From the Howard Hughes Medical Institute and Department of
Molecular Genetics, University of Texas Southwestern Medical School,
Dallas, Texas 75235
Synapsins I and II are abundant phosphoproteins
that are localized to synaptic vesicles and have essential functions in
regulating synaptic vesicle exocytosis. Synapsins contain a single
evolutionarily conserved, large central domain, the C-domain, that
accounts for the majority of their sequences. Unexpectedly, the crystal
structure of the C-domain from synapsin I revealed that it is
structurally closely related to several ATPases despite the absence of
sequence similarities (Esser, L., Wang, C.-R., Hosaka, M., Smagula,
C. S., Südhof, T. C., and Deisenhofer, J. (1998)
EMBO J., in press). We now show that the C-domains of both
synapsin I and synapsin II constitute high affinity ATP-binding
modules. The two C-domains exhibit similar ATP affinities but are
differentially regulated; ATP binding to synapsin I is
Ca2+-dependent whereas ATP binding to synapsin
II is Ca2+-independent. In synapsin I, the Ca2+
requirement for ATP binding is mediated by a single, evolutionarily conserved glutamate residue (Glu373) at a position where
synapsin II contains a lysine residue. Exchange of Glu373
for lysine converts synapsin I from a
Ca2+-dependent protein into a
Ca2+-independent ATP-binding protein. Our studies suggest
that synapsins I and II function on synaptic vesicles as ATP-binding
proteins that are differentially regulated by Ca2+.
 |
INTRODUCTION |
Ever since their discovery as major brain phosphoproteins and
abundant components of synaptic vesicles, synapsins have been intensely
studied (1, 2). In vertebrates, synapsins constitute a family of at
least four homologous proteins (synapsins Ia, Ib, IIa, and IIb;
collectively referred to as synapsins I and II) that are derived by
alternative splicing from the primary transcripts of two genes (3, 4).
The four synapsins are composed of common amino-terminal domains found
in all synapsins (the short A- and B-domains and the long C-domain) and
divergent carboxyl-terminal domains that are present in different
combinations in each synapsin (D- to I-domains) (3). The C-domains
account for more than half of the synapsin sequences and constitute
their most conserved domain. In addition to the vertebrate synapsins, a
synapsin gene was described in Drosophila; this gene also
produces two protein products that differ at the carboxyl terminus (5).
Only the C-domain is highly conserved between vertebrate and
invertebrate synapsins, suggesting that it is the central functional
domain.
The four vertebrate synapsins are co-expressed in most neurons (6).
They are substrates for multiple protein kinases. All synapsins contain
a phosphorylation site for Ca2+,
calmodulin-dependent protein kinase I, and protein kinase A at the amino terminus (3). In addition, synapsin I but not synapsin II
is phosphorylated by Ca2+, calmodulin-dependent
protein kinase II, Cdk5, and mitogen-activated protein kinase at more
carboxyl-terminal sites (7-9). The physiological roles of the
synapsins have remained elusive because their sequences are not related
to proteins of known functions and because they are sticky proteins
that bind to a number of possible targets (actin filaments,
microtubules, neurofilaments, spectrin, calmodulin, annexin, to name a
few of the binding proteins described; reviewed in Refs. 10-12). In
knockout mice, synapsins are essential for maintaining stable vesicles
and for normal short term synaptic plasticity, suggesting that they
constitute important regulatory molecules (13-15). These experiments
established that synapsins function in mature synapses but did not
reveal their actual activity in the nerve terminal.
Recently, the crystal structure of the C-domain of bovine synapsin I
was solved (16). The structure revealed that the C-domain constitutes a
large, independently folding domain that forms a stable dimer.
Surprisingly, data bank searches for three-dimensionally related
proteins demonstrated that the C-domain of synapsin I is very similar
to the structures of five ATP-utilizing enzymes: glutathione
synthetase, D-alanine:D-alanine ligase, biotin
carboxylase
-chain, succinyl-CoA synthetase
-chain, and
pyruvate,orthophosphate dikinase. No sequence similarity was detected
between these proteins and the C-domain of synapsin I, but more than
80% of the C
carbon atoms of the C-domain can be superimposed on
those of glutathione synthetase or
D-alanine:D-alanine ligase with a root mean
square deviation of 0.32 nm. The five enzymes to which synapsin I is structurally related bind ATP and transfer phosphate from bound ATP to
a substrate (17, 18), suggesting that synapsin I may also bind ATP and
be a phosphotransfer enzyme. In support of this, the structure of a
complex of the synapsin I C-domain with
ATP
S1 and Ca2+
was solved (16). In this structure, ATP was bound by residues similar
to the ATP-binding residues in the structurally related enzymes, and
Ca2+ was coordinated by the pyrophosphate moiety of ATP
S
and two glutamate residues (Glu373 and
Glu386).
The crystal structure of the synapsin I C-domain suggested the
possibility that ATP binding may be regulated by Ca2+, an
interesting hypothesis in view of the role of synapsins in Ca2+-regulated exocytosis (13-15). Furthermore, because
the C-domains of synapsins I and II are highly homologous, the results
with the synapsin I C-domain raised the question if synapsin II also binds ATP. We have now addressed these issues using recombinant C-domains from synapsins I and II. Our results show that all synapsins bind ATP; surprisingly, ATP binding is directly regulated by
Ca2+ only in synapsin I but not synapsin II. The distinct
regulatory properties of synapsins depend on a single, evolutionarily
conserved amino acid, indicating an evolutionary selection of their
separate regulatory properties. These data suggest that synapsins may
be differentially regulated phosphotransfer enzymes on the surface of
synaptic vesicles.
 |
MATERIALS AND METHODS |
Construction of Expression Vectors and Expression of Recombinant
Proteins--
Synapsin expression vectors in pGEX-KG (19) with wild
type or mutant C-domain sequences were obtained by polymerase chain reaction with oligonucleotide primers containing flanking restriction sites essentially as described using standard molecular biology techniques (20, 21). The following pGEX plasmids were used in the
current study: pGEXrSynI-C encoding residues 110-421 of wild type rat
synapsin I, pGEXrSynI-C/K269Q, pGEXrSynI-C/E373K, pGEXrSynI-C/E373S,
and pGEXrSynI-C/E166D encoding the mutant C-domain of synapsin I with
the indicated amino acid substitutions, and pGEXrSynII-C encoding
residues 113-421 of wild type rat synapsin II. Control plasmids used
were described previously (21, 22). The baculovirus expression vector
encoding full-length synapsin Ia fused at the carboxyl terminus with a
hexahistidine sequence was constructed in the
CspI/RsrII and KpnI sites of pFASTBAC1 (Life Technologies, Inc.) by polymerase chain reaction. All vectors were verified by DNA sequencing. Expression of GST fusion proteins was
performed essentially as described (20-22). For baculovirus expression, the Bac-to-Bac expression system (Life Technologies, Inc.)
was used according to the manufacturer's specification. Recombinant
protein was produced in High-five cells and purified on nickel-agarose.
All recombinant proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining and quantified using known
amounts of bovine serum albumin run on the gels.
[35S]ATP
S and [
-32P]ATP Binding
Measurements and ATPase Assays--
Purified GST fusion proteins
immobilized on glutathione beads were washed 3 times with buffer A (50 mM HEPES-NaOH, pH 7.4, 25 mM NaCl) containing 2 mM EGTA ± 2.1 mM Ca2+.
Aliquots of the beads with 5 pmol of recombinant protein were used in
0.1-ml binding assays containing buffer A with 10 nM
[35S]ATP
S and 2 mM EGTA ± 2.1 mM Ca2+ and the indicated additions of
nucleotides or Mg2+. For the determination of the
Ca2+ concentration dependence of [35S]ATP
S
binding, Ca2+/EGTA buffers were used in buffer A with 10 nM [35S]ATP
S, and the concentration of
free Ca2+ was calculated using the Chelator program (23).
After a 1-h incubation at room temperature, beads were washed three
times in the incubation buffer without [35S]ATP
S, and
the radioactivity bound to the beads was determined. Binding
measurements to full-length baculovirus synapsin I were performed
similarly using synapsin I immobilized to nickel-agarose beads, with
beads lacking synapsin I used as a control. Beads were washed with
buffer A, and binding measurements were performed as described before
for the GST fusion proteins except that all buffers lacked EGTA.
[
-32P]ATP binding experiments were performed
analogously. Binding data were analyzed using GraphPad PRIZM software
by nonlinear regression assuming a single binding site. ATPase assays
were performed with purified recombinant proteins as described
(24).
 |
RESULTS |
High Affinity ATP Binding by Synapsins I and II--
To study the
ATP binding properties of synapsins, we expressed their C-domains as
GST fusion proteins and used 35S-labeled ATP
S as a
non-hydrolyzable ATP ligand. We immobilized the GST fusion proteins on
glutathione-agarose beads, incubated them with
[35S]ATP
S under a variety of conditions, and washed
them with the respective incubation buffers. Bound
ATP
35S was then measured by scintillation counting, and
background binding was determined using GST alone or GST fusion
proteins of other synaptic proteins. With this method we found that the C-domains from both synapsin I and synapsin II avidly and specifically bound ATP
S, with background binding accounting for less than 0.1%
of the specific signal (Fig. 1).

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 1.
ATP binding to the C-domains of synapsins
(Syn) I and II. GST fusion proteins of the C-domains
of synapsin I (lanes 1-6) or synapsin II (lanes
7-12) or GST alone (lanes 13 and 14) were
immobilized on glutathione-agarose beads (5 pmol of total protein).
Beads were incubated in the presence of 2 mM EGTA and 10 nM [35S]ATP S with 0.25 mM ATP,
0.25 mM GTP, 2 mM Mg2+, and 2.1 mM Ca2+ in the indicated combinations and
washed in the incubation buffers. Bound ATP S was measured by liquid
scintillation counting. Data shown are means ± S.E. from a
representative experiment performed in triplicate and repeated multiple
times with comparable results.
|
|
Next we investigated the nucleotide specificity and effect of
Mg2+ on ATP binding to synapsins. Mg2+
inhibited but did not abolish ATP
S binding to both synapsin C-domains, similar to its effect on GTP binding to Rab proteins (Fig.
1) (25). Only ATP but not GTP competed for ATP
S binding. To evaluate
the relative ATP affinities of synapsins, we measured ATP displacement
curves. We observed an almost identical EC50 for ATP of
approximately 0.1 µM for both synapsins, indicating that
both proteins bind ATP with a similar high affinity (Fig. 2).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
ATP binding affinities of the C-domains from
synapsin (Syn) I and II. [35S]ATP S
binding to immobilized GST-C-domain fusion proteins from synapsin I
(A) or synapsin II (B) was measured as described
for Fig. 1 with or without Ca2+. Binding reactions were
carried out in the presence of increasing concentrations of unlabeled
ATP or GTP as shown. Data were fit to a single binding site, resulting
in the half-maximal inhibition constants (EC50) shown on
the right. Note that GTP does not compete for binding and
that ATP exhibits almost identical competition curves for both
synapsins but only synapsin I requires Ca2+ for
binding.
|
|
To test if full-length synapsin I also binds ATP
S, we purified
recombinant synapsin Ia produced in High-five cells as a fusion protein
with a hexahistidine sequence. Binding experiments were performed with
synapsin I immobilized on nickel-agarose beads as described for the GST
fusion proteins above, except that EGTA could not be added to the
incubations because it would have eluted the nickel from the column
matrix. We observed robust ATP binding with synapsin I beads but not
with control beads (Fig. 3A).
The ATP affinity of full-length synapsin was similar to that of the C-domain (EC50
0.12 µM). Much higher
concentrations of ADP than ATP were required to displace ATP
S
(EC50
6 µM), and GTP was again inactive
(Fig. 3B). Together these studies demonstrate that the
C-domains from both synapsins constitute ATP-binding modules with a
high nucleotide specificity and with similar ATP affinity and that ATP
binding is also a property of full-length synapsin.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
ATP S binding to full-length synapsin I. A, recombinant synapsin Ia was produced with a baculovirus
expression system in High-five cells as a fusion protein with a
carboxyl-terminal hexahistidine sequence. Nickel-agarose beads
containing immobilized synapsin Ia (Synapsin Ia) or control
protein from uninfected High-five cells (Control) were
incubated with 10 nM [35S]ATP S in the
presence of 2 mM Mg2+ and/or Ca2+
as indicated and washed, and bound ATP S was measured. B,
ATP S binding reactions with synapsin Ia and control beads were
carried out in the presence of increasing concentrations of unlabeled ATP, ADP, or GTP. Data were fit to a binding curve predicted for a
single binding site; half-maximal inhibition concentrations are shown
next to the curves.
|
|
Finally we investigated if the C-domains of synapsin I or II or
full-length synapsin I exhibit ATPase activity. No ATP hydrolysis by
synapsins was detected even at high protein concentrations under a
variety of conditions, such as the presence of different lipids or
various divalent cations (data not shown). Thus synapsins are not
constitutively active ATPases. This result is not unexpected if
synapsins are functionally similar to the enzymes that they resemble
structurally (glutathione synthetase,
D-alanine:D-alanine ligase, biotin carboxylase
-chain, succinyl-CoA synthetase
-chain, and
pyruvate,orthophosphate dikinase) (16). These enzymes transfer phosphates to a substrate during ATP hydrolysis and are unlikely to be
active in the absence of substrate.
Regulation of ATP Binding by Ca2+--
ATP
S bound
to the C-domain of synapsin I or to full-length synapsin I only in the
presence of Ca2+ (Figs. 1, 2A, and
3A), suggesting that synapsin I has a
Ca2+-binding site. To elucidate the apparent affinity of
this site, we measured ATP
S binding at different Ca2+
concentrations (Fig. 4). In the absence
of Mg2+, half-maximal ATP binding was observed at 5-7
µM free Ca2+, suggesting that a high affinity
binding site in the C-domain of synapsin I is involved. In the presence
of Mg2+, higher Ca2+ concentrations were
required. This indicates that Ca2+ and Mg2+
compete for the same binding site in synapsin I or that
Mg2+ binding to a separate site modulates Ca2+
binding. ATP binding to both rat and bovine synapsin I was
Ca2+ dependent, suggesting that the effect of
Ca2+ on ATP binding is evolutionarily conserved (data not
shown). To exclude the possibility that the Ca2+ dependence
of binding was an artifact of ATP
S, we also used radioactive ATP for
the binding experiments. Again we found that ATP binding required
Ca2+ (data not shown). Thus synapsin I is directly
regulated by Ca2+ in addition to its indirect regulation by
Ca2+-dependent kinases.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Ca2+ titration of
[35S]ATP S binding to the C-domain of synapsin I. [35S]ATP S binding to the immobilized GST fusion
protein of the synapsin I C-domain was measured as described above in
the presence of Ca2+/EGTA buffers to clamp the free
Ca2+ concentration. Binding reactions were carried out in
the absence of Mg2+ or in the presence of 0.1 mM Mg2+ as indicated.
|
|
What about the C-domain of synapsin II, which is homologous to the
C-domain of synapsin I (78% sequence identity) and binds ATP with a
similar affinity (Fig. 2)? Surprisingly, Ca2+ neither
inhibited nor enhanced ATP
S binding to synapsin II (Figs. 1 and 2).
To exclude the possibility that these results were artifacts caused by
sequence variations, we confirmed them with several independent
cDNA clones. Thus ATP binding to synapsins I and II is
differentially regulated by Ca2+ despite their high degree
of homology and similar ATP binding affinities.
A Single Residue Controls Ca2+ Regulation of ATP
Binding to Synapsins--
The result that the C-domains from synapsin
I and II exhibit differential regulation of ATP binding by
Ca2+ is surprising in view of their high degree of sequence
identity. The crystal structure of synapsin I showed that ATP is bound
by several residues that are conserved in the enzymes to which synapsin I is structurally homologous. These residues include
Lys225, Lys269, and Gly276.
Ca2+ is coordinated by the
- and
-phosphates of ATP
and by two glutamate residues (Glu373 and
Glu386). These residues are evolutionarily conserved in
human, rat, mouse, and bovine synapsin I, and the binding properties of
bovine and rat synapsin I are similar. Analysis of the sequence of
synapsin II shows that all of the residues involved in ATP binding are also conserved in agreement with their similar ATP binding properties. Of the Ca2+-coordinating residues, however, the residue
corresponding to Glu373 is a lysine (Lys374) in
synapsin II, whereas Glu386 is also a glutamate in synapsin
II. Similar to Glu373 in synapsin I, Lys374 is
evolutionarily conserved in synapsin II. This suggests that synapsins
were diversified in evolution in Ca2+-regulated and
Ca2+-independent forms by a single point mutation similar
to the diversification of synaptotagmins in Ca2+-regulated
and Ca2+-independent forms (26).
To test this hypothesis, we analyzed the structural determinants of ATP
binding to the C-domains of synapsin I by site-directed mutagenesis. We
expressed mutant synapsin I C-domains with single amino acid
substitutions and analyzed their ATP
S binding properties. The
different mutants did not exhibit increased instability, suggesting that there was no major impairment of the folding of the C-domain in
the different mutants (data not shown).
First we studied a mutation in Lys269 of synapsin I because
the crystal structure indicated that this lysine should be essential for ATP binding. Substitution of Lys269 for glutamine
completely abolished ATP
S binding as predicted (Fig.
5). This result confirms the
crystallographic model of ATP binding and validates the specificity of
the ATP
S binding assays. As a positive control, we analyzed a
substitution of Glu166 to aspartate, which had no effect on
binding. We then studied single amino acid substitutions in the
presumptive Ca2+ binding site of synapsin I. Exchange of
Glu373 in synapsin I for lysine had no effect on overall
ATP binding but eliminated the requirement for Ca2+ (Fig.
5). Thus the E373K substitution transformed the
Ca2+-dependent ATP binding activity of the
C-domain of synapsin I into a Ca2+-independent ATP binding
activity. However, when Glu373 was substituted for serine,
ATP
S binding was abolished, suggesting that a serine in this
position in synapsin I is not compatible with ATP binding. These data
demonstrate that substitutions of single amino acid residues either
transform its Ca2+ regulation of ATP binding or abolish ATP
binding altogether.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 5.
Ca2+-dependent
[35S]ATP S binding to wild type and mutant C-domains
from synapsin (Syn) I and II. The C-domains from
synapsin I and II were analyzed as wild type proteins (WT)
or with the indicated amino acid substitutions under three binding
conditions: Ca2+ alone, Ca2+ and
Mg2+, and no divalent cations. GST and a GST-synaptotagmin
fusion protein (Syt) were studied as controls.
|
|
 |
DISCUSSION |
Synapsins have been at the center of attention for many years
because of their abundance, stoichiometric phosphorylation by multiple
protein kinases, and strategic localization to synaptic vesicles. Few
proteins are associated with so many detailed mechanistic models and
postulated functions as synapsins. They have been implicated in
neurogenesis, gene expression, axonal extension, synaptogenesis, and
neurotransmitter release (for a review of these proposed extended functions, see Ref. 10). Analyses of knockout mice that lack synapsins
confirmed an essential role in regulating synaptic vesicle exocytosis
and suggested a primary, possibly exclusive, function for synapsins in
the mature nerve terminal (13-15). However, these and many of the
other studies on synapsins, although technically elegant, failed to
reveal the molecular nature of synapsin action because they suffered
from limitations that made precise functional definitions difficult.
These limitations include the use of synapsin fragments that contain
parts of domains instead of complete domains, the high surface activity
of synapsins (27), which may explain why synapsins bind with a high
apparent affinity to so many different proteins (especially
cytoskeletal elements (28)), and the use of complex biological systems
in which direct and indirect effects are difficult to distinguish
(13-15). We have now attempted to overcome these limitations by
correlating structural and functional approaches. Our data show that
synapsins are ATP-binding proteins that are differentially regulated by
Ca2+ and may serve as phosphotransfer enzymes.
The crystal structure of the C-domain from synapsin I demonstrated that
it constitutes an autonomous, independently folding domain (16). The
C-domain is a dimer in which the amino and carboxyl termini of each
subunit are in close proximity. Comparison of the C-domain structure
with data bases uncovered a high degree of structural similarity to a
group of enzymes: glutathione synthetase, D-alanine:D-alanine ligase, biotin carboxylase
-chain, succinyl-CoA synthetase
-chain, and
pyruvate,orthophosphate dikinase. All of these enzymes bind ATP and
transfer phosphate from bound ATP to a substrate (17, 18). The
crystallographic data raise a number of questions. Do synapsins bind
ATP? What are the relative ATP binding properties and affinities of
different synapsins? Is ATP binding to synapsins regulated by
Ca2+? We have now addressed these issues to explore the
functional implications of the structural observations. Our data showed
that the C-domains of both synapsins bind ATP, but not GTP, with
similar high affinity, thereby characterizing synapsins as
ATP-utilizing proteins on the vesicle surface.
Although we were unable to measure ATPase activity in synapsins, the
following data support the notion that ATP binding to synapsins is part
of an enzyme reaction. 1) All five proteins to which the
three-dimensional structure of C-domains is closely related are
ATPases. 2) In glutathione synthase and
D-alanine:D-alanine ligase, a flexible
catalytic loop with a central arginine/lysine residue is essential for
activity. The synapsin I C-domain has a flexible loop at the same
position that is 100% conserved in synapsin II and also contains a
central lysine residue. 3) Synapsins bind ADP with a much lower
affinity than ATP, indicating that after hydrolysis, ADP would be
exchanged for ATP. The fact that we could not detect ATPase activity in
synapsins is not surprising because the enzymes to which synapsins are
structurally homologous transfer the phosphate to the substrate and
would not be expected to be active in the absence of substrate.
Together our data suggested that synapsins are probably dimeric enzymes
of unknown substrate specificity on the vesicle surface.
This description paints a unitary view of synapsins in agreement with
their high degree of homology. Unexpectedly, however, we found that ATP
binding was differentially regulated in the two synapsins despite their
similar ATP affinity. Ca2+ was required for ATP binding to
synapsin I but not synapsin II. The difference in regulation could be
traced to a single amino acid difference between the two synapsins:
Glu373 in synapsin I, which corresponds to
Lys374 in synapsin II. Substitution of Glu373
to a lysine converted synapsin I into a Ca2+-independent
ATP-binding protein. This finding has functional and evolutionary
implications. Evolutionarily, it implies that synapsins I and II
co-evolved with the C-domain as the main functional domain and that at
some time a divergence in regulation occurred that is based on the
substitution of a single amino acid. A similar evolutionary switch
between Ca2+-dependent and
Ca2+-independent isoforms in a protein family by a change
in a single amino acid was recently observed in synaptotagmins (26).
Functionally, this observation suggests that synapsin II may be
constitutively active whereas synapsin I may only be activated upon
increases in Ca2+. This hypothesis agrees well with the
more severe phenotype of synapsin II knockouts than synapsin I
knockouts, despite the greater abundance of synapsin I (14). A view of
synapsins emerges from these studies in which the major central domain
of synapsins, the C-domain, represents a differentially regulated
ATP-binding domain that is flanked by amino- and carboxyl-terminal
extensions and may serve as a phosphotransfer enzyme of unknown
specificity.
We thank Drs. J. Deisenhofer and L. Esser for
advice, Drs. X.-S. Xie and D. K. Stone for help with the ATPase
assays, and Dr. K. Ichtchenko for producing the synapsin baculovirus
construct.