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ATCUN (amino terminal Cu(II) and Ni(II) binding) motifs chelate Cu(II) ions strongly. However, the impact of the phosphorylation of neighboring residues on such complexation has not been elucidated. The copper(II) dissociation constants of original and phosphorylated peptides from human histatin-1 and human serum albumin were compared using spectroscopic methods. Phosphorylation markedly weakened Cu(II) binding. Thus, these results indicate that phosphorylation may be a vital mechanism governing metal ion binding.
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Phosphorylation Impacts Cu(II) Binding by ATCUN Motifs
Tomasz Frączyk*
Cite This: Inorg. Chem. 2021, 60, 84478450
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sıSupporting Information
ABSTRACT: ATCUN (amino terminal Cu(II) and Ni(II) binding) motifs chelate Cu(II) ions strongly. However, the impact of
the phosphorylation of neighboring residues on such complexation has not been elucidated. The copper(II) dissociation constants of
original and phosphorylated peptides from human histatin-1 and human serum albumin were compared using spectroscopic
methods. Phosphorylation markedly weakened Cu(II) binding. Thus, these results indicate that phosphorylation may be a vital
mechanism governing metal ion binding.
More than 100 human extracellular proteins and peptides
contain an ATCUN motif,
1
which has an amino acid
sequence Xaa-Zaa-His, where Xaa is any amino acid residue with
a free N-terminal amine, and Zaa is any amino acid residue
except proline. Human histatin-1 and human serum albumin
(HSA) contain this motifAsp-Ser-His and Asp-Ala-His,
respectively. Cu(II) complexes with these molecules are
biologically relevant. Such peptides bind Cu(II) strongly, with
subpicomolar to femtomolar dissociation constants.
26
Phosphorylation of serine residues is one of the most frequent
post-translational modications (PTMs).
7,8
It impacts a range of
protein properties, e.g., molecular dynamics, interaction with
other molecules or neighboring residues, or the propensity to
change the cellular compartment.
8
However, there is no
information about the impact of such phosphorylation on
Cu(II) binding by the ATCUN motif.
Two hexapeptides, N-termini from human histatin-1
(DSHEKR-am) and HSA (DAHKSE-am), were chosen to test
the inuence of serine phosphorylation on Cu(II) binding by
the ATCUN motif. Spectrophotometric methods revealed the
dissociation constants for the original and modied peptides.
First, spectroscopic pH-metric titrations of Cu(II)/peptide
solutions in a 0.9:1.0 ratio with UVvis and circular dichroism
(CD) spectra registration were performed (Figures 1 and 2).
The appearance of ddbands characteristic of 4N square planar
complexes was detected by both methods. The pKavalues for the
formation of 4N complexes were calculated from the pH
dependence of the absorbance and CD signal (Figure 3), as
reported earlier (see also the Supporting Information).
9
Interestingly, phosphorylation caused an increase in such pKa
values by 0.3 pH units for both peptides (4.13 ±0.04 vs 4.37 ±
0.05 for DSHEKR-am and 4.53 ±0.04 vs 4.82 ±0.04 for
DAHKSE-am), strongly suggesting that serine phosphorylation
decreases the anity of these peptides for Cu(II).
According to a recently published approach,
2
the competition
of the hexapeptides and GGH tripeptide for Cu(II) ions was
observed by CD spectroscopy (Figure 4) to examine the
inuence of serine phosphorylation on Cu(II) binding by the
ATCUN motif. Briey, CD spectra of 0.67 mM hexapeptide and
0.60 mM Cu(II) with variable concentrations of GGH at pH 7.4
(equilibrated for 24 h) were recorded. Each spectrum was
decomposed to nd the component concentrations: Cu-
(hexapeptide) and Cu(GGH) complexes allowing me to
compute the relative strength of the hexapeptide with GGH as
a reference (Supporting Information).
In detail, the linear dependence is derived from the equation
K
K
Cu peptide GGH Cu GGH
Cu GGH peptide Cu peptide
()( ())
()( ( ))
Cu peptide
Cu GGH
f( ( ))
f( ( ))
T
T
=[][][]
[][][ ]
where [Cu(peptide)] and [Cu(GGH)] are measured by
decomposition of experimental spectra, and [peptide]Tand
[GGH]Tare the total concentrations of each ligand. The slope of
this dependence is equal to the number saying how many times
Received: March 26, 2021
Published: June 7, 2021
Figure 1. UVvis spectra of 0.67 mM peptide and 0.60 mM CuCl2in
the pH range of 3 (red) to 10 (violet). The diminishing (at 800 nm) and
appearing (at 530 nm) ddbands with increasing pH are shown for A)
DSHEKR-am, B) DpSHEKR-am, C) DAHKSE-am, and D)
DAHKpSE-am.
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the formation constant (Kf) for Cu(hexapeptide) is higher than
for Cu(GGH) (cf.Figure 5 and Supporting Information). The
dissociation constant of Cu(GGH) at pH 7.4 was recently
determined to be 609.5 fM (log Kf(Cu(GGH)) = 12.215 ±0.005).
2
Thus, one can multiply Kf(Cu(GGH)) by the relevant slope and
obtain the nal Kf(Cu(hexapeptide)). Based on this approach, the
dissociation constants are 2.2 fM (log Kf= 14.65 ±0.12) and
38.6 fM (log Kf= 13.41 ±0.03) for DSHEKR-am and
DpSHEKR-am, respectively. Analogously, 26.9 fM (log Kf=
13.57 ±0.05) and 60.8 fM (log Kf= 13.22 ±0.05) dissociation
constants were found for DAHKSE-am and DAHKpSE-am,
respectively. Thus, phosphorylation of nearby serine residues
markedly lowers the anity of the ATCUN motif for Cu(II)
ions. The eect is more pronounced for serine residues in the
second position in the sequence compared with the fth
position. This seems reasonable, as serine in the second position
is much closer to the coordination sphere.
Interestingly, copper anities of nonphosphorylated
DAHKSE-am and DSHEKR-am dier substantially from each
other. Nevertheless, the value for DAHKSE-am is close to that
obtained recently for human serum albumin whole protein (log
Kf= 13.02 ±0.05).
2
The anity for DSHEKR-am, in turn, is
equal to the one for DTHFPI-am, the N-terminal motif of
hepcidin, claimed to be the strongest Cu(II) chelator among
ATCUN motifs.
10
Both peptides have an Asp residue in the rst
position and a Ser or Thr residue in the second. The acidic
amino acid residue in the rst position and the hydroxyl-
containing one in the second position presumably ensure such
tight copper binding.
Human histatin-1 and human serum albumin are phosphory-
lated on serine residues tested in this work.
1113
However, about
20% of human histatin-1 is nonphosphorylated on Ser2.
14,15
Although phosphorylation of histatin-1 is benecial for its
binding to hydroxyapatite,
16
this peptide plays many other roles,
e.g., promotes angiogenesis
17
and exhibits antimicrobial
activity.
3,18
The involvement of phosphorylation in those
processes is not known yet. The binding of metal ions by
antimicrobial peptides leads to sequestration of this metal from
pathogens or is necessary for other antimicrobial modes of
action.
18
Contribution of copper to maintaining microbial oral
hygiene is probable as there is an inverse relationship of caries
status and Cu(II) concentration in the saliva of children.
19
Overall, Ser2 phosphorylation may aect metal-related
functions of histatin-1.
Figure 2. CD spectra of 0.67 mM peptide and 0.60 mM CuCl2in the
pH range of 3 (red) to 10 (violet). The appearance of ddbands at 490
and 570 nm with increasing pH is shown for A) DSHEKR-am, B)
DpSHEKR-am, C) DAHKSE-am, and D) DAHKpSE-am.
Figure 3. pH dependence of absorbance and circular dichroism of 0.67
mM peptide and 0.60 mM CuCl2for A) DSHEKR-am, B) DpSHEKR-
am, C) DAHKSE-am, and D) DAHKpSE-am. pKavalues (±SD) for
forming 4N square planar complexes calculated from the data are also
shown for each peptide.
Figure 4. CD spectra of 0.67 mM peptide and 0.60 mM CuCl2at pH 7.4
(50 mM HEPES) titrated with GGH. The redshifts of the ddbands
from the investigated Cu(hexapeptide) complex (violet) to Cu(GGH)
(black, dotted) during the titration are shown for A) DSHEKR-am, B)
DpSHEKR-am, C) DAHKSE-am, and D) DAHKpSE-am.
Figure 5. Determination of the relative Cu(II) binding strength of
investigated peptides and GGH, based on the competition experiment
shown in Figure 4, calculated for peptides from A) histatin-1 and C)
HSA, for nonphosphorylated (blue, dots) and phosphorylated (red,
circles) ones. The calculated slopes (±SD) reect the relative Cu(II)
binding anities. Speciation diagrams were obtained by decomposing
the CD spectra from Figure 4 for peptides from B) histatin-1 and D)
HSA. Shown are the data for investigated peptides (squares) and GGH
(diamonds), either for experiments with nonphosphorylated (blue, full
symbols) or phosphorylated versions (red, open symbols).
Inorganic Chemistry pubs.acs.org/IC Communication
https://doi.org/10.1021/acs.inorgchem.1c00939
Inorg. Chem. 2021, 60, 84478450
8448
Human serum albumin was found to be phosphorylated on
Ser5 by Fam20C kinase.
13
However, the extent of this
phosphorylation is not known. Although only 2% of ATCUN
in HSA is occupied by Cu(II) ions in blood plasma,
20
such
saturation is not known for other parts of the organism. Most of
HSA (6070%) is in the extravascular pool,
21
e.g., in skin, saliva,
and cerebrospinal uid.
22
Both saturation with Cu(II) ions and
phosphorylation on Ser5 may dier dramatically from the one in
the blood plasma. Such post-translational modication may
regulate Cu(II) binding by HSA locally or transiently.
The inuence of amino acid residue type on the stability of the
Cu(II) complex with the ATCUN motif has been thoroughly
studied. One of the most evident rules described in the literature
is the inverse linear dependence of the anity to metal ion on
the basicity of the N-terminal amine.
23,24
There is also a
hypothesis that the same dependence is valid for amide
nitrogens.
4
Interestingly, the presence of two Asp residues in
the ATCUN motif (Asp-Asp-His) is correlated with the highest
basicity of the N-terminal amine and, consequently, with the
lowest anity to Cu(II).
24
The basicity of amine in the free
serine amino acid is also heightened by phosphorylation of its
side chain (9.85 vs 9.25).
25,26
Therefore, the results presented in
this work suggest that phosphate moiety, similarly acidic as the
Asp side chain, may weaken the binding of Cu(II) ions by
stabilizing the protonated state of the amine and amide
nitrogens.
Based on the recent analysis of N-terminal sequences in
human proteins,
1
the selection of human proteins with the
ATCUN motif and serine or threonine residue within the rst
ve positions was performed and presented in Table S1. The
threonine residue was added to this analysis as its phosphor-
ylation probably shows the same impact on Cu(II) binding as
serine modication. There are 65 human proteins, either
secreted or with N-terminus exposed on the extracellular side
of the cell membrane, with ATCUN motifs containing at least
one Ser or Thr (Table S1). Seven proteins with such sequences
are localized in the endoplasmic reticulum or Golgi apparatus. It
sums up to 72 proteins localized in relatively oxidizing
compartments, thus, with a higher probability to meet divalent
copper ions.
1
Interestingly, more than 100 other proteins are
localized in compartments with a more reducing state (Table
S1), such as cytoplasm, nucleus, or mitochondrion.
1
The
binding of Cu(II) by those proteins is questionable (because
intracellularly Cu(I) is more prevalent than Cu(II)) but cannot
be excluded that may occur transiently or in pathological states.
The frequency of Ser or Thr in the sequence position 1, 2, 4, or 5
throughout the whole selection (223 proteins, Table S1)is
relatively uniform with a slight prevalence of serine residues
(Figure S7).
The results presented in this work indicate that phosphor-
ylation of serine residues markedly weakens the stability of
Cu(II) complexes with the peptides comprising N-terminal
sequences of human histatin-1 and human serum albumin. Thus,
phosphorylation is perhaps a mechanism of ne-tuning the
metal-related functions of these proteins. It may also be a general
mechanism valid for other proteins or peptides with ATCUN
motifs.
ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c00939.
Experimental section; details related to decomposition of
CD spectra and determination of Cu(II) binding anity;
and list of human proteins with ATCUN motif and serine
or threonine residue within rst ve positions (PDF)
AUTHOR INFORMATION
Corresponding Author
Tomasz Frączyk Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, 02-106 Warsaw, Poland;
orcid.org/0000-0003-2084-3446; Email: tfraczyk@
ibb.waw.pl
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.1c00939
Author Contributions
T.F. conceived, designed, performed the experiments and the
analysis, and wrote the article.
Notes
The author declares no competing nancial interest.
ACKNOWLEDGMENTS
The equipment used was sponsored in part by the Centre for
Preclinical Research and Technology (CePT), a project
cosponsored by the European Regional Development Fund
and Innovative Economy, The National Cohesion Strategy of
Poland.
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Link to Free Full Text: ............................................................................................................................................ https://onlinelibrary.wiley.com/share/author/J5YPUTRMDWJNMENJYC8K?target=10.1002/cbdv.202100043 ............................................................................................................................................ Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.
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Saliva is a key factor that contributes to the high efficiency of wound healing in the oral mucosa. This is not only attributed to physical cues but also to the presence of specific peptides in the saliva, such as histatins. Histatin-1 is a 38 aa antimicrobial peptide, highly enriched in human saliva, which has been previously reported to promote the migration of oral keratinocytes and fibroblasts in vitro However, the participation of histatin-1 in other crucial events required for wound healing, such as angiogenesis, is unknown. Here we demonstrate that histatin-1 promotes angiogenesis, as shown in vivo, using the chick chorioallantoic membrane model, and by an in vitro tube formation assay, using both human primary cultured endothelial cells, HUVECs, and the EA.hy926 cell line. Specifically, histatin-1 promoted endothelial cell adhesion and spreading onto fibronectin, as well as endothelial cell migration in the wound closure and Boyden chamber assays. These actions required the activation of the Ras and Rab interactor 2 (RIN2)/Rab5/Rac1 signaling axis, as histatin-1 increased the recruitment of RIN2, a Rab5-guanine nucleotide exchange factor (GEF) to early endosomes, leading to sequential Rab5/Rac1 activation. Accordingly, interfering with either Rab5 or Rac1 activities prevented histatin-1-dependent endothelial cell migration. Finally, by immunodepletion assays, we showed that salivary histatin-1 is required for the promigratory effects of saliva on endothelial cells. In conclusion, we report that salivary histatin-1 is a novel proangiogenic factor that may contribute to oral wound healing.-Torres, P., Díaz, J., Arce, M., Silva, P., Mendoza, P., Lois, P., Molina-Berríos, A., Owen, G. I., Palma, V., Torres, V. A. The salivary peptide histatin-1 promotes endothelial cell adhesion, migration, and angiogenesis.
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