaM. CaM contains four highly
conserved motifs, referred to as “EF-hands”, which show a typical
helix–loop–helix motif. CaM binds various metals to execute its
is more reactive when compared to GEE (Figure 2, compare b to c),
67 ± 3% BHD and 25 ± 3% GEE labeling.
2
+
3+
3+
2+
3+
2+
cellular functions, not only Ca , but also La , Tb , Pb , Sm , Sr ,
2
+
2+
2+
2+ 38
2+
Hg , Cd , Zn , and Mn . Among them, Ca binding is the most
2
+
important because Ca
binding induces an allosteric
communication of CaM to catalyze downstream enzymatic
reactions.
We evaluated the BHD reactivity with CaM by fixing the
concentration of EDC at 500 uM, 50 fold excess relative to CaM (10
uM). Because each CaM molecule contains 37 Asp and Glu, all Asp
and Glu on the protein surface have a chance to react. With
increasing amounts of BHD from 5-200 mM, the modification
extent of CaM increased from 43% to 65% (Figure S5). When the
concentration of BHD was increased further, the modification
extent did not significantly change, probably because EDC is no
longer the limiting reagent. Thus, we chose 200 mM as the optimum
concentration of BHD. We also tested the relative reactivity of
Figure 2. Mass spectra of modified CaM at +15 charge state: a) control:
2+
2+
2+
Ca -free CaM, b) Ca -free CaM labeled by GEE, c) Ca -free CaM
2+
o
5
labeled by BHD, d) Ca -free CaM labeled by d -BHD (25 C, 5 min).
“
light” and “heavy” BHD to CaM. Similar to cyclo(RGDFE), we
BHD as a footprinting reagent. Our next goal is to test BHD as a
footprinter to establish whether its reactions report the metal-
binding site for CaM. Isotope-encoding provided a “light” and
found that “light” and “heavy” BHD show nearly identical
modification extents for the intact CaM protein (Figure 2).
“
heavy” labeling of the two different states of CaM. In this protocol
To evaluate whether BHD improves the labeling efficiency with
respect to GEE, we compared the reactivity of both reagents. BHD
(Figure S6), Ca-free CaM was labeled by d -BHD in one Eppendorf
tube and Ca-bound CaM was
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Figure 3. a) EIC for BHD- (light blue) and d
5
-BHD- (dark blue) modified peptide 78-86, b) product-ion (MS/MS) spectrum for peptide 78-
5 0
86: unique mass shift of b) 123.0845 Da, and c) 118.0531 Da is consistent with d -BHD modified and d -BHD modification on E84.
2
+
labeled by d
0
-BHD in another tube. Following the footprinting, the
structures. CaM binds Ca at four, nonidentical sites that contain
the EF-hand structural motif, two in the N-domain (EF-1 and EF-
2) and two in the C-domain (EF-3 and EF-4). Each hand contains
tube contents were combined in a 1:1 ratio prior to trypsin digestion
and LC/MS/MS analysis. In this way, we could maintain all
experimental conditions (e.g., protein digestion, MS analysis) nearly
identical for both light and heavy samples following protein
modification.
2+
2+
an acidic, Ca -coordinating loop, or “EF-loop” where Ca is
chelated with seven oxygens from six residues in a pentagonal
bipyramidal fashion. 6 EF-1 chelates Ca with ligands D20, D22,
D24, and E31 from peptide 14-21, 22-30, 31-37. (Figure 5a and 5f).
For this binding site, the reagent is extraordinarily discriminating
such that the carboxylates of this region undergo little modification
when in the holo state (peptide 14-21, 22-30, 31-37).
,41
2+
Data processing showed 100% sequencing coverage of the tryptic
peptides. We took advantage of accurate-mass measurements to
obtain reliable EICs for integrating the signal intensities
representing (Figure 3a) modifications by d
(seen as two distinct masses, +123.0845 (C HD
(C ) on peptide 78-86, respectively (Figure 3b, 3c)). When
comparing their retention time, however, we did observe, as before,
a small isotope effect (Figure 3a) that d -BHD modified peptide 78-
6 eluted 15.6 s earlier than its d -BHD-modified counterpart. This
deviation is consistent with the previously reported effect of
5
0
-BHD and d -BHD
7
5 2
N
) and +118.0531
7
6 2
H N
5
8
0
3
6,37
deuteration.
In addition, we found that the labeled peptides are
more hydrophobic and eluted approximately 14 min later than their
unmodified counterparts (Figure S7). This chromatographic
pattern assists the identification of the modified peptides, sometimes
facilitating separation of isomeric peptides with modifications on
different residues.
Overall, we found that eight regions of the protein undergo large
conformational changes with modification extent difference over
40
5
0% and p-values less than 0.01. These regions are represented by
peptides 14-21, 22-30, 31-37, 38-74, 76-86, 78-86, 95-106, and 127-
2
+
148 (Figure 4), consistent with Ca -induced regional structural
transitions that dramatically alter the molecular surface of CaM. For
example, there is only trace amount of BHD-labeled peptides 22-30,
and 38-74 for the holo state, whereas modification extents of these
two peptides for the apo-state is > 8% (Figure 4, Table S1).
Figure 4. Comparison of the modification extents of constituent
peptides upon footprinting Ca -free CaM (dark blue) and Ca -bound
CaM (light blue).
2+
2+
To establish that these MS results are structurally meaningful, we
analyzed them with respect to the high-resolution NMR and X-ray
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+
2+
Figure 5. 3D structure of Ca -free CaM from the average NMR structure (PDB ID, 1CFD) and Ca -bound CaM from the X-ray crystal structure
2+
2+
2+
(
PDB ID, 1CLL), Asp and Glu shown as sticks. a) and f) region 20-31 in Ca -free CaM and Ca -bound CaM, b) and g) region 56-67 in Ca -free
CaM and Ca -bound CaM, c) and h) region 78-86 in Ca -free CaM and Ca -bound CaM, d) and i) region 93-104 in Ca -free CaM and Ca -
bound CaM, e) and j) region 129-140 in Ca -free CaM and Ca -bound CaM. (Gray sphere is the metal ion.)
2
+
2+
2+
2+
2+
2+
2+
Likewise, EF-2 region, which is represented by peptide 38-74 and
chelates Ca via D56, D58, D64 and E67 (Figure 5b and 5g), also
becomes unreactive in the holo state. Because Asp and Glu lose
nearly completely their chemical reactivity, the modification extent
difference for EF hand II reaches 97%.
2+
2
+
As pointed out earlier, the binding of Ca to the EF hand induces a
dramatic change of the linker region (peptide 76-86 and 78-86),
changing from a flexible loop (Figure 5c) to a rigid α-helix (Figure
h). Although the conformation of this linker region was under
debate that the formation of the α-helical conformation is a
crystallization artifact, many studies report that the Ca binding
does induce domain opening in CaM, indicating the linker region is
extended and becomes more rigid than in the apo-CaM. The
plasticity of the linker region in Ca -bound CaM allows EF-hand
domains to take up various orientations with respect to each other.44
Our footprinting results also reveal a significant modification
change, evidenced by two overlapping peptides that show
approximately 70% modification difference (see Table S1). The
D78, D80, E82, E83, and E84 on a flexible loop in holo-CaM can
easily react with BHD, whereas when this region becomes a rigid α-
helix region in the apo form, those residues are less reactive.
5
4
2
43
2+
2
+
Figure 6. Comparison of the modification extent of constituent
peptides from Mg -free CaM (dark blue) and Mg -bound CaM (light
blue).
2+
2+
EF-3, as represented by peptide 95-106 (Figure 5d), uses D95 and
E104 to chelate Ca (Figure 5i). Although the modification ratio for
this region in apo CaM is only 0.1%, there is negligible modification
for the Ca -bound CaM, indicating a large conformational change
of EF-3 hand. Finally, for EF-4 peptide 127-148 (Figure 5e) shows a
4% modification decrease upon binding. All the Asp and Glu in this
To investigate whether changes in SASA agree with our result, we
compared the residue-level quantification and the calculated SASA
2
+
2+
2+
of Ca -free and Ca -bound CaM (Figure S8). The modification
extent of all D and E residues at the EF-hands significantly decreases
2
+
2+
upon Ca binding. The results correlate well with the similar trend
that most D and E residues of Ca -bound CaM at the binding sites
dramatically lose their SASAs. Nevertheless, the decreased
modification extent of E82/E83/E84 does not correlate well with
the their theoretical increasing SASAs. This may be due to the
reaction in this region not only depends on solvent accessibility but
7
2+
2+
region, except a distant E139, are candidates for binding with Ca
(Figure 5j).
4
5
also on stereochemistry. Compared with previous GEE study,
BHD with 10% amount of GEE labels 27 D and E in CaM, showing
higher reactivity. The significant differences of labeling efficiency for
GEE and BHD are that the alpha effect in BHD increases the
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nucleophilicity of the nitrogen owing to the presence of adjacent
nitrogen atoms with nonbonding electron pairs, leading BHD to be
more reactive. In addition, BHD is more discriminating given that
it successfully reports four binding-sites and that the linker region
undergoes large conformational changes.
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Mg2+ Binding. In addition to Ca -binding, we extended the
2+
2
+
2+
approach to study Mg binding with CaM. Like Ca , divalent Mg
is also a “hard” ion and prefers “hard” ligands with oxygen being most
2+
preferred. In contrast to the well-characterized Ca -bound
structure, the binding of Mg to CaM is still unresolved.
Conclusions from microcalorimetric studies indicate four identical
Mg binding sites that do not completely overlap with the Ca -
binding sites in CaM. On the other hand, conclusions from flow
2
+
0
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0
2+
Figure 7. 3D structure of Mg -bound CaM (PDB ID, 3UCW). a)
2
+-
2+
Region 20-31, b) region 55-67. (Gray sphere is the metal ion; red sphere
is water molecule.)
2+
dialysis, NMR, and fluorescence indicate that Mg shares same
2+ 47
2+
2+
binding sites with Ca . Only recently, the structure of Mg -bound
Furthermore, the linker region of Mg -bound CaM does show
significant change, but the changes are not as large as those of Ca -
bound CaM. (Figure 6, peptide 76-86 and 78-86). The modification
extent differences of Mg -bound CaM are on the borderline (57%)
whereas the Ca -bound shows differences at 70%. One expects that
Ca -bound CaM should undergo larger conformation changes in
this region on the basis of previous studies.
6
2+
CaM was partially resolved by X-ray crystallography to show the N-
terminal Mg binding sites are similar to those of Ca .
Nevertheless, the full high-resolution structure of Mg -bound CaM
2+
2+
2+
2+
2+
remains unknown.
2+
Although previous reports disagree on details, they do indicate that
5,43,49
2
+
Mg indeed binds to CaM, likely at the calcium sites. When we
2
+
compared our footprinting results for Mg -bound with those of
Finally, we can evaluate EF-3 and EF-4 regions even though there is
no crystal structure for this part in Mg bound CaM. Like for Ca -
bound CaM, we detected trace amount of modification for peptide
95-106 (EF-3). Peptide 127-148, containing EF-4, undergoes
relatively less conformational change with Mg compared to Ca -
binding, with modification extent differences 58% vs. 74% for Ca -
binding. The ionic radius of Mg2
2
+
2+
2+
Ca -bound, we found that eight peptides regions have modification
extent difference more than 0.5 and p-value less than 0.01 (Figure 6
and Table S2). Those eight peptides regions belong to EF-1 to 4 plus
to the linker region. Our results support the conclusion that the
-
2+
2+
2
+
2+ 5,41,47
2+
binding site of Mg resembles that of Ca .
+ 50
is too small to allow some
Comparing the footprinting results for EF-1 and the X-ray structure,
2+
carboxylic acid residues to coordinate with Mg , eliminating any
large differences in the reaction of BHD with Mg binding.
2+
we found that although D20, D22, and D24 chelate Mg , E31 is too
2+
2+
far from Mg to contribute to the coordination. (Figure 7a) In
2+
contrast to pentagonal bipyramid coordination for Ca ,
In summary, peptide 14-21, 22-30, 31-37,76-86, 78-86, and 127-
148 of Mg -bound CaM experience conformational changes, but
they are less significant than those of Ca -bound CaM. This
indicates that Mg with smaller ionic radii binds to CaM without
chelating with the 12 residues of the EF-hand, consequently
causing smaller structural changes.
2
+
2+
octahedrally coordinated Mg does not directly bind to Glu12 of
the EF-hand. Footprinting results shows that E31 undergoes 0.008%
2+
2
+
2+
BHD modification in Mg -bound CaM, but undetectable
2
+
th
modification in Ca -bound CaM, correlating well with the
coordination geometry. Furthermore, the modification-extent
2
+
2+
differences for Mg are significant, but not as significant as for Ca -
2
+
2+
binding (89% for Mg - vs. ~100% for Ca - bound ones), suggesting
weaker binding for Mg . This difference is consistent with the
enthalpy changes upon Ca binding being exothermic, whereas
Conclusion
2
+
2
+
With this new footprinting reagent, we can readily identify the four
Ca binding sites in CaM and correlate them with 3-D structures. In
contrast to other irreversible labeling strategies that either monitor
the backbone or side chains around the metal-binding site, this
reaction directly footprints carboxyl groups at the binding site. For
2
+
endothermic, and thus unfavorable, for Mg binding to CaM.
2
+
2+ 48
Consequently, Mg does not bind to CaM as tightly as Ca .
2
+
For EF-2 regions, however, Mg shows a very different pattern than
2+
2
+
2+
Mg binding, which is weaker and more difficult to discriminate, the
footprinting still shows that Mg binds with four EF hands similar
Ca for binding with CaM. From the X-ray structure, Mg
2+
coordinates with two oxygens from T62 and a backbone carbonyl
group. (Figure 7b) The remaining four coordinating positions are
occupied instead, by four water molecules that form strong
hydrogen bonds with D58 and E67, allowing Asp and Glu residues
2+
to Ca .
This approach has several advantages and some disadvantages: 1)
BHD coupled with EDC enables improved chemical reactivity and a
stable modified product. 2) The isotope-encoded benzylic acid used
2+
to gain some protection. In other words, Mg directly binds to first-
shell water molecules, allowing it to bind in an indirect way to D58
and E67 (the binding is called a second-shell ligand interaction).
Our MS results show no modification extent difference in peptide
for d
high-yield d
synthesis. 3) The d
5
5
-BHD synthesis is commercially available and inexpensive;
-BHD can be obtained conveniently with a two-step
-BHD, incorporating five deuterium atoms,
5
2
+
38-74 with that in Ca -CaM even though there is an indirect
2
+
improves the resolution and accuracy of quantification, especially for
large proteolytic peptides. On the other hand, there is a small isotope
effect on LC elution when a long chromatographic gradient was used
for our custom-made C18 column. Further, the reagent is unable to
interaction between Mg and the nearby carboxylic residues. This
identical footprinting, however, also reminds us that a more sensitive
reagent is needed if we want to resolve the pattern between first-shell
and second-shell ligand binding.
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Page 8 of 12
differentiate first- and second-shell ligand interactions, and a more (7) Denessiouk, K.; Permyakov, S.; Denesyuk, A.; Permyakov, E.;
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discriminating labeling protocol or reagent are needed. For the
future, we wish to extend this new footprinting approach to other
hard metal-ion binding and, via a titration protocol, to test its ability
to determine metal-binding affinity.
Johnson, M. S., Two structural motifs within canonical EF-hand
calcium-binding domains identify five different classes of calcium
buffers and sensors. PLoS One 2014, 9, e109287.
(
8) Dudev, T.; Chang, L.; Lim, C., Factors governing the
3+
2+
2+
substitution of La for Ca and Mg in metalloproteins: A
ASSOCIATED CONTENT
Supporting Information
DFT/CDM study. J. Am. Chem. Soc. 2005, 127, 4091-4103.
(
9) Payandeh, J.; Pfoh, R.; Pai, E. F., The structure and regulation of
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10) Jesnsen, M.R., Petersen, G.; Lauritzen, C.; Pedersen, J.; Led, J.
1
The Supporting Information is available free of charge on the ACS
Publications website.
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(
J., Metal binding sites in proteins: Identification and
characterization by paramagnetic NMR relaxation. Biochemistry
5
Synthetic route and MS spectra for BHD and d -BHD; modification
reaction by BHD coupling reaction; condition screening for BHD;
workflow of isotope-encoded covalent labeling; EIC for unmodified
peptide 78-86; modification extent and calculated SASA of D and E
residues at the EF-hand; summary of carboxylic acid modification
2
005, 44, 11014-11023.
(
11) Zhang, Q.; Dai, X.; Cong, Y.; Zhang J.; Chen, D.; Dougherty,
M. T.; Wang, J.; Ludtke, S. J.; Schmid, M. F.; Chiu, W., Cryo-EM
structure of a molluscan hemocyanin suggests its allosteric
mechanism. Structure, 2013, 21, 604-613.
2
+
2+
2+
2+
percentage for Ca -free/Ca bound CaM and Mg -free/Mg -bound
CaM.
(
12) Kojetin, D. J.; Matta-Camacho, E.; Hughes, T. S.; Srinivasan,
S.; Nwachukwu, J. C.; Cavvet, V.; Nowak, J.; Chalmers, M. J.;
Marciano, D. P.; Kamenecka, T. M.; Shulman, A. I.; Rance, M.;
Griffin P. R.; Bruning, J. B.; Nettles, K. W., Structural mechanism for
signal transduction in RXR nuclear receptor heterodimers. Nat.
Commun. 2015, 6, 8013.
AUTHOR INFORMATION
Corresponding Author
*
7
E-mail: mgross@wustl.edu. Tel: +1-314-935-4814. Fax: +1-314-935-
484.
(13) Fast, C. S.; Vahidi, S.; Konermann, L., Changes in enzyme
structural dynamics studied by hydrogen exchange-mass
spectrometry: Ligand binding effects or catalytically relevant
motions? Anal. Chem. 2017, 89, 13326-13333.
Notes
Any additional relevant notes should be placed here.
(
14) Jensen P. F.; Comamala, G.; Trelle, M. B.; Madsen, J. B.;
JØrgensen, T. J. D.; Rand, K. D., Removal of N‑linked glycosylations
at acidic pH by PNGase A facilitates hydrogen/deuterium exchange
ACKNOWLEDGMENT
mass spectrometry analysis of N‑linked glycoproteins. Anal. Chem.
2016, 88, 12479-12488.
We acknowledge research supported by grants NIH P41RR103422, as
well as Dr. Gerstenecker from Washington University in St. Louis for his
advice for this project and Protein Metrics for software.
(
15) Wang, L.; Chance, M. R., Protein footprinting comes of age:
Mass spectrometry for biophysical structure assessment. Mol. Cell.
Proteomics 2017, 16, 706-716.
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