Results and
Discussion
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 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 β-strands, four α-helices, and four
310-helices (Figures 1 and 2). The core of the globular
fold is composed of a 5-stranded antiparallel β-barrel (strands 5, 11,
13, 14 and 15) and a 5-stranded antiparallel β-sheet (strands 1, 2, 3, 6
and 7) (Figure 2A). The β-barrel and β-sheet are packed closely
together. Other parts of the molecules, including the four α-helices,
four 310-helices, and five short β-strands (strands 4,
8, 9, 10, 12, 16, and 17) surround the β-core structure. A parallel
2-stranded β-sheet formed by the two short strands β10 and β17 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 S. japonicum andS. 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 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 α4-helix, β8,
β9, β10, β17 regions, as well as some connecting loops. In comparison
with the structure of E-PPase (PDB code 4UM4), the β-barrel, β6, β7,
α1-helix, α2-helix, and the N-terminal portion of α3-helix of human PPA1
have their counterparts in the E-PPase structure (RMSD=2.60 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 α3-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].
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 S. japonicum and S. cerevisiae (Sj-PPase and Y-PPase,
PDB codes 4QLZ and 2IHP). Formation of the human PPA1 homodimer buries
1860 Å2 of solvent accessible surface area (SASA) from
the two protomers, comparable to those in the Sj-PPase and Y-PPase
homodimers (1830 Å2 and 2030 Å2respectively). 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 β10 and the following loop), around Pro180
(linker between α1- α2 helices and n-terminus of α2-helix), and around
Trp283 (310 helix η4, strand β17, 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 β10 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 TbbVSP1,
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 TbbVSP1)
or has a C-terminal extension in different configurations (Pf-PPase).
The dimerization modes in Tg-PPase, TbbVSP1, 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 α-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].
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 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 β-barrel and the adjacent β6-strand, which are well
conserved and defined in all structures of soluble Family I PPases.
Tyr193 and Lys194 make the transition from the 310-helix
η2 to the α2-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
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 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 β-core and therefore relatively rigid and pre-organized. The outer
portion of the cavity is defined by residues from peripheral structures,
including the strands β8 and β9, the 310 helix η2 , the
loop connecting β6 and β7, and the loop connecting β12 and β13 (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.