In vivo, brain serine racemase is also activated by a multi-PSD-95⁄ discs large ⁄ ZO-1 PDZ domain glutamate receptor interacting protein GRIP that is usually coupled to the GluR2⁄ 3 subu
Trang 1multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP
Florian Baumgart1, Jose´ M Manchen˜o2and Ignacio Rodrı´guez-Crespo1
1 Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Ciencias Quı´micas, Universidad Complutense de Madrid, Spain
2 Grupo de Cristalografia Macromolecular y Biologı´a Estructural, Instituto Rocasolano, CSIC, Madrid, Spain
Serine racemase (SR) is a pyridoxal phosphate
con-taining enzyme that catalyses the conversion of the
naturally occurring amino acid l-serine into its
race-mic counterpart d-serine [1,2] In brain tissues,
d-serine occupies the so-called ‘glycine site’ within
the NMDA receptor together with the
neurotrans-mitter glutamate Initially, Wolosker and coworkers
performed elegant studies that resulted in the mole-cular cloning of the enzyme [3] and determined that
it was essentially expressed in astrocytes [4,5] The observation that certain neurones displayed signifi-cant levels of d-serine [6] was confirmed when the expression of SR was detected in neuronal cells as well [7,8]
Keywords
calcium activation; D -serine; GRIP; PDZ
domain; serine racemase
Correspondence
I Rodrı´guez-Crespo, Departamento de
Bioquı´mica y Biologı´a Molecular, Facultad de
Ciencias Quı´micas, Universidad
Complutense, 28040 Madrid, Spain
Fax: +34 91 394 4159
Tel: +34 91 394 4137
E-mail: nacho@bbm1.ucm.es
(Received 7 June 2007, revised 6 July 2007,
accepted 11 July 2007)
doi:10.1111/j.1742-4658.2007.05986.x
Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca2+ and
Mg2+, as well as by ATP and ADP In vivo, brain serine racemase is also activated by a multi-PSD-95⁄ discs large ⁄ ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2⁄ 3 subunits of the a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
Ca2+channel In the present study, we analysed the mechanisms by which ser-ine racemase becomes activated by GRIP, divalent cations and ATP We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production However, full-length GRIP does augment significantly enzymatic activity We expressed various GRIP shorter con-structs to map down the regions within GRIP that are necessary for serine racemase activation We observed that, whereas recombinant proteins con-taining PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly aug-ment its activity Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine race-mase On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg2+ ions Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca2+, ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms
Abbreviations
AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AMPpcp, phosphomethylphosphonic acid adenylate ester; GRIP, glutamate receptor interacting protein; hSR, human serine racemase; PDB, protein databank; PDZ, PSD-95 ⁄ discs large ⁄ ZO-1; PLP, pyridoxal
phosphate; SR, serine racemase.
Trang 2Sequence comparison studies have shown that SR is
homologous to other type II fold-pyridoxal phosphate
enzymes However, we observed that, unlike other
amino acid racemases, Ca2+, Mn2+, Mg2+ and other
divalent cations were able to activate recombinant
brain SR through a process that involved the
stabiliza-tion of the enzyme [9] The addistabiliza-tion of chelating
agents such as EDTA or EGTA almost completely
abrogated the synthesis of d-serine Further work
dem-onstrated that d-serine synthesis by a brain purified
SR was activated by ATP, ADP and GTP as well as
by divalent cations [10] Likewise, when SR was
expressed recombinantly in human embryonic kidney
cells and purified, both divalent cations and ATP or
ADP activated the enzyme [11] In addition, these two
reports showed that ATP was not hydrolysed during
turnover, hence suggesting a novel, allosteric role for
this nucleotide during d-serine synthesis
By means of the yeast two-hybrid approach, three
proteins have been shown to interact with brain SR:
glutamate receptor interacting protein (GRIP) [1,12],
PICK1 [13] and Golga3 [14] GRIP is a large protein
with seven PSD-95⁄ discs large ⁄ ZO-1 (PDZ) domains
that associates with the GluR2⁄ 3 subunits of the
a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid
(AMPA) subtype of glutamate receptors using PDZ4
and PDZ5 [15] PICK1, a protein that contains a
sin-gle PDZ domain, also binds to the GluR2⁄ 3 subunits
of the AMPA receptors as well as to protein kinase C
[13] Both GRIP and PICK1 bind to the four
carboxy-terminal amino acids of SR (-Ser-Val-Ser-Val-COOH
in the human enzyme and -Thr-Val-Ser-Val-COOH in
the mouse enzyme) [12,13] Interestingly, coexpression
of GRIP with SR caused a five-fold increase in the
d-serine released into the medium [1], whereas
infec-tion of astrocytes with a GRIP adenovirus resulted in
a two- to three-fold increase [12] Finally, Golga3
binds to the first 66 amino acids of brain SR and,
remarkably, this binding also results in an increased
d-serine synthesis However, activation of SR by
Golga3 takes place through a decreased ubiquitylation
of SR and diminished proteasomal degradation [14]
Because calcium activates SR [9,11], it is conceivable
that GRIP binding might target SR to the proximity
of the Ca2+-permeable AMPA channel Alternatively,
due to the promiscuous interaction of GRIP with
numerous cellular proteins, GRIP binding to SR might
bring it to the close proximity of some other protein
responsible for the observed increase in d-serine
syn-thesis Finally, GRIP might also activate brain SR
through a putative conformational change that might
result in an increased catalytic rate With that in mind,
we analysed in detail the mechanisms by which the
hepta-PDZ domain protein GRIP activates brain SR
We show that GRIP induces a conformational change
in SR responsible for the observed increase in d-serine synthesis
Results Binding of PDZ6 of GRIP to brain SR When SR is transfected in COS7 cells, high levels of
d-serine can be detected in the supernatant Conse-quently, we transfected brain SR in COS7 cells in the presence and absence of transfected PDZ6 domain of GRIP or full-length GRIP (Fig 1A) In the three cases, similar levels of transfected SR were obtained (Fig 1A, upper panel) Remarkably, although full-length GRIP was able to increase significantly the activity of SR, its PDZ6 domain was unable to increase the levels of
d-serine in the supernatant To investigate whether the PDZ6 domain of GRIP was efficiently transfected
in COS7, we performed an immunoblotting, which resulted in a positive band at the expected molecular mass (approximately 13 kDa; Fig 1A, upper right-hand panel) Next, to rule out the possibility that SR and the PDZ6 domain of GRIP might be unable to interact, we immunoprecipitated the transfected SR from the COS7 cells and investigated whether the PDZ6 domain of GRIP effectively associated with the carboxy-terminal end of SR As shown in Fig 1A, the PDZ6 domain of GRIP was found to be in associa-tion with SR, an observaassocia-tion that confirms the proper binding of both proteins
Next, we decided to analyse the binding of the PDZ6 domain of GRIP to brain SR using recombinant pro-teins We expressed recombinant brain SR in Escheri-chia coli using the pCWori vector as previously described [9,16], as well as the isolated PDZ6 domain
of GRIP (residues 665–768) Both recombinant pro-teins were purified to homogeneity (Fig 1B) Because the PDZ6 domain of GRIP is just a part of the entire protein, we confirmed by circular dichroism that the protein was properly folded (Fig 1B, right panel) The far-UV circular dichroism spectrum of the PDZ domain of GRIP indicates an abundance in b-sheet content, as confirmed from the crystal structure of this domain [17]
Once we had confirmed that the PDZ6 domain of GRIP was properly folded, we analysed the activity of
SR under different conditions in the presence and absence of this binding protein (Fig 1C) When SR is purified in the presence of divalent cations, the addition of extra Ca2+ or Mg2+ has a limited effect
on its catalytic activity The chelating agent EDTA
Trang 3significantly diminished catalytical activity, whereas
ATP significantly increased the racemase activity
(Fig 1C) When ATP plus Ca2+⁄ Mg2+ was added to
the reaction mixture, the synthesis of d-serine reached
almost a three-fold increase over basal activity levels
When recombinant PDZ6 domain of GRIP was added
to SR in a 2 : 1 molar ratio, no significant changes in
activity could be observed This clearly indicates that
the binding of the PDZ6 domain by itself is not
responsible for the previously reported increase in SR
activity [1,12]
Binding of brain SR to recombinant constructs of
GRIP that included additional PDZ modules
GRIP is a very large protein (1112 amino acids)
com-posed of at least nine protein modules The architecture
of this protein is depicted in Fig 2A Each PDZ
domain is comprised of approximately 100 amino acids
and they are conserved in terms of folding, although
each PDZ module is able to interact with a different
subset of target proteins [18,19] GRIP contains two
clusters of three PDZ domains, since PDZ1, PDZ2 and
PDZ3 are consecutive in the sequence as well as PDZ4,
PDZ5 and PDZ6 On the other hand, PDZ7 is close to
the carboxy-terminal end of the amino acid sequence
[15] The two clusters of PDZ domains, as well as the
second cluster and PDZ7, are separated by large
domains of unknown function referred to as GAP1 and
GAP2 (Fig 2A) Binding of GRIP to SR occurs
through the direct interaction of the PDZ6 domain of
GRIP and the final four amino acids of SR [12]
We performed the recombinant expression in E coli
and the purification of two larger fragments of GRIP
and incubated the purified proteins with SR to analyse
changes in activity A hexa-His tag was introduced at the N-terminal end and a FLAG tag was introduced at the carboxy-terminal end Expression and purification conditions were optimized to obtain a homogeneous band by SDS⁄ PAGE When the purified protein that contained the cluster PDZ4-PDZ5-PDZ6 (residues 468– 768) was incubated with SR in a 2 : 1 molar ratio,
we failed to observe the expected increase in d-serine
A
B
C
Fig 1 Binding of SR to the PDZ6 domain of GRIP (A) COS7 cells
were transfected with a SR plasmid and then, 24 h
post-transfec-tion, they were trypsinized, split into three flasks and transfected
with a PDZ6, a full-length GRIP or an empty plasmid The amount
of the released D -serine into the medium was determined in the
three cases (left panel) The efficient expression of SR (upper left
panel) and PDZ6 domain of GRIP (upper right panel) in the
trans-fected COS7 cells was determined by immunodetection by
wes-tern blot The association between the PDZ6 domain of GRIP and
SR was determined through the immunoprecipitation of SR and the
immunodetection of FLAG-tagged PDZ6 (bottom right panel).
(B) Coomassie Blue-stained SDS ⁄ PAGE gels of purified
recombi-nant SR (left) and the PDZ6 domain of GRIP (middle) The circular
dichroism spectrum of purified PDZ6 domain of GRIP is shown in
the right panel (C) D -Serine synthesis by recombinant SR in the
absence (black bars) and presence (grey bars) of a two-fold molar
excess of the PDZ6 domain of GRIP under different assay
condi-tions Data are representative of three independent experiments.
Trang 4synthesis (Fig 2B) Although a limited rise in activity was observed when both ATP and Ca2+were present, this increase is far from the expected five-fold [1] or
two-to three-fold [12] increase that was reported when both proteins were transfected in mammalian cells Remark-ably, when we incubated the purified protein that contained the PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end of GRIP (residues 468–1112) with SR in a 2 : 1 molar ratio, a significant increase in activity was obtained when no additional ATP was added (Fig 2C) This increase was over two-fold when purified SR lacked additional Ca2+ or ATP, as well as when additional
Ca2+had been included in the reaction mixture
Characterization of the activation of brain SR
by GRIP(468–1112)
We next analysed whether larger ratios of PDZ6 domain:SR might be able to increase the synthesis of
d-serine We tested up to a ratio of 23 : 1 using the recombinant PDZ6 domain of GRIP (residues 665–768) and recombinant SR (Fig 3A) Even at this large excess
of PDZ6, the increase in racemization activity was extre-mely limited However, when we tested larger ratios of recombinant PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end of GRIP (residues 468–1112), a significant increase in the racemase activity was observed (Fig 3B) Due to solu-bility problems, we could not go beyond a 5.8 ratio of GRIP fragment:SR but, at this point, we observed a 2.8-fold augmentation in racemase activity Hence, this result indicates that certain residues of GRIP that lie outside the binding site for SR present within PDZ6 are responsible for a complete activation of the enzyme Next, we maintained a 1 : 1 molar ratio of the recom-binant protein PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end to racemase and inspected the catalytic properties of the latter at increasing concentrations of the substrate l-ser-ine in the absence of additional ATP (Fig 3C) We were able to determine that binding of this fragment of GRIP
to SR changed both the Kmand the Vmaxof the reaction The Kmfor d-serine synthesis increased from 1.9 mm in the absence of GRIP fragment (filled circles) to 3.4 in its presence (empty circles), whereas the Vmax of the reaction augmented 1.65-fold (from approximately
115 ± 8 nmolÆmg)1Æmin)1 to 190 ± 7 nmolÆmg)1Æ min)1; Fig 3C, insert) Consequently, binding of PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end of GRIP induces a conformational change in recombinant SR that is responsible for its increased catalytic properties
We next considered whether the binding of GRIP
to SR might result in a shift in the response towards calcium In the presence of ATP, a preparation of recombinant SR that had been purified in the presence
D-Serine Synthesis (%) 50
100
150
200
250
300
100
200
300
400
+ ATP mock
+ EDTA + Ca
2+
+ Ca
+ ATP mock
+ EDTA + Ca
2+
+ Ca
C
GAP2 GAP1
PDZ7
PDZ4 PDZ5 PDZ6
PDZ3 PDZ2
468
665 768
1112
A
B
C
75 50 37 25
75 50 37 25
Fig 2 D -Serine synthesis by purified recombinant SR in the
pres-ence of the PDZ4-PDZ5-PDZ6 and
PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end modules of GRIP (A) GRIP is formed by seven PDZ domains
separated by two large GAP domains of unknown function The
residue numbering indicative of the beginning and end of each
domain is shown on the right D -Serine synthesis is measured
under various conditions in the absence (black bars) or presence
(grey bars) of GRIP constructs The effect of (B) PDZ4-PDZ5-PDZ6
and (C) PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end is shown together with
a Coomassie Blue-stained SDS ⁄ PAGE (inserts) Data are
represen-tative of three independent experiments.
Trang 5of divalent cations, but afterwards dialysed, was
incu-bated with increasing levels of Ca2+ in the absence
and presence of PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end
of GRIP (Fig 3D) Values above 2 mm Ca2+ could not be reached due to protein precipitation in the assay In both cases, recombinant SR became activated
in the 1–100 lm range of Ca2+ The activation curve was not displaced towards lower Ca2+concentrations,
a clear indication that GRIP binding does not result in
a higher sensitivity of SR towards this divalent cation
ATP binding does not involve significant changes either in the quaternary structure of SR or in its kinetic properties
At this point, we considered the mechanism by which the addition of ATP or ADP might augment the cata-lytic properties of SR It should be noted that the highly homologous threonine dehydratase, an E coli enzyme, binds to and becomes activated by AMP, in a process that results in the decrease of the Km for threonine from 70 mm in the absence to 5 mm in the presence of this cofactor [20] At the same time, binding of AMP to
E coli threonine dehydratase results in the association
of the protein monomers into tetramers [21] Accord-ingly, we inspected the catalytic properties of SR in the presence and absence of added ATP In the presence of
100 lm ATP, the enzyme becomes activated but, inter-estingly, the Vmax for d-serine synthesis increases 2.2-fold, whereas the Km of the reaction remains almost unchanged (approximately 3.2 mm in the absence against 3.0 mm in the presence of ATP) (Fig 4A) Remarkably, ATP binding is not involved in protein oligomerization We previously showed that brain SR is found in solution in a dimer–tetramer equilibrium [9], with the dimer eluting at approximately 12.8 mL in a Superdex-200 column Addition of 10 lm ATP plus
100 lm Mg2+to brain SR did not induce changes in the elution profile (Fig 4B) Although a slight increase in the population of high molecular mass oligomers eluting
at approximately 10 mL was observed, the population
of dimers remained the most abundant in solution
Molecular modelling of human SR (hSR) using the crystal coordinates of homologous enzymes from Schizosaccharomyces pombe and Thermus thermophilus
Although no mammalian serine racemases have been resolved to date, the structures of two homologous racemases have been recently solved and their atomic coordinates deposited in public databases The three-dimensional structure of SR from S pombe has been solved in the presence of Mg2+ as well as with the ATP analogue phosphomethylphosphonic acid adenyl-ate ester (AMPpcp) [protein databank (PDB) codes:
molar ratio (GRIP/SR) D-Ser synthesis (%) 50
100 150 200 250
molar ratio (GRIP/SR)
D-Ser synthesis (%) 50
100
150
200
250
[L-Ser] (m M )
A411 nm
0.00
0.01
0.02
0.03
0.04
0.05
0.06
1/[L-Ser]
-0.5 0.0 0.5 1.0 1.5
A411 nm
0 20 40 60 80
additional Ca 2+ added
100 n
M
1 µ
M
10 µ
M
100 µ
M
1 m
M
A411 nm
0.02
0.04
0.06
0.08
+ EDTA
A
C
D
B
Fig 3 Characterization of SR activation by the
PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end module of GRIP D -Serine synthesis by
recombi-nant SR in the presence of increasing molar ratios of (A)
the recombinant PDZ6 domain or (B)
PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end module of GRIP (C) The kinetic properties of D -serine
racemization were determined in the absence (filled circles) or
pres-ence (empty circles) of a two-fold molar excess of
PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end module of GRIP towards SR at increasing
substrate concentrations The Lineweaver–Burk plot is depicted in
the insert These assays were performed in the presence of 1 m M
Ca 2+ and in the absence of ATP (D) Increase in D -serine synthesis
at increasing Ca 2+ concentrations by recombinant SR in the
absence (filled circles) or presence (empty circles) of a two-fold
molar excess of the PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end module of
GRIP Data are representative of three independent experiments.
Trang 61V71 and 1WTC] Likewise, the atomic coordinates of
threonine deaminase from T thermophilus have been
obtained by X-ray crystallography in the presence of
Ca2+ (PDB code: 1VE5) With the aim of building a
full-atom 3D model of hSR, we carried out a tertiary
structure prediction approach by using three different
servers available on the Internet (see Experimental
pro-cedures) The results provided by the program were
fully consistent in that all of them rendered the same
hit with the highest score: the SR from the fission
yeast S pombe (40% sequence identity with hSR) In
the case of 3d-jury, the threading predictions were
stopped because it readily considered 1V71 as a
signifi-cant hit The other potential template provided by sp3
and 3d-shotgun with a similar but lower score is
thre-onine deaminase from T thermophilus (TtTD; PDB
code: 1VE5) (41% sequence identity with hSR) Both
proteins, which belong to the tryptophan synthase
b-subunit-like pyridoxal-phosphate-dependent enzymes
superfamily, share the same polypeptide chain fold
The 3D comparison of the Ca atoms of the two crystal
structures yielded a root mean square (rms) deviation
of 1.3 A˚ for 289 Ca atoms Thus, the template selected
for building a 3D model for hSR was the SR from
S pombe (SpSR) Although the 3D models provided
by both 3d-shotgun and sp3 are indistinguishable
(rmsd¼ 0.75 A˚ for 318 Ca-atoms), it should be noted
that the last server provides a model for the first four
residues of hSR and also for the last 12 amino acid
residues, which were not modelled by 3d-shotgun
Superimposition of the crystal structure of SpSR and the herein proposed full-atom model for hSR reveals that the pyridoxal phosphate (PLP) binding site
is conserved The coenzyme is located deep between the two subdomains The lysine residue Lys56 in hSR would be homologous to Lys57 in SpSR, Lys51 in TtTD, or Lys62 in threonine deaminase from E coli [22], which are covalently bound to PLP through the formation of a Schiff base Conversely, the microenvi-ronment of the phosphate moiety of PLP is essentially conserved, mainly interacting with the tetraglycine loop (Gly185 to Gly188), which is highly conserved in PLP-dependent enzymes Other residues interacting with the PLP coenzyme are Phe55, which embraces the pyridine ring, Ser313 and Asn154 The only significant difference in the vicinity of PLP between hSR and SpSR is precisely the presence of this last residue Asn54 instead of Tyr152 in SpSR
The crystal structure of both SpSR and TtTD revealed a common cation binding site, which in the first case is occupied by Mg2+ and in the second by
Ca2+(Fig 5A) In both cases, the metal is
hexavalent-ly coordinated The cation binding site is formed by two carboxylate-containing residues (Glu208, Asp214
in SpSR and Glu203, Asp209 in TtTD), a main chain carbonyl oxygen (Gly212 in SpSR and Ala207 in TtTD) and three well-ordered water molecules, which
in turn are hydrogen-bonded to main chain carbonyl groups Both the geometry and the distances perfectly agree with the coordination of these metals [23] The
Fig 4 Binding of ATP to SR changes the
Vmaxof the racemization reaction but not the oligomeric state of the enzyme (A) Michaelis–Menten representation and Lineweaver–Burk plot of the activation of brain SR by ATP Assays were performed in the presence of 1 m M Ca 2+ plus 1 m M
Mg 2+ (B) Gel filtration elution profile of recombinant SR (approximately 400 lg per run) in the absence (upper trace) and pres-ence (bottom trace) of 10 l M ATP plus
100 l M Mg 2+ Data are representative of four independent experiments.
Trang 7corresponding region of hSR is perfectly superimpos-able with the above crystal structures, indicating that the cation binding site is conserved, thus explaining the Ca2+-binding ability of hSR In this case, the homologous amino acid residues in hSR putatively involved in Ca2+ coordination are: Glu210, Asp216 and Ala214
Based on the crystal structure of SpSR (PDB code: 1V71), a putative model for the dimeric hSR can be easily built (Fig 5B) In this structure, two monomers related by a crystallographic two-fold axis tightly asso-ciate forming dimeric species It is worth noting that the presence of an extended C-terminal end in the model for hSR herein proposed would not preclude this association (Fig 5B) The putative interface between hSR monomers would be made up primarily
of hydrophobic residues essentially contributed by regular structure elements of the polypeptide chain Estimation of the hydrophobic surface area with nac-cess from the ligplot package reveals that approxi-mately 76% of the contact area is hydrophobic, which
is typical of obligate complexes Additionally, the crys-tal structure of SpSR in the presence of AMPpcp has also been deposited (PDB code: 1WTC), revealing a putative ATP binding site According to the present model for hSR, the ATP analogue would be located between hSR monomers in a shallow manner, mainly interacting with polar residues (Fig 5B) Remarkably, comparison of the crystal structures of SpSR in the presence and absence of AMPpcp shows two impor-tant aspects First, binding of the ATP analogue does not induce large conformational changes in the protein and, second, the ligand does not modify the dimeric state of the protein, which is in agreement with the results herein provided
Finally, the proposed structure for the dimeric hSR permits the visualization of a tentative model for the hSR:PDZ6 complex (Fig 5C) This model has been constructed assuming that the C-terminal eight resi-dues of hSR adopt an extended conformation similar
to that of the octapeptide of human liprin-a com-plexed with the GRIP1 PDZ6 domain [17], with an identical mode of interaction of PDZ6 Although this model should only be considered tentatively, it per-mits the identification of the regions of hSR that can
be directly affected by the binding of the PDZ domain
Discussion Fluorescence studies from numerous groups have dem-onstrated that astrocytes respond to neurotransmitters not with action potentials, like most neurones, but
A
B
C
Fig 5 (A) Calcium binding site of hSR The polypeptide chains of
hSR (yellow) and SpSR (blue) are superimposed Calcium is shown
as a green sphere; water molecules (wat) present in the crystal
structure of SpSR are shown as red spheres Labels for the
resi-dues are for hSR Carboxylate-containing resiresi-dues and carbonyl
oxy-gens involved in cation binding are shown as sticks The figure was
generated with PYMOL [32] Positioning of the Ca 2+ , ATP and PDZ6
domain binding sites in the model of hSR (B) 3D Model of the
dimeric hSR The relative position of the PLP (red sticks), Ca 2+
(magenta spheres) and AMPpcp (orange sticks) are shown The
N- and C-terminal ends of hSR are indicated as N and C,
respec-tively Magnesium ions complexed with the nucleotide present in
the crystal structure of SpSR (PDB code: 1WTC) are shown as
green spheres (C) Putative 3D model of the PDZ6:HSR complex.
The figure was generated with PYMOL
Trang 8with propagating waves of intracellular calcium ions.
In response to these calcium oscillations, glial cells
release ‘gliotransmitters’ such as glutamate, ATP and
d-serine Released glutamate and d-serine occupy
binding sites in the NMDA receptors of neurones
hence regulating neuronal function In this context, we
analysed how brain SR becomes activated by three
independent factors: divalent cations, nucleotides such
as ATP and the multi-PDZ domain protein GRIP
When an adenovirus that contained GRIP was used to
infect mice the cerebellar d-serine concentration was
augmented two-fold [12] One likely explanation for
this observation is that the PDZ4-PDZ5 domains of
GRIP might interact with the AMPA channel,
target-ing brain SR bound to the PDZ6 domain of GRIP
towards its proximity Under these circumstances,
brain SR might become activated due to the calcium
influx through the channel Conversely, brain SR
acti-vation might occur through a conformational change
induced upon GRIP binding Using purified
recombi-nant proteins, we have been able to establish that
GRIP binding to brain SR induces a conformational
change that increases the racemase activity by over
two-fold Although the PDZ6 domain of GRIP is
involved in the interaction with the last four
carboxy-terminal residues of SR, this association is not enough
to induce SR activation Our results indicate that
addi-tional PDZ modules are necessary to trigger the
observed activation In this regard, it must be noted
that multi-PDZ domain proteins are known to display
‘communication’ between individual modules For
example, neither the PDZ4, nor the PDZ5 domain of
GRIP bind to the GluR2 subunit of the AMPA
chan-nel independently, with the concerted action of both
being necessary for the association [15] Analysis of
the amino acid sequence of GRIP reveals the presence
of two GAP domains that separate the two three-PDZ
domain tandems, PDZ1-PDZ2-PDZ3 from
PDZ4-PDZ5-PDZ6 and the latter from PDZ7 Activation of
brain SR requires the presence of GAP2 together with
PDZ7 Interestingly, in the absence of additional ATP,
the activation induced by the presence of the
PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end of GRIP reached the
highest value The conformational change induce by
GRIP binding on brain SR results in changes in both
the Km and the Vmax for d-serine synthesis and the
response curve for activation by calcium remained
unaltered
Considering that the cellular concentration of ATP
(3–6 mm) is well above that needed for SR activation
[11], it is intriguing to elucidate what might be the
exact role of the nucleotide in vivo Because amino acid
racemization in PLP-containing racemases is not an
ATP-driven reaction and ATP is not hydrolysed dur-ing catalysis [10,11], the nucleotide could exert an allo-steric role Inspection of the 3D model of brain SR reveals that ATP is positioned in the monomer–mono-mer interface However, crystals of the homologous SpSR reveal that these enzymes establish identical monomer–monomer interactions in the presence and absence of the nucleotide According to our results, the absence of modulation of the oligomeric state
of the enzyme by the nucleotide is in clear contrast with the behaviour displayed by the homologous bacterial threonine deaminase [20,21] Our data also indicate that, in the absence of added ATP, brain SR
is more readily regulated by GRIP binding It is then conceivable that, in the microenvironments where SR
is present, cellular ATP levels might be low, hence per-mitting a tight regulation through ATP binding Our finding that SR, GRIP and the GluR2 subunit
of the AMPA channel are able to form a ternary com-plex appears to indicate that SR might be positioned close to this calcium channel, hence modulating its activity
Experimental procedures Chemicals and antibodies
Ultrapure l-serine was purchased from NovaBiochem (Laufelfingen, Switzerland) Horseradish peroxidase was obtained from Roche Molecular Biochemicals (Mannheim, Germany) SR monoclonal and GRIP antibodies were obtained from Transduction Laboratories d-Serine,
d-amino acid oxidase from porcine kidney and FLAG anti-body were purchased from Sigma (St Louis, MO, USA)
obtained from Sigma Using a campus facility, we injected pure recombinant brain SR and pure PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end GRIP fragment into rabbits Injections were performed every week over a 6-week period The anti-serum was then obtained and tested
Cloning of SR and the GRIP constructs PDZ4-PDZ5-PDZ6, PDZ6 and PDZ4-PDZ5-PDZ6-GAP2-PDZ7-end into pCWori and recombinant protein expression
The plasmid encoding the cDNA of mouse SR was a gener-ous gift from S H Snyder (Johns Hopkins University, Baltimore, MD, USA) and GRIP1 cDNA in pBK was kindly given to us by R Huganir (Johns Hopkins University)
We have previously described the recombinant expression and purification of brain SR [9] All the constructs were cloned in the pCWori plasmid that possessed a hexa-His tag
Trang 9at the 5¢-end in frame when the NdeI site was used
(N-termi-nus of the recombinant protein) [9,16] In addition, by PCR,
we introduced a SalI site at the 3¢-end of the amplified gene
followed by eight amino acids that encoded for the FLAG
epitope and a XbaI site (at the carboxy-terminal end of the
protein) Hence, all constructs were cloned between NdeI and
SalI to ensure that there would be a tag at each end of the
protein In constructs with PDZ4 at the 5¢-end, we performed
a PCR with GRIP as template using the oligonucleotide
5¢-GGACAGGTTGTTCATATGGAAACACA-3¢, which intro
duced the NdeI site (underlined sequence) as forward primer
The reverse oligonucleotide that annealed at the 3¢-end of the
gene and introduced the FLAG epitope was 5¢-GGTCT
AGAGCTTATCGTCATCGTCCTTGTAGTCGACTGTG
TTAGT-3¢ The SalI and XbaI sites are underlined For
PDZ6 cloning (320 bp) we used the oligonucleotide 5¢-GAT
GAGCATATGAGTTCCCGGGCG-3¢ as forward primer
(introducing a NdeI site) and 5¢-TGAGTCGACGGGGAT
GGGCAGCTT-3¢ (introducing a SalI site) as reverse primer
The amplification of the PDZ4-PDZ5-PDZ6 GRIP construct
was performed using the forward primer 5¢-GGACAGGT
TGTTCATATGGAAACCACA-3¢ (introducing a novel
NdeI site) together with 5¢-TGAGTCGACGGGGATGGG
CAGCTT-3¢ (introducing a novel SalI site)
The amplified DNA fragments were subcloned into a
pGEM-T vector (Promega, Madison, WI, USA) and
con-firmed by automated DNA sequencing Then, they were
double digested with NdeI plus SalI and ligated into the
corresponding sites of pCWori In all cases, the hexa-His
was at the N-terminal end of the protein and the FLAG
epitope at the carboxy-terminal end BL21
(DE3)-compe-tent cells (Novagen, Merck Chemicals Ltd, Nottingham,
UK) were routinely transformed with the respective
pCW-ori constructs and an overnight 10 mL culture was used to
inoculate a 2.8 L flask containing 0.75 L of 2· yeast ⁄
tryp-tone medium Typically, 1.5 L of cell cultures were grown
in the presence of 100 mgÆL)1 ampicillin at 37C to an
absorbance of 1.0 and induced adding 1 mm isopropyl
thio-b-d-galactoside The E coli cultures were then grown at
30C (to avoid degradation, especially of the PDZ
con-structs) at 220 r.p.m for 16–20 h before the cells were
har-vested by centrifugation at 5140 g for 30 min in an F10
rotor (Sorvall, Norwalk, CT, USA) The cell pellets were
frozen in plastic bags as thin films and stored at)80 C
Immunoblot and immunoprecipitation
Regarding immunoblot, immunoprecipitation and confocal
analysis, we followed standard cellular biology protocols, as
reported in previous studies performed by our group [24,25]
Cloning of PDZ6 into pCDNA3
The DNA of FLAG-tagged PDZ6 was amplified from the
pCWori-PDZ6 construct, using 5¢-ATGCACCATCACC
AGAATTCCCATATG-3¢ as forward primer and 5¢-CAT GTTTGACAGCTTATTCTAGAG-3¢ as reverse primer con taining EcoRI and XbaI restriction sites (underlined sequences) The PCR product was double digested with EcoRI plus XbaI and ligated into the corresponding sites of pCDNA3 Thus, the PCR product of PDZ6 that we obtained contained the sequence for a C-terminal FLAG tag for immunoprecipitation and immunodetection The PDZ6-pCDNA3 plasmid was subsequently confirmed by automated DNA sequencing
Determination ofD-serine concentration
A routine colorimetric assay with 200 lL of total sample was used with l-serine as a substrate, coupling the appear-ance of d-serine to commercial d-amino acid oxidase and horseradish peroxidase, plus the peroxidase substrate O-phenylenediamine The d-serine that was produced dur-ing the incubation period was degraded by d-amino acid oxidase, which specifically targets d-amino acids generating -keto acid, ammonia, and hydrogen peroxide The hydro-gen peroxide was quantified using horseradish peroxidase and O-phenylenediamine, which turns yellow upon oxida-tion The activity of serine racemase was determined in the presence of 20 mm Mops, pH 8.1, 10 lL of purified enzyme (5 lg approximately), 10 mm l-serine, 0.03 mm dithiothreitol, 5 lm PLP, 50 lgÆmL)1 O-phenylenediamine,
1 nm FAD, 0.2 mgÆmL)1 d-amino acid oxidase, and 0.01 mgÆmL)1 horseradish peroxidase The reactions (200 lL, final volume) were incubated at 37C for 2 h before measuring the absorbance (A411 nm) with a Beckman DU-7 spectrophotometer (Beckman Coulter Inc., Fullerton,
CA, USA) The d-serine present in a given sample was determined by correlating the absorbance (A411 nm) with
d-serine calibration curves d-Serine measurements were not influenced by the concentration of l-isomers present in the sample Because standard commercial l-serine prepara-tions contain trace amounts of d-serine, it was necessary to purchase ultrapure l-serine from NovaBiochem to achieve
a reasonable signal-to-noise ratio Once optimized, this three-enzyme assay was able to detect d-serine formed in
an unknown 100 lL sample in the linear range from 50 lm
to 1 mm (up to 0.1 lmol total of d-serine) All the measure-ments were performed in triplicate
To measure d-serine production by COS7 transfected with
SR, we used the second part of the colorimetric assay described above Transfected cells were incubated for approximately 8 h with phenol-red-free Dulbecco’s modified Eagle’s medium supplemented with 10 mm l-serine Col-lected supernatants were boiled for 10 min and centrifuged before the assay To estimate d-serine levels, we typically analyzed a 300 lL sample adding 10 nm FAD, 10 lgÆmL)1 horseradish peroxidase, 50 lgÆmL)1 O-phenylenediamine and 100 lgÆmL)1 d-amino acid oxidase in a final volume
of 400 lL Samples were incubated for 5 h at 37C,
Trang 10centrifuged in a table-top microcentrifuge at 16 000 g and
subsequently measured at 411 nm with a spectrophotometer
Circular dichroism measurements
CD spectra were recorded on a Jasco J-715
spectropolarime-ter (Jasco Inc., Easton, MD, USA) using a 0.1 cm path
length cell at 25C The temperature in the cuvette was
reg-ulated with a Neslab RT-111 circulating water bath (Neslab
Inc., Portsmouth, NH, USA) The buffer used was 50 mm
Tris, pH 7 A minimum of five spectra were accumulated
for each sample and the contribution of the buffer was
always subtracted The resultant spectra were smoothed
using j715 noise reduction software provided with the CD
spectrophotometer
Gel filtration analysis of recombinant SR in the
presence and absence of ATP
Generally, we followed a previously published protocol [9]
Aliquots of 200 lL of approximately 2 mgÆmL)1of
recom-binant racemase eluted from the Ni-nitrilotriacetic acid
affinity resin were injected into a GP 250 plus fast protein
liquid chromatography system equipped with two P-500
pumps and a Superdex HR200 column (Amersham
Bio-sciences, Piscataway, NJ, USA) Separation was performed
at 25C and protein detection was performed at 280 nm
The flow rate was kept at 1 mLÆmin)1 and the buffer
consisted of 50 mm Tris, pH 7.0, 50 mm NaCl, 1 mm
dithiothreitol in the presence or absence of 10 lm ATP plus
100 lm Mg2+ We were unable to use concentrations of
ATP above 10 lm due to the elevated absorbance signal
that we obtained
Modelling hSR
The servers used in this work for prediction of the tertiary
structure of hSR were: sp3 (http://sparks.informatics.iupui
edu⁄ hzhou ⁄ anonymous-fold-sp3.html) [26], 3d-shotgun
(INUB predictor; http://inub.cse.buffalo.edu) [27] and
3d-jury (BioInfoBank Meta server; http://meta.bioinfo.pl/
submit_wizard.pl) [28] Currently, these servers are
consid-ered among the best performers for structure prediction as a
result of the CAFASP4 experiment (Critical Assessment of
Fully Automated Structure Prediction) [28] Considering the
prediction results, a full-atom 3D model of hSR was built by
using the atom coordinates of SR from S pombe (PDB code:
1V71) because it was provided as the best template for the
human homologue Sequence alignments were analysed with
clustal w [29] The quality of the final structure was
assessed with the verify3d program [30] and also with
procheck[31] In the first case, the 3D profile score is high
(approximately 60), which is typical for correct structures
and, in the second case, the program indicated that the
stereochemistry of the model is correct, with 99.3% of amino acid residues in the allowed regions of the Ramachandran plot (data not shown) 3D Superposition of protein struc-tures and other analyses were performed with software o [23]
Acknowledgements
We would like to thank Dr Martı´nez del Pozo for many useful comments and corrections of the manu-script We are also grateful to Dr Galve-Roperh for numerous comments and suggestions This work was supported by grant BMC2006 05395 from the Spanish DGICYT
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