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 this knowledge
Mills et al. produced the first crystal structure of
SHIP2 in complex with the inhibitor. From this Figure 3: The hydrogen bonding, and electrostatic interactions of the
experiment the active site of SHIP2 and its inhibitor biphenyl 2,3,4,5,6-pentakisphosphate bound to the active
major catalytic residues were identified8. site of SHIP2. The Inhibitor binds to the surface of the enzyme
interacting with a large amount of solvent exposure to the back side.
The major hydrogen bond and salt bridge Hydrogen bonding interactions with Ser and Lys residues as well as salt
interactions of the protein-inhibitor complex bridges with multiple Asn residues stabilizes the complex. Bp-
are shown in figure 3. The crystal structure 2,3,4,5,6-5P inhibits SHIP activity by blocking substrate access to the
shows an active site localized to the protein catalytic His, Asp, Asn triad buried further down in the active site.
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 Mg2+ 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 Mg2+ 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 shown as 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 is likely 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 likelihood of complex formation as a fraction of 1.
Interacting Buried Surface iG # of Salt # of Disulfide
# of H-bonds CSS
Structures Area (2) (kcal/mol) Bridges Bridges
SHIP2 + Ligand 303.7 -6.7 13 0 0 0.1
SHIP2 + SHIP2 167.8 1 1 0 0 0

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 showing outliers (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. Other 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 residing in the corresponding electron density. In
addition to these observations, the protein appears to have multiple breaks in the polypeptide chain, resulting in several
C and N-termini.
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.

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 within close proximity to
each other 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
proposed that the inhibitor acts
competitively by blocking substrate access
to the active site. Figure 6: Rotamer and electron density fit analysis using WinCoot identified
several areas of poorly defined electron density. Multiple amino acid
Analysis of the electron density of SHIP2 residues possessed truncated side-chains such as the glutamate above.
using WinCoot was possible through the use Many of these residues reside near areas of no electron density, implying
of both double-difference and single- that completion of the side chain would result in negative electron density.
difference Fourier transform maps. The In addition many of the residues possess a relatively low probability of
single-difference Fourier transform (Fobs- existing in their current positions according to rotamer fit analysis.
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.