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\begin{document}

\title{Crystallographic and Modeling Study of the Human PPA1 (Inorganic
Pyrophosphatase 1): a Potential Anti-cancer Drug Target}



\author[1]{Haiying Niu}%
\author[2]{Jiang Zhu}%
\author[1]{Quanxin Qu}%
\author[2]{Xia Zhou}%
\author[2]{Xiaolan Huang}%
\author[2]{Zhihua Du}%
\affil[1]{Tianjin First Central Hospital}%
\affil[2]{Southern Illinois University Carbondale}%


\vspace{-1em}



  
  \date{June 15, 2024}


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\selectlanguage{english}
\begin{abstract}
Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of
pyrophosphate to phosphates. PPases play essential roles in growth and
development, and are found in all kingdoms of life. Human possess two
PPases, PPA1 and PPA2. PPA1 is present in all tissues, acting largely as
a housekeeping enzyme. Besides pyrophosphate hydrolysis, PPA1 can also
directly dephosphorylate phosphorylated JNK1. Upregulated expression of
PPA1 has been linked to many human malignant tumors. PPA1 knockdown
induces apoptosis and decreases proliferation. PPA1 is emerging as a
potential prognostic biomarker and target for anti-cancer drug
development. In spite of the biological and physiopathological
importance of PPA1, there is no detailed study on the structure and
catalytic mechanisms of mammalian origin PPases. Here we report the
crystal structure of human PPA1 at a resolution of 2.4 \selectlanguage{ngerman}Å. We also
carried out modeling studies of PPA1 in complex with JNK1 derived
phosphor-peptides. The monomeric protein fold of PPA1 is similar to
those found in other family I PPases. PPA1 forms a dimeric structure
that should be conserved in animal and fungal PPases. Analysis of the
PPA1 structure and comparison with available structures of PPases from
lower organisms suggest that PPA1 has a largely pre-organized and
relatively rigid active site for pyrophosphate hydrolysis. Results from
the modeling study indicate the active site of PPA1 has the potential to
accommodate double-phosphorylated peptides derived from JNK1. In short,
results from the study provides new insights into the mechanisms of
human PPA1 and basis for structure-based anti-cancer drug developments
using PPA1 as the target.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Abbreviations List:}

\textbf{HRV-3C:} Human Rhinovirus 3C Protease\textbf{JNK1:} c-Jun
N-terminal kinases 1\textbf{PPA1:} Inorganic Pyrophosphatase
1\textbf{PPA2:} Inorganic Pyrophosphatase 2

\textbf{Introduction}

Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of one
molecule of pyrophosphate (PPi) to two molecules of phosphates (Pi).
PPases are ubiquitous enzymes present in all cell types. PPases include
membrane-bound V-PPases (vacuolar H+-translocating PPases) and soluble
PPases. V-PPases use pyrophosphate (instead of ATP) as an energy source
to transport H+ or Na+ across membranes in plants and some bacteria,
archaea, and parasitic protists {[}1, 2{]}. Soluble PPases are divided
into three families that differ in their sequences and structures
{[}3-5{]}. PPases from families II and III are only found in
prokaryotes, whereas Family I PPases exist in all kingdoms of life.
Family I PPases are single-domain, Mg\textsuperscript{2+} dependent
enzymes that exist as hexamers in prokaryotes and dimers in eukaryotes.

PPi is a metabolic product that is generated by many biosynthetic
reactions for the synthesis of various important biomolecules, including
proteins, nucleic acids, polysaccharides, and fatty acids {[}6{]}. The
conversion of PPi to Pi by soluble PPases is an essential cellular
process. On the one hand, the reaction is critical for maintaining
cellular PPi homeostasis; on the other hand, the reaction provides a
thermodynamic pull for the PPi-generating biosynthetic reactions because
enzymatic hydrolysis of PPi is highly exergonic.

Soluble PPases are essential enzymes for growth and development. In E.
coli, the ppa gene for PPase is essential for cell growth {[}7{]}. In
fermenting yeast, PPase defects lead to cell cycle arrest and autophagic
cell death {[}8{]}. In C. elegans, the PPase enzyme PYP-1 is required
for larval development and intestinal function {[}9, 10{]}. In mammals,
the PPase enzyme PPA1 has been reported to have the potential to
regulate neurite growth in mouse neuroblastoma cells {[}11{]}, to induce
type I collagen synthesis and stimulate calcification by osteoblasts
{[}12{]}. Increased expression and activity of cytosolic PPA1 in the
liver of rat and mouse has been correlated with aging {[}13, 14{]}.

Human possess two Family I PPases, PPA1 (289 aa) and PPA2 (305 aa)
{[}15{]}. PPA2 is localized to the mitochondrion. The two human PPases
share \textasciitilde{}70\% homology. PPA1 is a ubiquitous enzyme found
in all tissues. It is found that PPA1 acts largely as a housekeeping
gene {[}15{]}, presumably due to its essential role in energy
metabolism. More recently, the role of PPA1 in tumorigenesis has emerged
{[}16-19{]}. Upregulated expression of PPA1 has been linked to many
human malignant tumors including colorectal cancer {[}20-22{]}, diffuse
large B-cell lymphoma (DLBCL) {[}23{]}, lung adenocarcinoma {[}17,
24{]}, prostate cancer {[}25{]}, hepatocellular carcinoma {[}19{]},
breast cancer {[}26{]}, gastric cancer{[}27, 28{]}, intrahepatic
cholangiocarcinoma (ICC) {[}29{]}, and ovarian cancer {[}24, 30, 31{]}.
In laryngeal squamous cell carcinoma (LSCC), metastases tumors showed an
intense increase of PPA1 compared to primary tumors {[}32{]}. These data
indicate that PPA1 protein is a potential prognostic biomarker for
certain cancers.

The exact mechanism for the involvement of PPA1 overexpression in
tumorigenesis is not fully understood. Dysregulated metabolism is an
important hallmark of cancer cells {[}33{]}. Metabolic alterations
caused by PPA1 overexpression may provide the extra energy and
biomaterials for the cancer cells to maintain their high proliferation
rate. In addition, it is found that PPA1 can directly dephosphorylate
phosphorylated JNK1 (c-Jun N-terminal kinase) {[}22{]}. Functioning as a
JNK phosphatase, PPA1 may regulate the activities of downstream
effectors (including p53, \selectlanguage{greek}β --\selectlanguage{english}catenin, Bcl-2, and Caspase-3) that
control cell proliferation and apoptosis. In consistence with this
speculated mechanism, we previously found that PPA1 overexpression in
epithelial ovarian carcinoma (EOC) promoted the dephosphorylation and
translocation of \selectlanguage{greek}β-\selectlanguage{english}catenin {[}31{]}. In another study on EOC {[}30{]},
it was found that PPA1 overexpression caused the decrease in p53 and
increase in \selectlanguage{greek}β-\selectlanguage{english}catenin expression level. Conversely, PPA1 knockdown led
to the increase of p53 and decrease of \selectlanguage{greek}β-\selectlanguage{english}catenin level.

While PPA1 overexpression enhances migration, invasion and proliferation
of cancer cells {[}27{]}, PPA1 knockdown (by RNAi mediated silencing) is
shown to induce apoptosis and decrease proliferation in various cancer
cells {[}22-24, 30, 31{]}. Results from these studies suggest that
strategies for PPA1 down-regulation may have therapeutic potential for
the treatment of certain cancers. PPA1 down-regulation might be achieved
by mRNA knockdown through RNAi or RNA-targeting CRISPR systems.
Alternatively, the PPA1 protein might represent a valuable target for
the development of specific small-molecule inhibitors as anti-cancer
drugs. Efforts had been made to target PPases from pathogenic organisms
for small-molecule drug development. Anti-PPase small molecules were
shown to have promising antibiotic activity against drug resistant
strains of Staphylococcus aureus {[}34{]}. Small molecule inhibitors of
the VSP1 (vacuolar soluble pyrophosphatase 1) protein from Trypanosoma
brucei, the causative agent of African trypanosomiasis (HAT), provided a
40\% protection from death in a mouse model of T. brucei infection
{[}35{]}. Inhibition of PPase activity could also be achieved by
targeting allosteric sites, as demonstrated by the discovery of
allosteric and selective inhibitors of PPase from Mycobacterium
tuberculosis {[}36{]}.

In spite of the biological and physiopathological importance of human
PPA1, there is no detailed study on the structure and catalytic
mechanisms of this enzyme. Extensive structural and biochemical studies
had been reported on Family I PPases from Escherichia coli (E-PPase)
{[}37-39{]} and Saccharomyces cerevisiae (Y-PPase) {[}40-45{]}. X-ray
crystallographic studies had also been carried out on soluble PPases
from other species, including Thermus thermophilus (T-PPase) {[}46{]},
Pyrococcus horikoshii (Pho-PPase) {[}47{]}, human parasites Plasmodium
falciparum (Pf-PPase) and Toxoplasma gondii (Tg-PPase) {[}48{]},
Mycobacterium tuberculosis (Mt-PPase) {[}36, 49{]}, Trypanosoma brucei
brucei (also called vacuolar soluble protein TbbVSP1) {[}50{]}, and S.
japonicum (Sj-PPase, not published).

In this paper, we present the crystal structure of human PPA1. We also
carried out modeling studies of PPA1 in complex with JNK derived
peptides containing phosphotyrosine and phosphothreonine residues.

\textbf{Materials and Methods}

\textbf{Molecular cloning, protein sample preparation and
crystallization}

A cDNA plasmid that contains a DNA fragment encoding the full-length
human PPA1 (GenBank NC\_000010.11) was purchased from DNASU Plasmid
Repository. The protein product of this cDNA corresponds to the human
PPA1 protein with GenBank accession No. NP\_066952. The DNA encoding the
full length PPA1 (residues 1-289) was amplified by PCR from the plasmid
with the forward primer 5'-\emph{CAAGGACCGAGCAGCCCCTCA}
atgagcggcttcagcaccg-3' and the reverse primer
5'-\emph{ACCACGGGGAACCAACCCTTA} CTAGTTTTTCTGGTGATGGAAC-3'. Each of these
primers contains a 5'-end specific sequence (21 nucleotides, underlined)
for ligation-independent cloning (LIC). The PCR product was purified by
agarose gel electrophoresis and processed by T4 DNA polymerase in the
presence of 2.5 mM dATP to generate 5'-overhangs at both ends. To find
an appropriate way to express the PPA1 protein in \emph{E. coli} in a
soluble form, we had tested a series of in-house developed LIC cloning
vectors containing different fusion tags, including the MBP, GST, TRX,
SUMO, HALO, NusA, and Fc tags, etc. The cloning vector with a HALO tag
{[}51{]} gave the best result. The cloning vector encodes the Halo tag,
His tag, and an eight amino-acid recognition sequence for human
rhinovirus (HRV) 3C protease in front of a specific LIC sequence. The
vector was linearized by SmaI digestion and subsequently processed by T4
DNA polymerase in the presence of 2.5 mM dTTP to generate 5'-overhangs
at both ends. The processed PCR insert and vector were mixed and
incubated at room temperature for 20 minutes. The mixture was used to
transform NEB-turbo\emph{Escherichia coli} competent cells. The
recombinant plasmid was transformed into NiCo21(DE3) \emph{E. coli}
cells (New England Biolabs). Cell culture was grown in LB media until it
reached an OD\textsubscript{600} of 0.6-0.8. Isopropyl
\selectlanguage{greek}β-\selectlanguage{english}D-1-thiogalactopyranoside (IPTG) was added to the culture (at 0.4 mM
final concentration) to induce protein expression. After induction, the
temperature was lowered to 288 K and the culture was allowed to grow
overnight before harvest.

The PPA1 protein was expressed in \emph{E. coli} as a fusion protein
with an N-terminal Halo tag and His tag, and a HRV 3C protease
recognition for cleavage motif. The fusion protein was purified by NTA
affinity resin. The fusion tags were then cleaved by HRV 3C protease.
After removal of the tags, the target protein contains an artificial
sequence GPSSP at the N-terminus as a cloning artifact. The cleaved tags
were separated from the target protein by a reverse IMAC (immobilized
metal-affinity chromatography) process with NTA resin. The purified
target proteins were concentrated to \textasciitilde{}10 mg/ml in a
buffer containing 25 mM Tris (pH 7.5), 200 mM NaCl, and 1 mM DTT.

Crystallization trials were carried out using 96-well-format plates. Ten
sets of in-house prepared screening solutions (each containing 96
different conditions with various PEGs as the precipitants) were used in
the trials. Multiple crystallization conditions were identified within a
few days. After optimizing the crystallization conditions, diffracting
crystals of the PPA1 were obtained at 295 K by hanging-drop vapor
diffusion against 50 \selectlanguage{greek}µ\selectlanguage{english}l well solution using 96-well format
crystallization plates. The crystallization drops contained 2 \selectlanguage{greek}µ\selectlanguage{english}l protein
sample mixed with 2 \selectlanguage{greek}µ\selectlanguage{english}l well solution. The well solution contains 20\%
PEG 3350, 100 mM Bis-Tris propane (pH 7.5), 200 mM KBr, and 10\% (v/v)
glycerol. Crystals were flash-cooled in liquid nitrogen.

\textbf{Data collection, data processing, and structure determination}

Data collection was carried out at beamline 21ID-D of LS-CAT at the
Advanced Photon Source (Argonne National Laboratory). Data were
processed, integrated, and scaled with the programs Mosflm and Scala in
the CCP4 package {[}52{]}. Phasing was achieved by using the molecular
replacement method, which was carried out by using the program Phaser
{[}53{]} that is implemented in the PHENIX package {[}54{]}. The
monomeric structure of the E. coli PPase (PDB code 4um4) was used as a
search model. This structure is chosen as a search model because All
plausible space groups of the P222 family were tried. A single solution
was found in the space group P212121. Interactive model building was
carried out with Coot {[}55{]}. Structure refinement was carried out by
PHENIX. Structure determination statistics are shown in Table 1. The
program UCSF Chimera {[}56{]} was used to generate all of the molecular
graphic figures. Figures for electrostatic surface rendering were
generated by Chimera using the Coulombic Surface Coloring tool with the
default settings.

Sequence alignments of the eukaryotic soluble PPases with known
structures were carried out by Clustal Omega {[}57{]}. Molecular
interactions at the PPA1 homodimerization interface were depicted by
using the program LIGPLOT {[}58{]}. Atomic coordinates and diffraction
data have been deposited in the Protein Data Bank with accession code
6C45.

\subsection*{Structural modelling of PPA1 with various putative
substrates}

{\label{structural-modelling-of-ppa1-with-various-putative-substrates}}

The determined crystal structure of human PPA1 does not contain
substrate at the putative active site. To gain insights into the
possible catalytic mechanism of human PPA1, modelling studies were
carried out to place various putative substrates at the active site. At
first, a pyrophosphate molecule was docked into the active site by using
AutoDock Vina {[}59{]}. Chimera was used to prepare the PPA1 receptor
structure for docking and to visualize the docking results. To explore
whether the PPA1 active site is capable of accommodating peptide
substrates containing phosphorylated tyrosine and/or phosphorylated
threonine residues, a series of phosphotyrosine and/or phosphothreonine
containing peptides were modelled into the PPA1 active site by using
Chimera (including energy minimization of the bound peptide at the
active site). The phosphotyrosine and/or phosphothreonine containing
peptides were initially derived from the phosphorylated MAP kinase Erk2
(PDB code 2erk) {[}60{]}. Two Erk2-derived tetrapeptides were used:
184-EYpVA (superscript p indicates phosphotyrosine) and 181-FLTpE
(superscript p indicates phosphothreonine), to model a Yp and a Tp at
the active site respectively. To prepare peptides corresponding to the
JNK1 sequence for modelling studies, the 181-FLTpEYpV sequence in the
Erk2 structure were mutated to JNK1 sequence 181-MMTpPYpV by Chimera. A
pentapeptide 182-MTpPYpV was used to model the Yp at the active site. A
hexapeptide 181-MMTpPYpV was used to model the Tp at the active site.

\section*{Results and Discussion}

{\label{results-and-discussion}}

\subsection*{The overall structure of human PPA1 is similar to other
Family I
PPases}

{\label{the-overall-structure-of-human-ppa1-is-similar-to-other-family-i-ppases}}

The crystal structure of human PPA1 was determined at a resolution of
2.39 \selectlanguage{ngerman}Å using the molecular displacement (MR) method. A monomeric
structure of E-PPase was chosen as the search model to minimize bias on
the structure determination. The sequence of human PPA1 is much longer
than that of E-PPase (289 residues vs 176 residues). The two sequences
are only marginally homologous (residues 44-194 in human PPA1 matches
residues 16-143 with 27\% identity, 41\% similarity, and 18\% gap). It
is also known that prokaryotic and eukaryotic PPases assume different
oligomerization states in the crystal structures. The fact that the use
of a monomeric E-PPase structure as a search model for MR readily leads
to a clear solution of the structure of human PPA1 indicates that the
PPA1 and E-PPase share highly homologous core sgtructures.

The polypeptide chain of human PPA1 adopts a single domain globular fold
consisting of seventeen \selectlanguage{greek}β-\selectlanguage{english}strands, four \selectlanguage{greek}α-\selectlanguage{english}helices, and four
3\textsubscript{10}-helices (Figures 1 and 2). The core of the globular
fold is composed of a 5-stranded antiparallel \selectlanguage{greek}β-\selectlanguage{english}barrel (strands 5, 11,
13, 14 and 15) and a 5-stranded antiparallel \selectlanguage{greek}β-\selectlanguage{english}sheet (strands 1, 2, 3, 6
and 7) (Figure 2A). The \selectlanguage{greek}β-\selectlanguage{english}barrel and \selectlanguage{greek}β-\selectlanguage{english}sheet are packed closely
together. Other parts of the molecules, including the four \selectlanguage{greek}α-\selectlanguage{english}helices,
four 3\textsubscript{10}-helices, and five short \selectlanguage{greek}β-\selectlanguage{english}strands (strands 4,
8, 9, 10, 12, 16, and 17) surround the \selectlanguage{greek}β-\selectlanguage{english}core structure. A parallel
2-stranded \selectlanguage{greek}β-\selectlanguage{english}sheet formed by the two short strands \selectlanguage{greek}β\selectlanguage{english}10 and \selectlanguage{greek}β\selectlanguage{english}17 helps to
anchor the C-terminus of the polypeptide to the protein fold.

Among the soluble PPases with known structures, human PPA1 share the
highest sequence homology with the PPases from \emph{S. japonicum}
and\emph{S. cerevisiae} (about 70\% similarity, see Figure 1). The
structures of the PPases from these three species are similar.
Superimposition of the monomeric human PPA1 structure to the monomeric
structure of PPase from S. japonicum (PDB code 4QLZ) and S. cerevisiae
(PDB code 2IHP) gave a RMSD of 0.98 \selectlanguage{ngerman}Å (based on 3032 common atoms) and
0.97 Å (based on 2981 common atoms) respectively. Figure 2B shows a
superimposition of the monomeric structures of human PPA1 and Y-PPase.
The two structures superimpose well in most parts of the molecules,
including the active site. Some differences exist in the \selectlanguage{greek}α\selectlanguage{english}4-helix, \selectlanguage{greek}β\selectlanguage{english}8,
\selectlanguage{greek}β\selectlanguage{english}9, \selectlanguage{greek}β\selectlanguage{english}10, \selectlanguage{greek}β\selectlanguage{english}17 regions, as well as some connecting loops. In comparison
with the structure of E-PPase (PDB code 4UM4), the \selectlanguage{greek}β-\selectlanguage{english}barrel, \selectlanguage{greek}β\selectlanguage{english}6, \selectlanguage{greek}β\selectlanguage{english}7,
\selectlanguage{greek}α\selectlanguage{english}1-helix, \selectlanguage{greek}α\selectlanguage{english}2-helix, and the N-terminal portion of \selectlanguage{greek}α\selectlanguage{english}3-helix of human PPA1
have their counterparts in the E-PPase structure (RMSD=2.60 \selectlanguage{ngerman}Å over 640
common atoms). These portions of the structure are common in all known
structures of soluble Family I PPases.

Human PPA1 and other three eukaryotic PPases (Sj-PPase, Y-PPase, and
Pf-PPase) have a C-terminal extension (after the \selectlanguage{greek}α\selectlanguage{english}3-helix) sequence
compared to other Family I PPases (Figure 1). This C-terminal extension
sequence assumes a similar structure in human PPA1, Sj-PPase, and
Y-PPase (Figure 2B), and is involved in the homodimerization of the
PPases (see below). The C-terminal extension in Pf-PPase assumes a
different structure that is not involved in the homodimerization of
Pf-PPase {[}48{]}.

\subsection*{Human PPA1 forms a dimeric structure that is conserved in a
subset of Family I
PPases}

{\label{human-ppa1-forms-a-dimeric-structure-that-is-conserved-in-a-subset-of-family-i-ppases}}

In the crystal, human PPA1 exists as a homodimer. The two protomers in
the homodimer assume a relative orientation that is analogous to an
identical twin standing arm in arm, facing opposite directions (Figures
2C and 2D). Similar homodimers are observed in the structures of PPases
from \emph{S. japonicum} and \emph{S. cerevisiae} (Sj-PPase and Y-PPase,
PDB codes 4QLZ and 2IHP). Formation of the human PPA1 homodimer buries
1860 \selectlanguage{ngerman}Å\textsuperscript{2} of solvent accessible surface area (SASA) from
the two protomers, comparable to those in the Sj-PPase and Y-PPase
homodimers (1830 Å\textsuperscript{2} and 2030
Å\textsuperscript{2}respectively). The three crystals have different
space groups (P21 21 21, P32, and P1 21 1). The presence of similar
homodimers in the structures of the three PPases crystallized in
different space groups and the large buried SASA of dimerization
indicate that formation of the homodimer is unlikely due to crystal
packing artifact.

The homodimerization of human PPA1 is driven by multiple factors
including shape complementarity of the dimerization interface,
hydrophobic contacts, intermolecular hydrogen bonds, and electrostatic
interactions. A large number of residues, including Arg52, Trp53, Asn83,
Phe85, Pro86, Lys88, Ser127, Val129, Asp165, Lys179, Pro180, Gly181,
Tyr182, Ala185, Asp281, Lys282, Trp283, His285, and His286 are involved
in homodimerization (Figures 3 and 4). These residues are scattered in
six different regions within the primary sequence, with the three
regions around Phe85 (strand \selectlanguage{greek}β\selectlanguage{english}10 and the following loop), around Pro180
(linker between \selectlanguage{greek}α\selectlanguage{english}1- \selectlanguage{greek}α\selectlanguage{english}2 helices and n-terminus of \selectlanguage{greek}α\selectlanguage{english}2-helix), and around
Trp283 (3\textsubscript{10} helix \selectlanguage{greek}η\selectlanguage{english}4, strand \selectlanguage{greek}β\selectlanguage{english}17, and the following
loop) having most of the residues (Figure 1). Importantly, residues
within the C-terminal extension play an important role in dimerization
by not only directly participating in the interfacial interactions but
also holding the \selectlanguage{greek}β\selectlanguage{english}10 region in position.

A large portion of the interface molecular surface is defined by the
sidechains of the hydrophobic residues and aliphatic parts of the
sidechains of other residues (white to orange red surface areas in
Figure 4). Hydrophobic contacts in the dimerization interface are
extensive (Figure 3). Some examples include the contacts mediated by
Phe85, Pro86, and Trp283 (Figure 4), which are conserved among the three
eukaryotic PPases (human PPA1, Sj-PPase, and Y-PPase, see Figure 1).

There are several hydrogen bonds at the dimerization interface. Except
the one between the backbone oxygen of Asp281 and sidechain of Arg52,
all other hydrogen bonds are mediated by structured water molecules
(Figure 3). Two electrostatic interactions are observed, between the
sidechains of the Asp165-Lys282 and Asp281-Arg52 pairs. The Asp281-Arg52
pair is conserved in human PPA1, Sj-PPase, and Y-PPase (Figure 1).

Soluble Family I PPases are known to exist in different oligomeric
states. Prokaryotic PPases form hexamers under physiological conditions
{[}37-39, 46, 47, 49{]}. All but one know eukaryotic PPases structures
form dimers {[}40-45, 48{]}. The exception is Tb\textsubscript{b}VSP1,
which forms a tetramer (dimer of dimer) {[}50{]}. The crystal structure
of human PPA1 reveals a dimerization mode that is conserved in the
Sj-PPase and Y-PPase. The C-terminal extensions in these PPases are
critically involved in dimerization. Other eukaryotic PPases either do
not have a C-terminal extension (Tg-PPase and Tb\textsubscript{b}VSP1)
or has a C-terminal extension in different configurations (Pf-PPase).
The dimerization modes in Tg-PPase, Tb\textsubscript{b}VSP1, and
Pf-PPase are different from each other, and different from the conserved
mode among human PPA1, Sj-PPase, and Y-PPase {[}50{]}. The available
crystal structures of eukaryotic PPases show that diverse modes of
dimerization exist in eukaryotic soluble Family I PPases. Previously, it
was proposed (based on sequence conservation) that soluble Family I
PPases from animal and fungi might share a similar mode of dimerization
{[}50{]}. The crystal structure of human PPA1 provides a strong piece of
evidence to support this proposal.

Although it is now generally believed that soluble Family I PPases exist
in a multimeric state, the functional roles of multimerization and its
structural diversity on PPase function, if any, are not known. In the
case of human PPA1 (and the homologous Sj-PPase and Y-PPase),
dimerization places the two active sites (one from each protomer) on
opposite molecular surface of the dimer, far away and isolated from each
other (Figures 2C and 2D). Catalytic reactions at the two active sites
should be independent to each other. Residues involved in dimerization
are also far away from the active site in the monomeric structure,
dimerization should only have allosteric effect, if any, on the active
site. Of course, even if dimerization is not required for the
phosphatase function of human PPA1 under physiological condition, it may
still be relevant to other PPA1 function(s) that is not known currently.

A very outstanding feature of the human PPA1 dimeric structure is the
presence of a large cleft at the dimerization interface (Figure 2D). The
size of the cleft can easily accommodate a 4-turn \selectlanguage{greek}α-\selectlanguage{english}helix. Whether this
cleft represents a functional site of human PPA1 is not known and
deserves further studies. From the perspective of structure-based drug
development suing human PPA1 as a target, the dimerization interface
cleft may serve as a useful allosteric target site. In the case of
Mt-PPase, inhibitors bind in a non-conserved interface between monomers
of the hexameric structure were identified, which block the hydrolysis
reaction in an uncompetitive and allosteric manner {[}36{]}.

\subsection*{Human PPA1 has a largely pre-organized active
site}

{\label{human-ppa1-has-a-largely-pre-organized-active-site}}

Extensive studies had been carried out on the active site structures and
catalytic mechanisms of Y-PPase {[}40-45{]}. These previous knowledges
are critical for analyzing the active site of human PPA1 in the current
structure.

The human PPA1 structure does not contain the substrate at the putative
active site. To gain insights into how the speculative active site
residues (inferred from structure-based sequence alignment with Y-PPase)
orient in relative to the pyrophosphate substrate, the substrate was
modeled into the human PPA1 structure by superimposition of the
structure with a substrate-bound, fluoride-inhibited Y-PPase structure
(PDB code 1RE6A) {[}45{]}. The overall structures superimposed well with
a small RMSD of 0.98 \selectlanguage{ngerman}Å (Figure 2B). The portions of structures that
define the active site show only very slight difference in backbone and
sidechain conformations (Supplementary Figure S1). The superimposed
coordinates of the pyrophosphate were merged with the PPA1 coordinates
to generate the structure of PPA1 with a pyrophosphate at the active
site.

Figure 5A shows the active site structure of human PPA1 with a modeled
pyrophosphate substrate. The 14 human PPA1 active site residues
(matching the established active site residues in Y-PPase) are Glu49,
Lys57, Glu59, Arg79, Tyr94, Gly95, Asp116, Asp118, Asp121, Asp148,
Asp153, Lys155, Tyr193, Lys194 (Y-PPase residue numbers are one less
than the shown human PPA1 residue numbers). Most of these residues
locate in the \selectlanguage{greek}β-\selectlanguage{english}barrel and the adjacent \selectlanguage{greek}β\selectlanguage{english}6-strand, which are well
conserved and defined in all structures of soluble Family I PPases.
Tyr193 and Lys194 make the transition from the 3\textsubscript{10}-helix
\selectlanguage{greek}η\selectlanguage{english}2 to the \selectlanguage{greek}α\selectlanguage{english}2-helix. The active side residues can divided into two groups
based on relative spatial relationship to the pyrophosphate substrate.
The first group consists of all of the negatively charged residues and
Gly95. These residues cluster on one side of the active site, close to
the P2 phosphorus atom. The second group includes all of the positively
charged residues and the two tyrosines. These residues largely locate on
the other side of the active site, close to the P1 phosphorus atom
(Figure 5A). Studies on Y-PPase reveal that the active site carboxylates
are responsible for coordinating with 4 metal ions and activating a
water molecule as the reaction nucleophile, while the positively charged
sidechains stabilize the transition state and leaving group {[}40-45{]}.

Due to the fact that most of the active site residues are located in the
rigid core of the structure, their positions (locations of the backbone
atoms) should have little changes upon substrate binding and during the
reaction. Of course, conformations of the sidechains (especially the
longer sidechains of lysine and arginine residues) could be changed.
Comparison of the apo-structure of human PPA1 and the substrate-bound,
fluoride-inhibited Y-PPase structure shows that the active site residues
superimpose well with some conformation differences in the sidechains of
Lys57, Arg79, and Lys194 (Supplementary Figure S1). Low B-factor values
for residues at the active site also suggest relatively lower
flexibility of these residues (Figure 5B). These data and analysis
indicates that the active site of human PPA1 is largely pre-organized,
which would minimize the need for conformational reorganization during
catalysis.

The active site of human PPA1 has the potential to accommodate
double-phosphorylated peptides from JNK1

{\label{the-active-site-of-human-ppa1-has-the-potential-to-accommodate-double-phosphorylated-peptides-from-jnk1}}

While it is well established that PPases catalyze the hydrolysis of
pyrophosphate, several recent studies reveal that PPA1 may also function
as a protein phosphatase {[}11, 22, 61{]}. It was found that human PPA1
could directly dephosphorylate phosphorylated JNK1 in both
phosphor-peptide and phosphor-protein levels, while no catalytic
activity towards pERK or p-p38 was detected {[}22{]}. PPA1-silencing
significantly down-regulated colon cancer cell proliferation. This
antiproliferation effect is impaired by JNK inhibitor, indicating that
the role of PPA1 in colon cancer is at least partially related to
regulation of JNK activity. Evidence for the function of PPA1 as a pJNK
phosphatase was also obtained in studies on neuronal differentiation in
mouse, rat, and chick embryo {[}11, 61{]}. It was shown that PPA1
knockdown or overexpression led to increased or decreased JNK
phosphorylation level, while no alteration of JNK phosphorylation level
was detected after treatment with a catalytically inactive PPA1 mutant.
PPA1 may play a role in neuronal differentiation via JNK
dephosphorylation. JNK is a member of the mitogen-activated protein
(MAP) kinase family. JNK regulates the activity of numerous downstream
molecules, including c-Jun, p53, and Bcl2, by phosphorylation. JNK is
activated by a dual phosphorylation of Thr183 and Tyr185 within a
180-FMMTPYVV motif. Dephosphorylation of JNK by protein phosphatases
inactivates the enzyme {[}62{]}.

So far, structural and mechanistic studies on soluble Family I PPases
only concerned inorganic pyrophosphate as the substrate. Given the
emerging role of PPA1 as a JNK phosphatase, we carried out modeling
studies to investigate whether the known pyrophosphate binding active
site could also accommodate phosphor-peptides.

Four structural models were constructed, each with a short
phosphor-peptide bound at the known pyrophosphate binding active site.
The phosphorus atom of the phosphortyrosine or phosphorthreonine residue
within the phosphor-peptide is located at the same position as the P2
phosphorus atom of a pyrophosphate substrate. The sequences of the four
phosphor-peptides match either JNK1 or the MAP kinase Erk2. A
JNK1-derived pentapeptide 182-MTpPYpV was used to model the Yp at the
active site (Figure 6A). A JNK1- derived hexapeptide 181-MMTpPYpV was
used to model the Tp at the active site (Figure 6B). Two Erk2-derived
tetrapeptides, 184-EYpVA and 181-FLTpE, were used to model the Yp and
the Tp at the active site respectively (Supplementary Figure S2).

All of the phosphor-peptides can be accommodated in the active site
pocket, without steric crash between the phosphor-peptide and PPA1. The
PPA1 structures with a bound phosphor-peptide showed only very minor
conformational changes (during energy minimization in Chimera) for
residues on the outer portion of the active site pocket.
Superimpositions of the phosphor-peptide bound PPA1 structures with the
apo-structure give RMSD values less than 0.2 \selectlanguage{ngerman}Å. The (putative) active
site of human PPA1 that would bind the pyrophosphate substrate is
located at the inner and deep portion of a cavity (Figure 5B. The
location of the active site is indicated by the black arrow. The cavity
is indicated by the white oval). The outer portion of the cavity becomes
much larger, expanding downward and leftward while getting shallower.

The pyrophosphate binding site is mostly defined by residues from the
inner \selectlanguage{greek}β-\selectlanguage{english}core and therefore relatively rigid and pre-organized. The outer
portion of the cavity is defined by residues from peripheral structures,
including the strands \selectlanguage{greek}β\selectlanguage{english}8 and \selectlanguage{greek}β\selectlanguage{english}9, the 3\textsubscript{10} helix \selectlanguage{greek}η\selectlanguage{english}2 , the
loop connecting \selectlanguage{greek}β\selectlanguage{english}6 and \selectlanguage{greek}β\selectlanguage{english}7, and the loop connecting \selectlanguage{greek}β\selectlanguage{english}12 and \selectlanguage{greek}β\selectlanguage{english}13 (Figure
5A). Since many of these residues are surface exposed, their sidechains
(especially the longer sidechains in Lys63, Lys74, Lys75, Glu149,
Glu151, and Lys199) are expected to have large flexibilities. The
B-factor surface rendering may also reflect the different flexibilities
of atoms in different areas of the cavity (Figure 5B). These properties
of the cavity explain how the cavity can accommodate various
phosphor-peptides in the modeling studies. While the phosphor-residue is
reaching into the inner (and deeper) portion of the cavity, the flanking
residues are accommodated by the outer, shallower, and more flexible
portion of the cavity.

These modeling results indicate that the active site pocket known for
pyrophosphate binding and catalysis has the potential to accommodate
various phosphor-peptides with single- or double-site phosphorylation.
It should be pointed out that the models only minimized steric crash
while keeping good geometry of the peptides and protein. No effort was
made to optimize the inter-molecular interactions (such as hydrogen
bonds, hydrophobic contacts, electrostatic interaction, etc). When these
interactions come into play in reality, the PPA1 active site pocket
might have substrate specificity for certain phosphor-peptides. Of note,
the six charged residues Lys63, Lys74, Lys75, Glu149, Glu151, and Lys199
are placed in different areas in the pocket (in Figure 5B, Lys 63 at
bottom left, Lys74 and Lys75 at top left, Lys199 at top right, Glu149
and Glu151 on the left). The peculiar distribution of charged residues
may have implications in peptide substrate specificity. Of course, the
exact structural details of peptide substrate recognition by human PPA1
can only be revealed by high resolution structures of PPA1 in complex
with phosphor-peptide or phosphor-protein substrates. It is known that
human PPA1 can directly dephosphorylate pJNK1 but cannot dephosphorylate
pERK or p-p38 {[}22{]}. Structures of the PPA1-pJNK1 complexes are
needed to reveal the molecular basis of this substrate specificity. The
structures will also be very helpful for the development of peptide or
peptide-like inhibitors of human PPA1.

\section*{Conclusion}

{\label{conclusion}}

Prior to our study, many crystal structures of soluble Family I PPases
from lower organisms are available. But no structural study on PPases
from higher organisms has been reported. Given the emerging role of
human PPA1 in tumorigenesis and the potential value of human PPA1 as a
target for anti-cancer drug development, it becomes essential to obtain
an atomic resolution structure of human PPA1. Such a structure is
presented in the current study. The structure, in conjunction with
results from modeling studies and previous knowledge on PPase
structures, sheds new insights into the mechanism of PPA1 functions. The
structure also provides the basis for structure-based drug discovery by
using \emph{in silico} virtual screening of small molecule compound
libraries, which is being carried out in our lab.

\textbf{Data Availability}

The atomic coordinates and diffraction data for the structures used to
support the findings of this study have been deposited in the Protein
Data Bank with accession code 6C45.

\textbf{Conflicts of Interest}

The author(s) declare(s) that there is no conflict of interest regarding
the publication of this paper.

\textbf{Funding Statement}

The work is supported by grant from The Tianjin First Central Hospital
of China (yuanCM201810).

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\textbf{Tables}

\textbf{Table 1. Data collection and refinement statistics.}\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Data collection & Native\tabularnewline
\midrule
\endhead
Space group & P21 21 21\tabularnewline
Unit-cell parameters &\tabularnewline
a (\selectlanguage{ngerman}Å ) & a = 50.27\tabularnewline
b (\selectlanguage{ngerman}Å ) & b = 137.04\tabularnewline
c (\selectlanguage{ngerman}Å ) & c = 216.44\tabularnewline
\selectlanguage{greek}α \selectlanguage{english}(\selectlanguage{ngerman}°) & 90\tabularnewline
\selectlanguage{greek}β \selectlanguage{english}(\selectlanguage{ngerman}°) & 90\tabularnewline
\selectlanguage{greek}γ\selectlanguage{english}(\selectlanguage{ngerman}°) & 90\tabularnewline
Resolution (\selectlanguage{ngerman}Å ) & 84.94--2.39\tabularnewline
No. of unique reflections & 60418\tabularnewline
Completeness (\%)\textsuperscript{a} & 99.9 (98.9)\tabularnewline
R\textsubscript{merge} (\%)\textsuperscript{b} & 11.8\tabularnewline
\textless{}I/\selectlanguage{greek}σ\selectlanguage{english}(I)\textgreater{} & 8.1\tabularnewline
CC\textsubscript{1/2} & 0.99\tabularnewline
Multiplicity & 4.5\tabularnewline
Anomalous completeness (\%) & N/A\tabularnewline
Anomalous multiplicity & N/A\tabularnewline
Refinement &\tabularnewline
Resolution (\selectlanguage{ngerman}Å ) & 84.94--2.39\tabularnewline
No. of reflections & 60418\tabularnewline
R\textsubscript{work}/R\textsubscript{free}++ (\%)\textsuperscript{c} &
19.3/25.1\tabularnewline
No. of atoms &\tabularnewline
Protein & 1135\tabularnewline
Water & 526\tabularnewline
B factors (\selectlanguage{ngerman}Å \textsuperscript{2}) &\tabularnewline
Protein & 39.19\tabularnewline
Water & 38.6\tabularnewline
R.m.s.d., bonds (\selectlanguage{ngerman}Å ) & 0.008\tabularnewline
R.m.s.d., angles (\selectlanguage{ngerman}°) & 1.248\tabularnewline
Ramachandran plot &\tabularnewline
Favored (\%) & 96.6\tabularnewline
Outliers (\%) & 0 .7\tabularnewline
\bottomrule
\end{longtable}

\textsuperscript{a}Values in parentheses are for the highest-resolution
shell

\textsuperscript{b}\emph{R} \textsubscript{merge} =
\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{hkl}}
\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{i}}\textbar{}\emph{I\textsubscript{i}} (\emph{hkl}
) - \textless{}\emph{I} (\emph{hkl} )\textgreater{} \textbar{}
/\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{hkl}} \selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{i}I\textsubscript{i}}
(\emph{hkl} ), where \textless{}\emph{I} (\emph{hkl} )\textgreater{} is
the average intensity ofreflection \emph{hkl} .

\textsuperscript{c}\emph{R} \textsubscript{work} =
\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{hkl}}|\textbar{}\emph{F}
\textsubscript{obs}\emph{\textbar{}} -\emph{\textbar{}F}
\textsubscript{calc}\emph{\textbar{}} |
/\selectlanguage{greek}Σ\selectlanguage{english}\emph{\textsubscript{hkl}\textbar{}F}
\textsubscript{obs}\emph{\textbar{}} , where \emph{F}
\textsubscript{obs} and \emph{F} \textsubscript{calc} are the observed
and calculated structure factors respectively.\emph{R}
\textsubscript{free} is calculated as for\emph{R} \textsubscript{work}
but only use a randomly selected subset of data (6\%) which were
excluded from refinement.

\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Sequence alignment of eukaryotic PPases with known
structures.} The secondary structures present in human PPA1 are
indicated above the human PPA1 sequence, with \selectlanguage{greek}α, η, \selectlanguage{english}and \selectlanguage{greek}β \selectlanguage{english}for \selectlanguage{greek}α \selectlanguage{english}helix,
3\textsubscript{10} helix, and \selectlanguage{greek}β \selectlanguage{english}strand respectively. Numbering is for
the human PPA1 sequence. Residues involved in human PPA1 dimerization
are indicated with a red italic letter \emph{i} above the sequence.
Active site residues important for catalysis in the yeast PPase are
indicated by a filled triangle. The sequence of \emph{P.
falciparum}PPase has a N-terminal extension of 76 residues (not shown).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Overall structure of the human PPA1 protein.} A) The
single domain monomeric structure. B) Superimposition of the monomeric
structures of human PPA1 (magenta) and yeast PPase (blue, PDB code
1E6A). C) The dimeric structure of human PPA1 in two different viewing
angles. The two monomers are colored cyan and magenta respectively. D)
Electrostatic surface representation of~the human PPA1 homodimeric
structures. The surface areas are colored according to electrostatic
potentials, from blue for the most positive, to white, to red for the
most negative regions.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Ligplot presentation of molecular interactions in the
human PPA1 dimerization interface.} Hydrogen bonds are designated with
green dashed lines (the distance between the two heavy atoms is
indicated by a number in angstrom). Hydrophobic interactions are
represented as starbursts.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. A graphic view of the human PPA1 homodimerization
interface.} The two protomers are rendered differently in surface mode
and cartoon mode respectively. The surface areas are colored according
to amino acid hydrophobicity on the Kyte-Doolittle scale, from dodger
blue for the most hydrophilic, to white, to orange red for the most
hydrophobic. The side-chains for some of the key residues belonging to
the protomer rendered in cartoon mode are shown in sticks. Hydrogen
bonds are represented as yellow sticks. Structured water molecules are
shown as spheres.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. The putative active site of human PPA1.} A) The active
site with a modeled pyrophosphate (rendered by transparent surface mode
in yellow). The side-chains are shown in stick mode (colored by atom
type) for residues corresponding to the active site catalytic residues
in yeast PPase. B) The active site pocket on the surface of PPA1. The
surface is colored according to B factor values of the atoms.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6. Structural models of the human PPA1 active site with
potential peptide substrates containing a phosphotyrosine and a
phosphothreonine.} A) A phosphotyrosine (p-Y185), within the context of
a pentapeptide 182-MT\textsuperscript{p}PY\textsuperscript{p}V, is bound
at the active site. B) A phosphothreonine (p-T183), within the context
of a hexapeptide 181-MMT\textsuperscript{p}PY\textsuperscript{p}V, is
bound at the active site. The phosphorylated peptides are rendered in
stick mode and space-filling mode in the two panels of the figures.

\selectlanguage{english}
\FloatBarrier
\end{document}

