A Crystallographic Structure-Function Analysis of SH2
Domain Containing Inositol Polyphosphate 5-Phosphatase
2 (SHIP2)
Abstract:_______________________________________________________________________________
___
SH2 domain containing inositol polyphosphate 5-phosphatases (SHIPs) are Mg 2+ dependent
phosphatase enzymes involved in the regulation of phosphoinositide signalling pathways. SHIPs
are responsible for cleaving the 5-phosphate of phosphatidylinositol(3,4,5)P 3, a phospholipid
involved in a variety of proliferative signal events. Recent studies have indicated the potential of
SHIPs as therapeutic targets for multiple diseases such as cancer, obesity, and diabetes.
However, the poor bioavailability of current SHIP inhibitors has troubled their clinical use. To
facilitate the rational design of clinically viable inhibitors, high resolution crystal structures of the
enzyme and in-depth knowledge of enzyme mechanism are required. In this paper, a recent
crystal structure of the enzyme SHIP2 is analyzed using various computational techniques to gain
greater understanding of the underlying structure-function relationship of SHIPs, and to
determine the accuracy of current crystal structure evidence.
Introduction:___________________________________________________________________________
____
Phosphoinositides (PtdIns) are a large family of phospholipids
ubiquitous to organismal cell membranes. The compounds consist of a
glycerol backbone linking two polyacyl lipid chains and a six carbon
inositol sugar. The inositiol sugar exists in nine possible stereoisomeric
forms common and may be phosphorylated on any of the five free
alcohol substituents (Figure 1). Phosphoinositides play a major role in
various intracellular signaling processes. The phosphoinositide
PtdIns(4,5)P2, or PIP2, which is phosphorylated at the 4 and 5 positions
of the inositol ring, is a substrate for phospholipase-C (PLC), which is
activated by stimulation of various G-protein coupled receptors.
Activated PLC cleaves PtdIns(4,5)P2 into inositol triphosphate (IP3) and
diacylglycerol (DAG). Inositol triphosphate is known to release
intracellular Ca2+ through its interaction with the IP3 receptor on the
endoplasmic reticulum, leading to various cellular responses 1. In
addition to the IP3/DAG signalling pathway, PIP2 plays a crucial role in
other signaling pathways. Certain receptor tyrosine kinases such as the
epidermal growth factor receptor (EGFR) stimulate activation of
phosphoinositide 3-kinase (PI3K), which phosphorylates PIP 2 at the 3
position to produce PtdIns(3,4,5)P3, or PIP3 . PIP3 has the potential to
recruit various downstream signal effectors through interaction with
protein PH domains. A highly important effector in this process is
protein kinase B (Akt) which is recruited to the cell membrane
through interaction with PIP3 and is activated upon phosphorylation Figure 1: The
by protein dependent kinase (PDK)2. Activation of Akt induces a generalized structure of
variety of proliferative and anti-apoptotic events, and over activity of phopshoinositide
this protein has been implicated in a variety of malignancies such as molecules, containing:
multiple myeloma and diabetes mellitus type II. two polyacyl chains (A),
a glycerol backbone (B),
Due to the therapeutic importance of PIP3, current research is aimed a bridging phosphate
at understanding the process of in vivo PIP3 degradation. Currently (C), and an inositol
there are two known enzymes which to degrade PIP 3, the 3- sugar (D). The carbons
phosphatase PTEN and the 5-phosphatase SHIP. The two enzymes of the inositol ring are
degrade PIP3, but their unique mechanisms produce different products
and, as a result, divergent signal pathways. While PTEN reproduces PtdIns(4,5)P 2, SHIP produces
the isomer PtdIns(3,4)P2. As both proteins degrade PIP 3, both proteins were initially implicated as
tumor suppressors. The tumor suppressor activity of PTEN is well characterized 3; however, recent
studies have shown that inhibition of SHIP induces apoptosis in cancer cell lines 4. Based upon
these recent discoveries, it is now commonly believed that SHIPs play a major role in cancer cell
proliferation, and inhibition of this enzyme is a major target for therapeutic intervention. In order
to develop suitable inhibitors for SHIP, high resolution crystal structures alongside in depth
knowledge of enzyme mechanism are required.
SHIP2 is a 155kDa multidomain protein
ubiquitously expressed in human cell
lines. The protein possesses three
domains crucial to function, an SH2
homology domain, a 5-phosphatase
domain, and a SAM domain5. Upon
suitable growth factor stimulation, SHIP2
is recruited to active receptors through
SH2 domain interactions and is
phosphorylated at Ser-132, Thr-1254, and
Ser-1258, inducing enzyme activity6.
SHIP2 then binds to PIP3 through plekstrin
homology (PH) domain interactions and
catalyzes dephosphorylation to produce
PtdIns(3,4)P2. Experiments by Vandeput
et al. determined biphenyl 2,3,4,5,6-
pentakisphosphate as a potent
competitive SHIP2 inhibitor7. Using Figure 3: The hydrogen bonding, and electrostatic
this knowledge Mills et al. produced interactions of the inhibitor biphenyl 2,3,4,5,6-
the first crystal structure of SHIP2 in pentakisphosphate bound to the active site of SHIP2. The
complex with the inhibitor. From this Inhibitor binds to the surface of the enzyme interacting
experiment the active site of SHIP2 with a large amount of solvent exposure to the back side.
and its major catalytic residues were Hydrogen bonding interactions with Ser and Lys residues
identified8. as well as salt bridges with multiple Asn residues
stabilizes the complex. Bp-2,3,4,5,6-5P inhibits SHIP
The major hydrogen bond and salt activity by blocking substrate access to the catalytic His,
bridge interactions of the protein-
inhibitor complex are shown in figure 3. The crystal structure shows an active site localized to the
protein surface with several positively charged arginine and lysine residues interacting with the
negatively charged phosphate groups. There is no current consensus on the exact catalytic
mechanism of 5-phosphatases; however, bioinformatic studies discovered strong sequence
homology between 5-phosphatases such as SHIP2 and with AP-endonucleases involved in DNA
base-excision repair9. This sequence homology implies a conserved enzymatic mechanism
between the two families. Based on this homology the catalytic mechanism of SHIP is assumed
to be conferred by active site His and Asp residues. The His residue alongside other positively
charged amino acids interact with the 5-phosphate of the lipid while the active site Asp
deprotonates a water molecule for nucleophilic attack at phosphorus. The coordination of
inositol bound oxygen to Mg 2+ polarizes the bond allowing expulsion of the phosphate group
(Figure 4).
Figure 4: A generalized mechanism of 5-phosphatase activity. Positively charged His and Asn
residues hold the 5-phoshate in place, while the active site Asp residue deprotonates water for
nucleophilic attack at phosphorus. Coordination of oxygen to Mg 2+ polarizes the P-O bond, allowing
cleavage and expulsion of the two products. This mechanism is theorized from sequence
homology to the AP endonuclease protein family.
From the information published by Mills et al., an analysis of the current SHIP2 crystal structure
was carried out, including a description of poorly-defined electron densities, strained bond
angles, and side-chain rotamer outliers. This information is presented below.
Results:________________________________________________________________________________
___
The asymmetric unit of SHIP2, identified by the initial crystal structure, was determine to be a
dimer (Figure 5). However, as the asymmetric unit showed only one protein forming an inhibitor
complex, it was likely that the biological unit existed in a monomeric form.
a) b)
Figure 4: (A) The asymmetric unit of SHIP2 in complex with biphenyl 2,3,4,5,6-
pentakisphosphate. The asymmetric unit presents as a dimer. However, as only one protein is
inhibitor bound, the biological unit is likely a monomer (B).
Thermodynamic analysis of the biological unit of SHIP2 using the PDBePISA assembly analysis
provided further evidence that no stable quaternary structures form in solution, and the protein
acts as a monomer. The thermodynamic data of possible biological assemblies is shown in Table
1. The table shows a single favorable interface interaction between a single SHIP2 protein and a
single inhibitor ligand.
Table 1: Thermodynamic analysis of the potential biological unit content of SHIP2. The free energy
gained upon solvation (iG) indicates the thermodynamic favorability of complex formation. From
the information it is indicated that formation of a SHIP2 dimer is thermodynamically unfavorable;
however, inhibitor binding is favorable. The CSS score, which is valued from 0 to 1, indicates the
Following determination of biological unit content, the crystal structure and electron density
maps were analyzed to determine strained bond angles, side-chain rotamer outliers, and areas of
poorly defined electron density. Using Molprobity, a Ramanchandran plot was created (Figure 5).
The plot shows Trp-506 as the single outlier. Molprobity also showed a total of 17 serious steric
clashes, and a total of 4 rotamer outliers.
To further analyze steric clashes and rotamer outliers WinCoot was used to observe the single
difference and double difference Fourier transform electron density maps. Based on single
difference Fourier maps calculating positive and negative electron density, several areas of
poorly defined density were identified. Multiple amino acid residues, such as Glu-726, possessed
incomplete atom counts. Many amino acid residues with incomplete atom counts are in areas of
no positive electron density, implying a weak electron density fit. Furthermore, analysis of
unusual rotamers indicated a large portion of amino acid residues with low probability (<10%) of
Buried iG
Interacting # of H- # of Salt # of Disulfide
Surface Area (kcal/m CSS
Structures bonds Bridges Bridges
(2) ol)
SHIP2 +
303.7 -6.7 13 0 0 0.1
Ligand
SHIP2 +
167.8 1 1 0 0 0
SHIP2
residing in the corresponding electron density. In addition to these observations, the protein
Figure 5: A Ramachandran plot of all amino
acid residues in SHIP2. The single outlier is
Trp-506, which possesses and angles of
59.4 and 94.2 respectively.
appears to have multiple breaks in the polypeptide chain, resulting in several C and N-termini.
Discussion:
_______________________________________________________________________________________
Based on calculations of the free
energy of solvation it is presumed
that SHIP2 acts as a monomer
during catalysis. This is supported
by the research from Mills et al.
Binding of the inhibitor biphenyl
2,3,4,5,6-pentakisphosphate to
SHIP2 buries a total of 303 2
surface area, including the residues
Asn-432, Asp-607, and His-718. As
these three residues are located
near the inhibitor binding site and in
close proximity to one another they
are likely the catalytic residue triad
involved in 5-phosphatase activity,
acting through a mechanism
analogous to the AP endonuclease
family of proteins. The residues
remain unchanged in the bound an
unbound forms. It is therefore Figure 6: Rotamer and electron density fit analysis using
proposed that the inhibitor acts WinCoot identified several areas of poorly defined electron
competitively by blocking substrate density. Multiple amino acid residues possessed truncated
access to the active site. side-chains such as the glutamate above. Many of these
residues reside near areas of no electron density, implying
Analysis of the electron density of that completion of the side chain would result in negative
SHIP2 using WinCoot was possible electron density. In addition many of the residues possess a
through the use of both double- relatively low probability of existing in their current positions
difference and single-difference Fourier transform maps. The single-difference Fourier transform
(Fobs-Fcalc, calc) where F and are the amplitudes and phases of the diffracted x-rays respectively
is used to compare the true structure against the currently modeled structure, misattributed
electron density shows as either positive or negative, indicating the extent of error in the model
structure11. The evidence provided by Molprobity and the electron density calculations by
WinCoot casts doubt on the accuracy of the current crystal structure model for SHIP2. Multiple
amino acid residues are truncated and possess missing side chain atoms (Figure 6); it is possible
that addition of these atoms into the protein structure with likely further distort the density fit
analysis. Furthermore, the current protein structure model possesses multiple C and N termini.
Biological assembly analysis from PDBePISA determined the only likely formation to be a
monomer, indicating a major inconsistency in the crystal structure data.
From the data presented above, it is evident that crystallographic studies of SHIP2 and their
inhibitors promise to further elucidate the structure and mechanism of 5-phosphatase enyzmes.
However, current crystal structure data provides, at best, a beginning to this research. In future
higher resolution structures are required to gain accurate data on SHIP2 structure and function.
Research on the topic has the potential to drive development of competitive and mechanistic
inhibitors of SHIP activity for the treatment of various ailments.
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