From the European Molecular Biology Laboratory, 6 rue
Jules Horowitz, B.P.181, 38042 Grenoble Cedex 9, France, the
§ Max Planck Institute for Brain Research,
Deutschordenstrasse 46, 60528 Frankfurt/Main, Germany, and the
¶ Institute for Plant Biochemistry, Eberhard Karls University,
Corrensstrasse 41, 72076 Tübingen, Germany
Received for publication, March 2, 2001, and in revised form, April 23, 2001
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ABSTRACT |
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Gephyrin is a ubiquitously expressed protein
that, in the central nervous system, forms a submembraneous
scaffold for anchoring inhibitory neurotransmitter receptors in the
postsynaptic membrane. The N- and C-terminal domains of gephyrin are
homologous to the Escherichia coli enzymes MogA and MoeA,
respectively, both of which are involved in molybdenum cofactor
biosynthesis. This enzymatic pathway is highly conserved from
bacteria to mammals, as underlined by the ability of gephyrin to
rescue molybdenum cofactor deficiencies in different organisms. Here we
report the x-ray crystal structure of the N-terminal domain (amino
acids 2-188) of rat gephyrin at 1.9-Å resolution. Gephyrin-(2-188)
forms trimers in solution, and a sequence motif thought to be involved
in molybdopterin binding is highly conserved between gephyrin and the
E. coli protein. The atomic structure of gephyrin-(2-188)
resembles MogA, albeit with two major differences. The path of the
C-terminal ends of gephyrin-(2-188) indicates that the central and
C-terminal domains, absent in this structure, should follow a similar
3-fold arrangement as the N-terminal region. In addition, a central
Gephyrin, a protein of 93 kDa, was identified originally by
copurification with the inhibitory glycine receptor from rat spinal cord (1, 2). Biochemical and gene targeting experiments have revealed a
crucial role for gephyrin in the clustering of both the glycine
receptor and related In addition to its synaptic function in the central nervous
system, gephyrin has been shown to be involved in the
biosynthesis of the molybdenum cofactor
(Moco),1 which is a crucial
component of different enzymes catalyzing important redox reactions
(16). This was first highlighted by the homology of gephyrin to the
Escherichia coli enzymes MogA and MoeA, which are known to
be implicated in Moco biosynthesis (12). Homology lies within the N-
and C-terminal domains of gephyrin, which are separated by an
intervening region of ~170 amino acids that harbors multiple protein
interaction domains (17). Similar multi-domain proteins comprising both
MogA and MoeA homologous domains are found in Drosophila
melanogaster (cinnamon) (18) and Arabidopsis thaliana
(Cnx1) (19), in which they have also been shown to be implicated
in Moco biosynthesis.
Moco consists of mononuclear molybdenum coordinated by the dithiolene
moiety of tricyclic pyranopterins such as molybdopterin. In
E. coli, Moco is synthesized by multiple enzymes (20, 21) that catalyze the conversion of GTP via precursor Z to molybdopterin (MPT) followed by the insertion of molybdenum (22-24). Gephyrin has
been shown to bind MPT with high affinity and to restore Moco biosynthesis in E. coli MogA mutants as well as in
Moco-deficient L929 fibroblasts and plants (25). These complementation
assays indicate that the N-terminal domain of gephyrin is functionally equivalent to MogA in catalyzing the transfer of molybdenum to MPT
(25). The MoeA-like C-terminal domain, in contrast, seems either
essential for the conversion of precursor Z to Moco, because MoeA
mutants of E. coli accumulate this precursor (26), or may have a function in the transformation of molybdenum into thiomolybdate (27). Thus, in gephyrin, gene fusion has allowed the recruiting of two
independent enzymatic activities and a membrane protein scaffolding
function into a single polypeptide.
The crucial role played by gephyrin in Moco biosynthesis was revealed
previously by the analysis of gephyrin knockout mice, which in addition
to an impaired synaptic localization of inhibitory neurotransmitter
receptors, shows only basal levels of Moco-dependent enzyme
activities in peripheral organs (5, 7). Notably, hereditary Moco
deficiencies in humans cause symptoms that are similar to those
observed upon the loss of inhibitory neurotransmission in gephyrin
knockout mice (5). Both are characterized by severe neurological
abnormalities such as increased muscle tone, mental retardation,
microcephaly, myoclonus, and tonic-clonic seizures (28). Moreover, a
microdeletion in the gephyrin gene has been identified recently in a
Moco-deficient patient (29). However, the lack of molybdoenzyme
activities such as sulfite oxidase in affected patients as well as in
gephyrin knockout mice (5) could cause neurological disorders through
the accumulation of toxic metabolites (28, 30). Therefore, it is not
yet clear whether the role of gephyrin in Moco biosynthesis and
receptor clustering at the postsynaptic membrane are two interdependent or completely separate processes.
Here we present the three-dimensional structure of the
N-terminal domain of gephyrin from rat and show that it forms a trimer both in solution and in the crystalline state. Overall, the structure of gephyrin displays high homology to that of E. coli MogA.
Differences include a MogA insertion of unknown function as well as
different paths of the C-terminal ends. The predicted orientations of
the C-terminal linker regions of gephyrin suggest 3-fold symmetry for
the entire molecule. A highly conserved region is discussed as being
the putative active site based on sequence homologies and mutagenesis data.
Gephyrin Cloning, Expression, and Purification--
Gephyrin
cDNA encoding residues 2-188 was amplified by polymerase chain
reaction using synthetic oligonucleotides and cloned into the
expression vector pRset (Invitrogen) using the restriction sites
NheI and EcoRI. This strategy placed a His tag
derived from the vector on the N terminus. The correct sequence of the
construct was verified by DNA sequencing. Expression was achieved in
E. coli BL21/codon+ cells (Invitrogen) following standard
protocols. Expression of gephyrin-(2-188) was induced with 1 mM isopropyl- Chemical Cross-linking Analysis--
Gephyrin-(2-188) was
incubated with glutaraldehyde concentrations as indicated (Fig. 1) in a
buffer containing 50 mM Hepes, pH 8.0, and 100 mM NaCl at room temperature for 30 min. The reaction was
quenched by the addition of 50 mM Tris, pH 8.0, for 30 min at room temperature. Samples were separated by 10% SDS-polyacrylamide gel electrophoresis under reducing conditions, and the bands were visualized by Coomassie Brilliant Blue staining.
Detection of Molybdopterin Bound to
Gephyrin-(2-188)--
Molybdopterin bound to gephyrin was
analyzed according to Johnson and Rajagopalan (26) with minor
modifications. 1 mg of gephyrin-(2-188) was oxidized over night at
room temperature by the addition of acidic iodine solution (1%
I2/2% KI/1 M HCl). Residual iodine was reduced
by the addition of ascorbic acid, and 1 M Tris base was
added to adjust the pH to 8.5. After centrifugation, alkaline
phosphatase (2 units, Sigma-Aldrich) and 7 mM
MgCl2 were added to the supernatant for dephosphorylation
(4 h at 37 °C). The sample was applied to a 0.5-ml QAE-Sephadex
(acetate form) column and washed (5 ml of water), and form A dephospho
was eluted with 5 ml of 10 mM acetic acid. High pressure
liquid chromatography was performed using a C18 column (Hypersil ODS, 5 µm, 25 cm) coupled to a fluorescence detector (370/450 nm). 1 ml of
the QAE-Sephadex eluate was injected and eluted with 50 mM
ammonium acetate/7% methanol at 1 ml/min. The lower limit for
quantitative measurement of the form A dephospho derivative of
molybdopterin was 0.03 pmol/injection corresponding to about 0.3 pmol of MPT/sample.
Crystallization, Data Collection, and Data
Processing--
Gephyrin-(2-188) was crystallized at a concentration
of 18 mg/ml using the hanging-drop vapor diffusion method. Crystals
grew in drops of a mixture of 1 µl of protein solution and 1 µl of reservoir solution containing 25% (w/v) polyethylene glycol 1500, 0.1 M sodium citrate, pH 5.6, and 10% isopropanol.
Cryotreatment was performed by a quick transfer of the crystals
into a solution containing 30% (w/v) polyethylene glycol 1500, 0.1 M sodium citrate, pH 5.6, 10% isopropanol, and 20%
2-methyl-2,4-pentanediol followed by flash cooling in a gaseous
nitrogen stream. Data to 1.9 Å were collected at the European
Synchrotron Radiation Facility (Grenoble) beamline ID14 EH2 at a
wavelength of 0.933 Å, with a 2° oscillation range. X-ray data were
processed using the program packages DENZO (31), SCALEPACK (31), and
TRUNCATE (32). The crystals belong to the rhombohedral space group R3
and contain 1 molecule/asymmetric unit (2.32 Å3/Da,
Matthews coefficient (33)). The unit cell dimensions in the R3
hexagonal setting are a, b = 65.77 Å and
c = 114.40 Å. The data collection statistics are
summarized in Table I.
Structure Solution and Refinement--
The structure of gephyrin
was solved by molecular replacement using the E. coli MogA
structure (34) as a search model, in which nonidentical residues were
exchanged to alanine. The program package AMoRe (35) was employed using
data between 15 and 4.5 Å. This resulted in a clear peak with a
correlation coefficient of 38.7 and an R-factor of 45.0 after rigid
body refinement, which corresponded to the correct solution. Automated
model building and refinement were performed with the program ARP/wARP
(36) and REFMAC (32), and 7% of the reflections were selected at random and set aside for the R-free calculation (37). Model building was completed by tracing missing areas (helix 1, residues 25-33; 43-52, helix 8) manually with the program TURBO (38) in
several steps alternated with cycles of automated refinement using data
to 1.9 Å. The automated refinement included both positional and
restrained B-factor refinement applying bulk solvent correction with
CNS (39, 40). Solvent molecules were included in the last stages of
interactive model correction and automatic refinement. The final model
was refined to an R-factor of 19.9 (Rfree = 21.9) (Table II). 91.6% of all residues lie in the most favored
regions of the Ramachandran plot, and no residues are located in
disallowed areas, as determined with the program PROCHECK (41).
Oligomerization State of Gephyrin-(2-188)--
The N-terminal
domain of gephyrin (residues 2-188) expressed in E. coli
forms trimers in solution as detected by gel filtration chromatography.
The recombinant protein (calculated molecular mass of 21.2 kDa)
eluted at a volume of 13.2 ml from a Superdex 200 gel filtration column
compared with a volume of 13.6 ml determined for bovine serum albumine
(molecular mass of 66 kDa) (data not shown); this is consistent with an
oligomeric structure. Chemical cross-linking experiments corroborate
this conclusion. Incubation of gephyrin-(2-188) with increasing
concentrations of the cross-linking reagent glutaraldehyde caused the
appearance of two adducts, the sizes of which corresponded to dimeric
and trimeric forms of gephyrin-(2-188) (Fig.
1).
Structure Determination--
Rat gephyrin-(2-188) expressed in
E. coli was crystallized in the rhombohedral space group R3.
The crystals diffracted x-rays to 1.9-Å resolution (Table
I) and contain 1 molecule/asymmetric unit. The three-dimensional structure of gephyrin-(2-188) was solved
by molecular replacement using the E. coli MogA structure (34) as a search model. The final model has been refined to a
crystallographic R-factor of 19.9 (Rfree = 21.9)
with good stereochemistry (Table II) and
includes residues 13-181 and 90 water molecules. The N-terminal His
tag and the first 12 residues as well as C-terminal residues 182-188
were disordered in the crystal, and the last two residues of helix 8 were built as alanines.
General Architecture--
The N-terminal domain of gephyrin forms
an
A side view of the trimeric gephyrin-(2-188) molecule (Fig.
2b) shows that the connection to the intervening region lies
underneath the trimer formed by the N-terminal domain (bottom part of
the gephyrin trimer). This connection includes the linker residues 173-181 that extend from the main structure by forming a short helix 8 (Figs. 2, b and c, and 3). Clear electron density
for this region was present in the map calculated after automatic model
building using ARP/wARP (36). The orientation of the connecting helix
is determined by a network of hydrogen bonds that are provided mostly
by water molecules and by a salt bridge from Lys-177 to Glu-76 (Fig.
2c). The fixed orientation of the linker residues implies
that the intervening region will form a second layer of domains that
may associate in a fashion that most likely will obey the same 3-fold
symmetry. The lack of clear electron density for the last seven
residues that are followed by a proline-rich region
(189-PPPPLSPPPP-198) suggests that the intervening regions may be
linked via a long spacer sequence. This may account for possible
interdomain flexibility that in turn may be crucial for interactions
with other effector molecules (15).
Comparison to the Structure of MogA--
Superposition of the
C-
However, despite of the overall similarity, there are three regions of
major divergence. (i) MogA contains 12 additional residues that form a
The structural differences mediated by the sequence insertion in MogA
as well as the different directions of the C-terminal ends result in
major differences in the exposed surfaces of the trimers (Fig. 4,
yellow arrows). The differences in the electrostatic surfaces of gephyrin-(2-188) and MogA noted on the top of the molecule
can be attributed to the insertion of the
The high degree of sequence similarity and the conserved function of
the N-terminal domain of mammalian gephyrin and E. coli MogA
suggest that the homologous domains from Caenorhabditis
Q23O69, Drosophila cinnamon, and Arabidopsis Cnx1
may have similar overall architectures (Fig. 3). Significant structural
differences can therefore only be expected at the respective N- and
C-terminal ends that are longer or form linkers to additional domains.
The sequence alignment shown in Fig. 3 highlights this conservation and
indicates a set of conserved residues that may be implicated in the
enzymatic function of these proteins.
Proposed MPT Binding Site--
The highest sequence conservation
among all known MogA-related polypeptides (Fig. 3) maps to a loop
region connecting
A second class of mutations in Cnx1, V557G (equivalent to gephyrin
Val-103) and N597L (equivalent to gephyrin Asn-143) (Figs. 3 and
5b), causes a dramatic decrease in MPT binding (42). Val-103 (
Interestingly, in the trimer the proposed MPT binding site of one
monomer is also close to Differential Splicing Produces Three Variants of the N-terminal
Domain--
Several splice variants of gephyrin that result from
insertion or deletion of different exonic sequence "cassettes" of
the highly mosaic gephyrin gene have been identified (17, 43, 44). The
structure of gephyrin-(2-188) is derived from a splice variant
containing cassette 2 (exon 3) within the N-terminal domain and
cassette 6 (exon 19) within the C-terminal MoeA-like domain; most of
the data available on the postsynaptic function of gephyrin have been
obtained with this specific variant. Two other splicing products with
possible insertions in the N-terminal domain analyzed here have been
described: cassette 1 (exon 2) is spliced in between residues Thr-21
and Val-22 and cassette 5 (exon 13) is spliced in between residues
Glu-98 and Ala-99, respectively (Figs. 2 and 3). Thr-21 locates to the
end of A soluble form of the N-terminal domain of gephyrin comprising
residues 2-188 was produced in E. coli and crystallized.
The data presented here show that gephyrin forms trimers in solution with a highly conserved trimer interface that closely resembles the one
observed in E. coli MogA (34). This strongly indicates that
trimer formation may be important for the enzymatic function of both
proteins. Interestingly, the proposed MPT binding site is formed by a
conserved loop region containing the residues GGTG, which are part of a
cavity of one monomer that is close to the Comparison of the crystal structures of the N-terminal domain of
gephyrin and E. coli MogA reveals a very high conservation of the basic protein fold from bacteria to mammals, a finding that is
consistent with the conservation of Moco biosynthesis. Both structures
reveal a similar molecular surface involved in MPT binding and
implicate the same key residues in their enzymatic function as deduced
from mutagenesis data (34, 42). Functional conservation has been also
demonstrated via complementation of MogA function in Moco-deficient
E. coli by gephyrin (25) or Cnx1 (42). Likewise, the
expression of gephyrin rescues the Cnx1 mutant of Nicotiana
plumbaginifolia (25). Moco synthesis is a multi-step
pathway (20, 21) that ultimately catalyzes the conversion of GTP via a
precursor Z to MPT (22, 23). Both the MogA and the MoeA domains from
E. coli as well as their counterparts from mammals
(gephyrin) and plants (Cnx1) have been shown to bind MPT, and there is
evidence that both domains must act interdependently to catalyze the
efficient transfer of molybdenum to MPT (25). This may explain the
genetic fusion of both activities in eukaryotic proteins such as
Drosophila cinnamon, Arabidopsis Cnx1, and
mammalian gephyrin.
The structures of gephyrin-(2-188) and E. coli MogA show
three structural differences, namely an insertion of a Gephyrin has dual functions as an enzymatic protein in Moco
biosynthesis, explaining its wide tissue distribution (17), and as a
scaffolding protein at postsynaptic membranes in the central nervous
system. There it regulates inhibitory receptor clustering by forming a
subsynaptic protein scaffold, which anchors inhibitory receptors to the
cytoskeleton. In addition, gephyrin may be involved in signal
transduction processes that control translation at synaptic sites (15).
Sequencing of different gephyrin cDNAs and analysis of the genomic
gene structure have revealed extensive alternative splicing that may
account for the functional diversity of gephyrin at the level of
protein subdomain organization (17, 43, 44). The gephyrin isoform that
contains cassettes 2 and 6 has been shown to bind to the The structure of the trimeric N-terminal domain of gephyrin reported
here serves as a starting point to understand this multifunctional protein. Further structural analysis will be required to fully understand the function of gephyrin in Moco biosynthesis and at the
synapse, including a precise mapping of molecular interactions of
molecules such as tubulin, profilin, PIN, RAFT1, and collybistin (reviewed in Ref. 15) with the different subdomains of gephyrin. Such
experiments should also unravel whether the N-terminal domain of
gephyrin analyzed here may have functions other than a role in Moco biosynthesis.
-hairpin loop found in MogA is lacking in gephyrin-(2-188). Despite
these differences, both structures show a high degree of surface charge
conservation, which is consistent with their common catalytic function.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminobutyric acid type A receptors at the
postsynapse (3-7). Gephyrin has been shown to anchor glycine receptors
to the subsynaptic cytoskeleton (8) by interacting with both
polymerized tubulin (9) and the cytoplasmic loop region of the glycine
receptor
-subunit (10, 11). Similar interactions are also thought to
mediate the synaptic localization of
-aminobutyric acid type A
receptors (12). A high concentration of gephyrin at the cytoplasmic
face of inhibitory postsynaptic membrane specializations (13, 14) is
consistent with gephyrin acting as a synaptic receptor scaffolding
protein (reviewed in Ref. 15).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside for
3 h. The cells were harvested by centrifugation and lysed by
sonication in a buffer containing 50 mM Tris, pH 8.8, 100 mM NaCl, and 5 mM
-mercaptoethanol. The
supernatant was cleared by centrifugation and loaded onto a
Ni2+-Sepharose (Amersham Pharmacia Biotech) column
pre-equilibrated in lysis buffer. The column was then extensively
washed with lysis buffer, and a 0-500 mM imidazole
gradient in lysis buffer was applied to elute gephyrin-(2-188).
Fractions containing pure gephyrin-(2-188) were concentrated and
loaded onto a Superdex 200 (Amersham Pharmacia Biotech) column in a
buffer containing 20 mM bicine, pH 9.3, and 100 mM NaCl. The total yield of gephyrin-(2-188) was 60 mg
from 1 liter of the E. coli culture.
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES
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Fig. 1.
Chemical cross-linking of
gephyrin-(2-188). Gephyrin-(2-188) was incubated with increasing
concentrations of glutaraldehyde: lane 1, none; lane
2, 0.1 mM; lane 3, 1 mM;
lane 4, 5 mM; lane 5, 10 mM; lane 6, 100 mM. The cross-linked
gephyrin-(2-183) was separated on a 10% SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Brilliant Blue. Molecular
mass markers (kDa) are shown.
X-ray data collection statistics
hkl
i|Ii
(hkl)
<I(hkl)>|/
hkl
i Ii
(hkl).
Refinement statistics
/
structure that contains a central predominantly parallel
-sheet with flanking
-helices.
-strands 1-4 and 6 form a
parallel
-sheet that is interrupted by the antiparallel
-strand 5 (Fig. 2a). The trimer axis
coincides with the crystallographic 3-fold axis and includes three
layers of interactions. The top layer (viewed from the opposite end of
the connection to the intervening domain in Fig. 2) is characterized by
a hydrophobic core made up of residues from helix 5 (Figs.
2a and 3). This is followed by hydrophobic and polar interactions contributed by a 3/10 helix and the linker to
-strand 5. The opposite site of the interface is characterized by polar interactions involving residues from the linker region preceding helix
4 and
-strand 4 (Figs. 2a and 3). The most prominent is a
salt bridge between Lys-101 and Glu-107 of a neighboring
molecule. In addition, this region also forms a number of polar
contacts with water molecules. Interestingly, the bottom surface is
more hydrophilic than the top surface, and it connects the N-terminal domain to the intervening region (Figs. 4, a and
c, and 2b).
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Fig. 2.
Ribbon diagram of gephyrin-(2-188).
a (top view), secondary structure elements are labeled for
one monomer. The positions of proposed active sites (MPT) are indicated
by black arrows. Cter, C terminus;
Nter, N terminus. b (side view), shows the path
of the C-terminal -helix 8 connecting to the intervening domain of
gephyrin. Positions of possible sequence insertions in differentially
spliced gephyrin variants are indicated by yellow arrows
(1, cassette 1; 5, cassette 5). C
(close-up view), the linker sequence in stereo. Hydrogen bonds are
shown as dashed lines. Figs. 2 and 5b were
generated using the program MOLMOL (46).
atoms of the MogA structure with the gephyrin model revealed an
overall root-mean-square deviation of 3.7 Å. If the MogA
-hairpin
insertion (Fig. 3) and the C-terminal ends (regions beyond residue 170 in both structures) of both
gephyrin-(2-188) and MogA are excluded, the root-mean-square deviation
is 1.24 Å (Fig. 5a). Both molecules form a very similar
trimer interface, and most of the residues within van der Waals contact
are conserved between gephyrin-(2-188) and MogA (Fig. 3).
Interestingly, a small hydrophilic cavity lies on top of the MogA
trimer interface, whereas the corresponding region in gephyrin is
mostly hydrophobic (Fig. 4, a
and b, white arrows).
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Fig. 3.
Sequence alignment of the Mog-A like domains
from gephyrin (Rattus norvegicus),
Caenorhabditis elegans protein (data base code Q23069,
35.1% homology to gephyrin), cinnamon (D. melanogaster, 41.6% homology), Cnx1 (A. thaliana, 41.97% homology), and MogA (E. coli, 28.5% homology). Secondary
structure elements are shown for rat gephyrin above the
sequence and for E. coli MogA below the
sequence (including the respective numbering). Residues conserved in
all proteins are boxed, and residues involved in trimer
interface contacts are highlighted in green. The potential
insertions of two alternatively spliced sequence cassettes are
indicated by arrows after residues Thr-21 (cassette 1, ALRAMSFLPGTAFFV SHKFGC) and Glu-98 (cassette 5, KFPTFPFCGLQKG).
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Fig. 4.
Electrostatic potential maps of
gephyrin-(2-188) (a and c) and MogA
(b and d). Regions where
electrostatic potential < 30 kBT are shown in red
and those > +30 kBT are shown in blue
(kB, Boltzmann constant; T, absolute temperature). The
surface changes between the gephyrin-(2-188) and the E. coli MogA structures caused by the insertion of a loop in MogA and
the different paths of the C-terminal ends are indicated by
yellow arrows. The surfaces viewed from the top
(a and b) and bottom (c and
d). White arrows indicate slight differences in
the trimer interface as seen from the top.
-hairpin (
-strands 7 and 8) that, if presented in the gephyrin
structure, would be positioned C-terminally to
-helix 6 (Figs. 3 and
5a). (ii) The loop region
connecting helix 2 and
-strand 2 (Fig. 3) is organized differently
in both molecules. In gephyrin-(2-188), Pro-46 is at the beginning of
a turn where the carbonyl oxygen of the previous residue Asn-45
hydrogen bonds to the backbone nitrogen of Leu-48. Instead, the same
region is extended in MogA and makes hydrogen bond interactions with a
C-terminal segment (residues 175 and 177). (iii) The C termini of both
molecules show completely different chain directions. At residue 170 (same residue number in both molecules), there is a dramatic change in
main chain orientation, as indicated by a
dihedral value of
53.8
for gephyrin and 67.4 for MogA. This results in opposite spatial
placements of these regions in both structures. In MogA, the turn at
residue 170 keeps the main chain antiparallel to helix 6 in an extended
conformation, which reverses direction at positions 179 and establishes
interactions with the MogA-specific
-hairpin insertion (
-strands
7 and 8). In gephyrin-(2-188), in contrast, the corresponding linker
segment between helix 7 and the C-terminal end extends into the
opposite direction away from the N-terminal domain and ends in a short
helix 8 (Figs. 2b and 5a).
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Fig. 5.
a, a stereo view of the superposition of
the C- atoms of gephyrin-(2-188) (yellow) and
corresponding residues of E. coli MogA (green)
shows an overall root-mean-square deviation of 3.7 Å. Superposition of
matching residues, excluding the
-hairpin loop (MogA residues
145-156) and the C-terminal ends (gephyrin residues 171-188) results
in an overall root-mean-square deviation of 1.24 Å. b, a
close-up view of the proposed MPT binding site of gephyrin. Conserved
residues 86-GGTG-89 and residues shown to be important for Moco
biosynthesis by mutagenesis are shown with side chains. Note the
position of conserved residues Asp-94 and Asp-61.
-hairpin loop as well as
the different paths of the C termini. Gephyrin shows a prominent
negatively charged surface as well as small positively charged islands
on the top of the trimer (Fig. 4a) as compared with a
reduced negative surface charge of MogA (Fig. 4b).
Interestingly, the bottom of the trimer that forms the interface to the
intervening domains of gephyrin is characterized also by a mostly
negatively charged surface and closely resembles the electrostatic
profile of the corresponding surface of MogA (Fig. 4, c and
d).
-strand 3 and helix 4 (Fig. 5b). The
conserved sequence motif GGTG in this loop region, which connects back
to
-helix 4, forms the base of a 15 × 14 × 23-Å wide
cavity flanked by residues from the tip of
-strand 1,
-helix 1, and the loop connecting
-strand 2 and
-helix 3 (Fig.
5b). This cavity has been hypothesized as forming the MPT
binding site because of its stringent conservation and the occurrence
of residual density in close proximity to the GGTG motif in MogA (34).
In the present structure, no residual density that could be attributed
to MPT binding was found in this area. This is in agreement with no
measurable detection of any form A dephospho derivative of
molybdopterin by spectroscopic methods in the sample used for
crystallization (data not shown; see "Experimental Procedures").
However, mutagenesis experiments strongly implicate this region in MPT
binding, and three mutations in the MogA-like domain of
Arabidopsis Cnx1 have been reported recently to affect Moco
biosynthesis. First, mutation of Asp-515 to Asn (equivalent to gephyrin
Asp-61), a residue that is conserved among all MogA-like sequences
(Figs. 3 and 5b), has been shown to increase MPT binding and
apparently block insertion of molybdenum into MPT (42). In the gephyrin
structure, Asp-61 is at the base of the proposed MPT binding site and
points toward Gly-87, which is consistent with substitutions at this
site affecting MPT binding. In addition, Asp-61 is surrounded by two
highly conserved aspartate residues, Asp-24 and Asp-94 (Figs. 3 and
5b). Notably, the MogA mutations D49A and D82A (equivalent
to gephyrin Asp-61 and Asp-94) have also been suggested to bind MPT
tighter than the wild-type protein. Collectively, these data underscore
the important role of aspartate residues in the catalytic mechanism
that transfers molybdenum to MPT (34, 42).
-helix 4) points into a hydrophobic pocket surrounded by the residues Leu-70, Ile-71, and Ile-141. Its position is also determined by hydrogen bonding of Arg-36 to the backbone oxygen of Val-103. Asn-143 is part of the central parallel
-sheet and locates to
-strand 6 next to the antiparallel
-strand 5. Therefore,
substitutions at these positions may have secondary effects on MPT
binding by affecting the overall structure of the binding region.
-helix 7 of a neighboring molecule (Fig.
2a, black arrow). There, the tip of the loop
region containing the conserved GGTG motif is coordinated by hydrogen
bonds between Arg-106 from one monomer and the backbone carbonyls of
Phe-90 and Ala-91 of the adjacent monomer. Phe-90 is one of the
conserved trimer interface residues (Fig. 3), which packs into a
hydrophobic pocket contributed by residues from two molecules. A
similar coordination is seen in the MogA structure, in which Arg-94
(gephyrin homologue Arg-106) contacts the backbone carbonyl of Gly-77
from a neighboring monomer. Similarly, Pro-78 (gephyrin homologue
Phe-90) packs into a hydrophobic pocket at the same site, and the
residues involved in these interactions are conserved among MogA-like
sequences (Fig. 3). These shared features may provide a possible
explanation for the conserved trimeric structure of these enzymatically
active polypeptides.
-strand 1, which is sandwiched between
-strands 2 and 3 at
a central position of the structure (Fig. 2, a and
b). The structure of gephyrin-(2-188) shows that the
insertion of cassette 1 could be accommodated by extending the loop
region connecting
-strand 1 and
-helix 1 without disturbing the
present topology, including trimer formation. However, it is
conceivable that local changes at the end of
-strand 1 could affect
the adjacent sequence GGTG that forms part of the proposed binding site
for MPT. The second splicing site, Glu-98, is solvent-exposed in
-helix 4, and an insertion at this position (cassette 5; Fig. 2,
a and b) may be achieved without global
structural changes simply by extending the linker region between
-helix 4 and
-strand 4. Again, local changes induced by this
insertion may also alter the structure of the MPT binding site (Figs. 2
and 5b). Insertion of cassette 5 into
-helix 4 will lead
to an exposed extension at the lower surface of the triangular
oligomer, which in turn could influence the orientation of the spacer
region connecting to the intervening domain as well as the interface
between the N-terminal and intervening domains.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix 7 of a neighboring
molecule. Conserved residues are found to employ similar interactions
to maintain the structural coordination of this loop by two neighboring
monomers in both gephyrin-(2-188) and E. coli MogA
structures. It is conceivable therefore that the conserved function of
MogA-like domains in Moco biosynthesis, namely the binding of MPT and
the transfer of molybdenum, involves a catalytic center formed at the
sites of intersubunit contact within the homotrimeric protein.
Presently, mutants of the trimer interface, which would generate
monomeric gephyrin or E. coli MogA, are not available to
test the role of oligomerization for enzymatic function. However,
oligomerization of native gephyrin has been observed previously in
sedimentation studies (45) and suggested to play a role in the
clustering of inhibitory receptors at developing postsynaptic membrane
specializations (15).
-hairpin loop
in MogA with unknown function, changes in the loop region connecting
-helix 2 and
-strand 2, and a different orientation of their
C-terminal ends. The C-terminal end of gephyrin-(2-188) indicates that
the N-terminal domain is connected to the intervening region via a
short helix in an arrangement suited to maintain the 3-fold symmetry
for the entire molecule. Interestingly, the bottom surface of
gephyrin-(2-188) reveals an overall negatively charged electrostatic
surface that closely resembles that of MogA. The conservation of this
surface is remarkable, because it seems solvent-exposed in MogA but
part of the interface in gephyrin. The disorder of the last seven
residues in the gephyrin structure (2) as well as the presence of
an adjacent proline-rich sequence also indicate that the
intervening domain may not be tightly packed against the N-terminal
domain. This may be important to accommodate the functions of gephyrin
at the postsynaptic membrane (15).
-subunit of
the glycine receptor (7, 11). Insertion of cassette 5 into the N-terminal domain abolishes this interaction (44), implicating the
N-terminal MogA-like domain indirectly in glycine receptor subunit
clustering. However, we could not detect any binding of a 51-amino acid
peptide corresponding to most of the M3 loop of the glycine inhibitory
receptor (11) to the N-terminal domain of gephyrin-(2-188) in
vitro (data not shown). This suggests that the N-terminal domain
of gephyrin may not be involved directly in glycine receptor
clustering. Therefore, the splicing of cassette 5 into
-helix 4 most
likely results in an exposed extension at the bottom part of the
gephyrin-(2-188) oligomer and may thus influence the linker region
connecting the N-terminal domain to the intervening domain; this in
turn may alter the conformation required for glycine receptor binding.
Two other isoforms, both of which lack the first 114 residues (43), do
not bind the glycine receptor
-subunit loop sequence (44). Again,
the resulting different fold of the residual N-terminal domain may
influence the conformation of the spacer or linker sequences or even
the entire intervening region. No data on the possible insertion of cassette 1 into
-strand 1 are yet available, and its effects on the
receptor-binding properties and/or Moco biosynthesis activity of
gephyrin have yet to be determined.
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ACKNOWLEDGEMENTS |
---|
We thank all members of the European Molecular Biology Laboratory/European Synchrotron Radiation Facility Joint Structural Biology Group (Grenoble) for support at the European Synchrotron Radiation Facility beamlines, Dagmar Magalei for technical assistance, and Maren Baier for secretarial help.
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FOOTNOTES |
---|
* This work was supported in part by Bundesministerium für Forschung und Technologie and Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1IHC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed. Tel.:
33-476-207281; Fax: 33-476-207199; E-mail:
weissen@embl-grenoble.fr.
Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.M101923200
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ABBREVIATIONS |
---|
The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; bicine, N,N-bis(2-hydroxyethyl)glycine.
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