Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity
Crystal Structure of Human Myotubularin- Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity
Seoung Min Bong 0 1
Kka-bi Son 1
Seung-Won Yang 0 1
Jae-Won Park 0 1
Jea-Won Cho 0 1
Kyung-Tae Kim 0 1
Hackyoung Kim 1
Seung Jun Kim 1
Young Jun Kim 1
Byung Il Lee 0 1
0 Research institute, National Cancer Center , Goyang, Gyeonggi 10408 , Republic of Korea, 2 Department of Biomedical Chemistry, Konkuk University , Chungju 27478 , Republic of Korea, 3 Medical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology , Daejeon 34141 , Republic of Korea
1 Editor: Wenqing Xu, University of Washington , UNITED STATES
Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dualspecificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)5R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P2 over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.
Phosphatidylinositol polyphosphates (PIs; also called phosphoinositides) regulate diverse
cellular functions, including membrane trafficking, cell growth/survival, cell division, and cellular
]. The formation of seven different PIs is regulated by various kinases and
phosphatases, including phosphatase and tensin homolog (PTEN), phosphatidylinositol-3-kinases
(PI3Ks), phosphatidylinositol phosphate kinases (PIPKs), and myotubularin-related (MTMR)
phospholipid phosphatases [
]. Given the cellular functions of PIs, it is not surprising that PI
dysregulation is associated with human diseases. Many mutations have been found in the
kinases and phosphatases involved in PI interconversions [
]. For example, mutations in
inositol 5-phosphatase (OCRL) are associated with Lowe Syndrome and Dent Disease [
an inactivating mutation in PIP kinase type 1γ (PIPKIγ) is associated with Lethal Contractural
Syndrome Type 3 (LCCS3) , and mutations in Fab 1, YOTB, Vac 1, and EEA (FYVE)
finger-containing phosphoinositide kinase (PIKFyve) are associated with François-Neetens
(http://www.ncc.re.kr) was funded by Ministry of
Health and Welfare (http://www.mohw.go.kr).
Mouchetée Fleck Corneal Dystrophy [
]. Moreover, inactivating mutations of PTEN and
activating mutations of PI3Ks have been identified in various cancers because of their association
with enhanced PI(3,4,5)P3 signaling [
]. Mutations in the myotubularin (MTM) family are
also associated with human diseases. For instance, mutations in the MTM1 gene cause
Xlinked centronuclear myopathy and myotubular myopathy [
]. Additionally, mutations in
myotubularin-related protein 2 (MTMR2) and MTMR13 cause Charcot-Marie-Tooth Disease
MTMRs are part of a large group of tyrosine/dual-specificity phosphatase superfamily
(PTP/DUSP) members in eukaryotes [
]. They are the primary enzymes involved in PI
metabolism. The human MTMR family comprises 14 members. The eight MTMRs (MTM1,
MTMR1–4, and MTMR6–8) that share the C(X)5R motif are catalytically active, and the other
six MTMRs (MTMR5 and MTMR9–13), which do not possess the C(X)5R motif, are inactive
]. The inactive MTMRs interact with the active MTMRs and regulate the
enzyme activity, subcellular localization, and substrate specificity of the active MTMRs [
]. Typically, the active MTMRs catalyze the dephosphorylation of phosphatidylinositol
3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) to
produce PI and PI(5)P, respectively [
]. Most MTMRs possess Pleckstrin homology
(PH)GRAM and coiled-coil (CC) domains in addition to the phosphatase (PTP) domain, and some
MTMRs contain extra domains, such as differentially expressed in normal and neoplastic cells
(DENN), PH, and FYVE domains [
]. The PH-GRAM domain has been predicted to function
in intracellular protein-protein or lipid-binding interactions [
1, 16, 17, 21, 25
]. The CC domain
mediates interactions between MTMRs and enables the homo- and/or heterodimerization of
]. Human MTMR1, which is located on the X chromosome (similar to
MTM1), was the first identified MTMR . MTMR1 is associated with myotonic dystrophy
and the efficient dephosphorylation of PI(3)P and PI(3,5)P2 in vitro [
MTMR1 was one of the earliest identified MTMRs, it is a relatively poorly studied protein. The
crystal structures of MTMR2 , MTMR6 (Structural Genomics Consortium, unpublished
work), and MTMR8 [
] provided detailed information regarding their substrate specificity
and regulatory mechanisms based on their dimerization, protein-protein interactions and
membrane binding. Thus, determining the 3-dimensional structure of MTMR1 would be
indispensable for understanding the structural basis of its substrate specificity and regulatory
mechanism. These biochemical data for MTMR1 could reveal the cellular mechanism
underlying myotonic dystrophy.
In this study, we determined the crystal structure of the PH-GRAM and active PTP domains
of human MTMR1 and investigated its substrate specificity through enzymatic and
mutagenesis assays. We also determined the structural aspects of substrate recognition, the catalytic
mechanism, and the regulatory mechanism of human MTMR1.
Materials and Methods
Di-C8 phosphatidylinositol 3-phosphate (di-C8 PI(3)P), di-C8 phosphatidylinositol
4-phosphate (di-C8 PI(4)P), di-C8 phosphatidylinositol 3,4-bisphosphate (di-C8 PI(3,4)P2), di-C8
phosphatidylinositol 3,5-bisphosphate (di-C8 PI(3,5)P2), di-C8 phosphatidylinositol
4,5-bisphosphate (di-C8 PI(4,5)P2), and di-C8 phosphatidylinositol 3,4,5-triphosphate (di-C8
PI(3,4,5)P3) were purchased from Cayman Chemical (USA). Malachite green reagent was
prepared as previously described [
]. The crystallization screening reagents were purchased from
Hampton Research (USA), Microlytic (USA), and Molecular Dimensions (UK).
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Purification, crystallization, and X-ray analysis
The cloning of the gene and expression, purification, and crystallization of the human MTMR1
protein have been described elsewhere [
]. Briefly, the MTMR1 gene (residues 95‒608, C438S
mutant) was inserted into a modified pET32 vector (Novagen) using the NdeI/XhoI restriction
enzyme sites. The resulting MTMR1 protein was fused with an octa-polyhistidine tag at its C
terminus. The recombinant protein was overexpressed in Escherichia coli Rosetta 2(DE3) cells
(Novagen). The protein was purified using Ni-NTA (Qiagen) and HiLoad 16/600 Superdex
200 prep-grade columns (GE Healthcare). The purified protein was concentrated to 13.5 mg/
ml and stored in a buffer containing 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM
Na2HPO4, and 5 mM dithiothreitol. The crystals were grown using the sitting drop vapor
diffusion method. Each sitting drop was prepared by mixing 1 μl of purified protein and 1 μl of
reservoir solution of 100 mM Na-HEPES (pH 7.0) and 15% (v/v) polyethylene glycol 20,000 in a
96-well crystallization plate. The reservoir solution supplemented with 25% (v/v) glycerol was
used as a cryoprotectant solution. The X-ray diffraction data were collected using an ADSC
Q315r detector at the Beamline 5C of the Pohang Light Source (Pohang, South Korea). The
Xray diffraction data were collected at 2.0 Å resolution. The data sets were processed and scaled
using HKL2000 [
]. The detailed data and statistics are summarized in Table 1.
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Structure determination and homology modeling
The crystal structure of MTMR1 was solved by molecular replacement methods using the
MOLREP program [
] in the CCP4 program package [
]. The human MTMR2 structure
(PDB entry 1LW3) was used as the search model. The model was manually constructed using
the Coot program [
]. The REFMAC 5 [
] and PHENIX [
] programs were used for model
refinement. The final model for MTMR1 yielded Rfactor and Rfree values of 22.0 and 27.4%,
respectively, with good stereochemistry. The refinement data were validated by the
PROCHECK program [
]. The detailed statistics for refinement are also summarized in Table 1.
Homology modeling for other MTMRs (MTMR3, 4, 5, 7, 8, 9, 10, 11, 12 and MTM1) was
performed using the Schrödinger program package or SWISS-MODEL [
MTMR1 constructs including or lacking the CC domain (95‒665 or 95‒607) were cloned into
the p3xFLAG-CMV-10 (Sigma-Aldrich) or pcDNA3-HA (Invitrogen) vectors, respectively.
HEK293 cells were cotransfected with FLAG- and influenza hemagglutinin (HA)-tagged
MTMR1 expression vectors using polyethylenimine (PEI; Sigma-Aldrich). The cells were
harvested 40 hours after transfection. The immunoprecipitation experiment was performed using
an anti-FLAG-M2 affinity gel (Sigma-Aldrich). The proteins were separated via 10% sodium
dodecyl sulfate-polyacrylamide electrophoresis SDS-PAGE, and the western blot was
performed with an anti-HA antibody (Cell Signaling Technology) and an anti-FLAG antibody
For the malachite green assay with di-C8 PIs, 4 μL of 1 mM di-C8 PIs was added to 15 μL of
assay buffer (50 mM Tris-HCl, pH 8.0, 10 mM dithiothreitol (DTT), and 0.5% NP-40). After
prewarming at 37°C for 30 min, the reactions were initiated by the addition of ~2 μg of
MTMR1. The reactions were quenched after 5 min by the addition of 20 μL of 0.1 M
N-ethylmaleimide and centrifuged at 18,000 x g for 10 min. Then, 25 μL of the supernatant was added
to 50 μL of the malachite green reagent and vortexed. The samples were incubated for 30–40
min for color development prior to measuring the absorbance at 620 nm.
Results and Discussion
The MTMR1 structure was determined by molecular replacement using the MTMR2 structure
as the search model and refined to 2.07 Å resolution. The 97 flexible N-terminal residues
(residues 1–97) and the C-terminal CC domain region (residues 606–665) were excluded during
gene cloning and were not modeled. The refined model included four MTMR1 molecules (A,
B, C, and D chains), 652 water molecules, and 6 phosphate ions in the asymmetric unit. The
overall conformations of the four molecules in the asymmetric unit were similar (S1 Fig).
Three loops (140–143, 161–170 and 187–193) were not modeled because of their invisible
electron density maps. The MTMR1 structure was refined to a crystallographic R-factor of 22.0%
(Rfree = 27.4%). The refined MTMR1 monomer structure was composed of 14 β-strands (β1
−β14) and 17 α-helices (α1−α17) that formed two individual domains: the PH-GRAM domain
(residues 98–205) and the PTP domain (residues 226–605) (Fig 1A). These two domains were
connected by a linker that contained two short 310-helices (residues 206–225). The PH-GRAM
domain, which was originally implicated in protein or lipid binding during
membrane-associated processes, exhibited a β-sandwich structure formed by seven β-strands (β1−β7) and one
α-helix (α1) that was similar to the MTMR2 structure [
]. The second domain of MTMR1
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Fig 1. Overall structure of MTMR1. (A) A ribbon diagram of MTMR1. The PH-GRAM domain is depicted in pink, the PTP domain is in light blue, the linker is
in orange, and the C(X)5R motif is in green. Two phosphate molecules are drawn. (B) The surface potential representation of MTMR1 with charge levels from
-3kT/e (red) to +3kT/e (blue). The active site pocket containing the two phosphate molecules is indicated as a yellow circle. The surface potential
representation was generated using PDB2PQR and APBS [
]. The figures were drawn using PyMol (Schrödinger, LLC). (C) Immunoprecipitation and
western blot analysis with FLAG- and HA-tagged MTMR1. A strong interaction was observed between FLAG-MTMR1 and HA-MTMR1 (95–665), suggesting
the formation of dimers, but no interaction was found with CC domain-truncated MTMR1 (95–607) constructs.
was a large PTP domain composed of seven β-strands (β8−β14) and sixteen α-helices (α2
−α17). The central twisted β-sheet was flanked by several α-helices forming a large globular
domain. The MTMR PTP domain was larger than other PTP domains, including those in
PTEN, MKPs, and VHR [
]. Similar to other members of the MTMR protein family, the
PTP domain of MTMR1 contained a SET-Interaction Domain (SID; α12−α14) that might
facilitate protein-protein interactions . The residues of the C(X)5R motif in the active site
(Cys438 (here, mutated to Ser438 for crystallization), Ser439, Asp440, Gly441, Trp442,
Asp443, and Arg444) were aligned in a loop between β14 and α9 (Figs 1 and 2). The
electrostatic surface potential showed a highly positive patch around the active site (Fig 1).
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Fig 2. Alignments of the amino acid sequences of the MTMRs. The C(X)5R motif and residues
corresponding to the WPD motif of the PTPs are indicated in green letters. The catalytically important
residues are marked in blue circles. Residues involved in the dimeric interfaces for MTMR2 and MTMR8 are
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marked with pink (MTMR2) and yellow (MTMR8) triangles, respectively. This figure was drawn using Espript
The molecular packing of MTMR1 in the crystal did not show any oligomeric association.
The results of a PISA analysis, used to estimate the oligomeric states, did not indicate an
oligomeric association [
]. The largest calculated interface area located between chains A and D or
chains B and C was approximately 950 Å2 and had a zero Complex Formation Significance
Score (CSS). In this study, the purified recombinant MTMR1 protein existed as a monomer
based on the dynamic light scattering estimation (data not shown). However, MTMRs usually
exist as dimers in solution [
]. This discrepancy suggested that the CC domain of MTMR1
might be essential for its dimerization. Notably, the association of MTMR2 with MTMR5 or
itself through the CC domain is necessary for its enzymatic activity and cellular localization
]. This result indicates the importance of the CC domain for the homo- or
heterodimerization of MTMRs. Immunoprecipation experiments using FLAG- and HA-tagged MTMR1 with
or without the CC domain showed that only CC domain-containing MTMR1 could exist as a
dimer, suggesting the importance of the CC domain in the dimerization of MTMR1 (Fig 1).
However, in MTMR8, only the PTP domain can exist as a dimer in solution, and its crystal
structure corresponds to that of the MTMR8 dimer without the CC domain [
]. The dimer
interfaces in MTMR8 are usually formed by hydrophobic residues in the α6−α7 loop, in α7,
and in the α16−α17 loop [
]. However, the corresponding residues are less conserved in
MTMR1, which may increase the contribution of the CC domain to the dimerization of
MTMR1 compared with MTMR8 (Fig 2).
We compared the structure of human MTMR1 with the structures of MTMR2 (PDB code
1LW3 or 1ZVR), MTMR6 (PDB code 2YF0), and MTMR8 (PDB code 4Y7I). The MTMR1,
MTMR2, and MTMR6 structures contain both the PH-GRAM and PTP domains, whereas the
MTMR8 structure contains only the PTP domain [
30, 31, 46
]. MTMR1, MTMR2, MTMR6,
and MTMR8 have a high degree of amino acid sequence conservation, and all of these MTMRs
are catalytically active phosphatases [
] (Fig 2). The MTMR2 structure was easily
superimposed with MTMR1, and its root-mean-square (r.m.s.) deviation was only 0.9 Å for the 513 Cα
atom pairs comprising the PH-GRAM and PTP domains. In the MTMR1 and MTMR2
structures, the PH-GRAM domain bound tightly to the large and shallow cleft of the PTP domain
formed by residues in and around three helices (α6, α13 and α17) and a loop located between
α11 and α12 (Figs 1 and 3A, S1 Table). The MTMR6 structure exhibited a large conformational
change (Fig 3A). The MTMR6 PH-GRAM domain was detached from the shallow cleft of the
PTP domain, and the crystallographic symmetry operation revealed a domain-swapped
dimeric formation of MTMR6 (S2 Fig). The domain-swapped PH-GRAM domain interacted
with the PTP domain of an adjacent subunit. However, the relative orientation of these two
domains of MTMR6 differed markedly from the MTMR1 and MTMR2 structures [
Specifically, the residues involved in the interactions between these two domains were less conserved
in MTMR6. Only three residues out of 21 interface residues in the PH-GRAM domain and
seven residues out of 27 residues in the PTP domain in MTMR6 were conserved (Fig 2 and S1
Table). Although a large conformational change was observed in MTMR6, the overall fold of
the individual domains of MTMR6 was similar to that of MTMR1 or MTMR2. The PH-GRAM
and CC domains of MTMR6 bind to another component in the plasma membrane and inhibit
the Ca2+-activated K+ channel KCa3.1. Interestingly, other MTMRs cannot inhibit KCa3.1
]. This difference may be due to the different orientation of the MTMR6 domains compared
with other MTMRs.
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Fig 3. Comparison of the MTMR1, MTMR2, MTMR6, and MTMR8 structures. (A) Overall comparison of the MTMR1, MTMR2, and MTMR6 structures that
contain the PH-GRAM and PTP domains. The sequence identities to MTMR1 are 67.6% for 678 MTMR2 residues, 36.9% for 694 MTMR6 residues, and
39.7% for 638 MTMR8 residues. (B) Comparison of the structures of the α5 helix, which is thought to be important for membrane association. Each MTMR is
colored aqua, pink, lime, or yellow. The chain A molecule of each MTMR was used for the structural comparisons.
In our structure, one or two phosphate molecules were observed in the active site pocket of
each MTMR1 molecule. The phosphate molecules were clearly visible in the electron density
map of the active site (Fig 4A). These two phosphates correspond nicely with the D1 and D3
sites of PI(3,5)P2 bound to MTMR2 (PDB code, 1ZVR). Among them, one phosphate molecule
(D3 phosphate site of PI) was positioned in the deep cleft of the active site pocket and was
mainly trapped by its interaction with the main chains of the six residues (Ser(Cys)438, Ser439,
Gly441, Trp442, Asp443, and Arg444) from the C(X)5R motif. Only Asp440 in the C(X)5R
motif did not interact with this phosphate. This D3 phosphate ion is observed in all four
MTMR1 chains in the asymmetric unit. The side chains of Asp440 and Asp443 are positioned
away from the active site cavity because of the formation of hydrogen bonds with the side
chains of Lys285 and Arg484, respectively, creating sufficient space for the phosphate ion or
D3 portion of the substrate (Fig 4B). The side chain conformation of Arg444 is restrained by
the main chains of Ile374 and His375, enabling this residue to interact with the phosphates.
The second phosphate molecule, which is positioned at the D1 site of PI and only found in two
MTMR1 molecules (chains A and B) in the asymmetric unit, interacts with the side chains of
Asn351, Ser439, and Arg444. Asn351 lies on helix α5 and is strictly conserved across the
MTMRs (Fig 2), suggesting its importance in binding PI. Interestingly, in contrast to the other
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Fig 4. Active sites of the known MTMR structures. (A) Active sites of individual MTMRs. The residues on
the C(X)5R loop and other important residues are shown using green stick models. The phosphates (in
MTMR1 and MTMR8), sulfate (in MTMR6), or PI(3,5)P2 (in MTMR2) molecules are also depicted as stick
models. The electron density map for the phosphate molecules in MTMR1 is shown with a red mesh. Black
arrows point to the cysteine residue in the C(X)5R motif of the MTMRs that is mutated to serine in the MTMR
structures, except for MTMR6, for crystallization purposes. (B) Illustrated representation of the
phosphatebound active site in the MTMR1 structure. The hydrogen bond distances between residues in the C(X)5R
motif and the phosphate ions were approximately 2.5–3.0 Å.
MTMR structures, the MTMR6 structure contains only one sulfate ion in the active site at the
D3 position (Fig 4). Although the amino acid sequence is still highly conserved, the helix
corresponding to α5 in MTMR1 is missing in the MTMR6 structure, indicating its flexibility. It is
unclear whether the missing structure of this helix is the result of the crystal packing or another
reason. Additionally, it should be noted that α5 has been thought to be important for
membrane association in MTMR2 and MTMR8 [
]. The well-conserved hydrophobic residues
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(Val325, V328, and Ala332 in MTMR2, and Leu245, Met248, and Ala252 in MTMR8) are
exposed to the surface, facilitating membrane association (Fig 3). However, in MTMR1, the
corresponding residues are usually hydrophilic (Ser346, Asp349, and Thr350), suggesting a
different mode of membrane association (Fig 3). The phosphate binding in the active site could
provide insight into the substrate-binding mode and enable us to construct a model for
PIbound MTMR1 (S3 Fig). Many MTMR structures do not contain any phosphate ions at the D5
phosphate site of PI. The MTMR2 structure in complex with PI(3,5)P2 shows that the D5
phosphate site is solvent-exposed and has fewer contacts than the other phosphate sites [
may explain the absence of the phosphate ion at the D5 phosphate position in the active site.
Therefore, it is not surprising that MTMRs dephosphorylate the D3 phosphate from PI(3)P
or PI(3,5)P2 and do not recognize the D5 phosphate of the substrates. Unlike PTEN, MTMRs
cannot dephosphorylate PIs that are phosphorylated at the D4 position. This difference may
be due to the presence of bulky tryptophan and arginine residues (Trp442 and Arg484 in
MTMR1) in the C(X)5R motif, as previously discussed [
Generally, the C(X)5R motif loop in the active site builds the framework of the
substratebinding pocket and determines the substrate specificity depending on its depth [
]. Similar to
MTMR2, the membrane-proximal surface of the active site of MTMR1 is highly positive (Fig
1B) and is relatively wider and deeper than the sites of phospho-Ser/Thr/Tyr-specific PTPs,
allowing it to accommodate relatively bulky substrates, such as PIs [
]. The cysteine residue
(Cys438 in MTMR1) in this motif attacks the phosphorous atom of the substrate as a
nucleophile, generating a phosphoenzyme intermediate and an aspartic acid (Asp443 in MTMR1)
that acts as both a general acid and base for the dephosphorylation reaction [
16, 18, 46
]. In our
structure, the side chain of Asp443, which is tethered by the Arg484 side chain, is positioned
within possible hydrogen bonding distance of a phosphate ion (Fig 4). Canonical PTPs position
their general acid/base (usually an aspartate) on the WPD-loop of the PTP domain. However,
in MTMRs, this aspartate residue lies on the C(X)5R instead of the WPD-loop (Fig 2). This
feature is one that distinguishes MTMRs from other PTPs. When we constructed homology
models of MTM1 and all of the MTMRs for which crystal structures had not been determined, all
of the models included the PH-GRAM and PTP domains (S4 Fig). Moreover, although most of
the catalytically active MTMRs show positive surface charges around the active sites, all of the
catalytically inactive MTMRs (with the exception of MTMR9) have a negative patch in the
corresponding region. However, the inactive MTMRs possess relatively large positive patches on
their dead active site faces, thereby facilitating membrane binding.
Next, we tested the substrate specificity of MTMR1 toward di-C8 PIs. MTMR2 exhibits
phosphatase activity against both PI(3)P and PI(3,5)P2 [
]. However, previous studies
have reported different results regarding its substrate specificity against PI(3)P and PI(3,5)P2
. In this study, the purified recombinant MTMR1 exhibited phosphatase activities against
both di-C8 PI(3)P and di-C8 PI(3,5)P2 (Fig 5A). We also tested whether the active site residues
affected the phosphatase activity. As expected, three MTMR1 mutants (C438S, D443A, and
R444A) showed no (or reduced) phosphatase activity. As explained previously, Cys438
functions as a nucleophile, and Asp443 is a general acid and base. Therefore, these two residues are
essential for the phosphatase activity. In the MTMR1 structure, Arg444 plays an important
role in binding to the phosphate group of the substrate, which explains the reduced
phosphatase activity of the mutant. Then, we produced additional MTMR1 mutants and checked their
enzymatic activities (K285A, K484A, R480A, and R444A/R480A). Here, K285 is a
hydrogenbonding partner with Asp440, and Arg484 interacts with Asp443 (Fig 4). R480 corresponds to
R459 in MTMR2, which interacts with the D5 phosphate of PI(3,5)P2. The mutation of Lys285
had little effect on the phosphatase activity, whereas the R484A mutant exhibited no
phosphatase activity. Arg484 may play critical roles in contributing to the structural restraints in the
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Fig 5. Enzymatic activity of MTMR1. (A) Specific activity of MTMR1 against di-C8 PIs. (B) Comparison of the specific activities of wild-type and mutant
(C438S, D443A, R444A, K285A, R484A, R480A, and R444A/R480A) MTMR1 against di-C8 PI(3)P and di-C8 PI(3,5)P2. The reactions were performed in
triplicate, and the specific activities are presented as moles of phosphate released per minute per mole of enzyme ± SD.
C(X)5R loop of MTMR1 in its enzymatic reaction. As expected, although the R480A mutation
did not affect phosphatase activity with PI(3)P, a ~ 25% reduction in phosphatase activity was
observed for PI(3,5)P2. Interestingly, a partial recovery of the specific activity was observed in
the reaction of the R444A mutant with PI(3,5)P2 because of the contribution of the D5
phosphate instead of the D3 phosphate. The R444A/R480A double mutant did not show any
enzymatic activity toward either PI(3)P or PI(3,5)P2.
In summary, we determined the crystal structure of human MTMR1. The MTMR1
structure contained two phosphate ions in the active site pocket that could promote substrate
binding. Moreover, the phosphatase assay showed high substrate selectivity for both PI(3)P and PI
(3,5)P2, similar to MTMR2.
S1 Fig. Structural superposition of four molecules in the asymmetric unit of the MTMR1
crystal. (A) Stereo view of the superposition of each MTMR1 molecule in the asymmetric unit.
(B) Stereo view of the active site using four superposed MTMR1 molecules in the asymmetric
unit (C) Calculated r.m.s.d. values between each MTMR1 molecule. Chain A, Chain B, Chain
C, and Chain D are shown in green, cyan, magenta, and yellow, respectively.
S2 Fig. Domain-swapped structure of MTMR6 (PDB code 2YF0). Each subunit was drawn
in red or cyan.
S3 Fig. Modeled structure of PI(3,5)P2-bound MTMR1. The structure of MTMR2 in
complex with PI(3,5)P2 (PDB code, 1ZVR) was used for modeling. The electron density map of two
phosphates in MTMR1 is shown.
S4 Fig. Comparison of the structures of MTM1 and 13 MTMRs. Electrostatic surface
potential of MTM1 and the MTMRs. All structures include the PH-GRAM and PTP domains.
MTMR1, MTMR2, and MTMR6 are crystal structures. MTMR8 is a partially modeled
structure. The other MTMRs and MTM1 are homology-modeled structures. All structures are
drawn in the same orientation, and the yellow arrows indicate the active site (C(X)5R loop) for
each PTP domain. Active MTMRs are indicated in red, and inactive MTMRs are indicated in
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green. The MTMR6 structure was used to model MTMR7 and MTMR8, and the MTMR2
structure was used to model the other MTMRs. The modeled structures for MTMR7 and
MTMR8, which have relatively high amino acid sequence similarities to MTMR6, adopt
conformations similar to that of MTMR6. However, no experimental evidence indicates that these
proteins will adopt such conformations.
S1 Table. Interface residues involved in the PH-GRAM and PTP interactions in MTMR1.
We thank the staff at the Pohang Light Source (Beamline 5C) for their assistance in collecting
the synchrotron data.
Conceived and designed the experiments: SMB YJK BIL. Performed the experiments: SMB KS
SWY JWP JWC KTK HK. Analyzed the data: SMB SJK YJK BIL KTK SJK. Contributed
reagents/materials/analysis tools: SJK. Wrote the paper: SMB YJK BIL.
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Crystallogr D Biol Crystallogr. 2015; 71(Pt 7):1528–39. Epub 2015/07/07. doi: 10.1107/
S139900471500927X PMID: 26143924.
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