From the UMR 6098 CNRS, 31 chemin Joseph Aiguier,
13402 Marseille Cedex 20, France and ¶ UMR 8619 CNRS,
Université Paris-Sud, Bâtiment 430, 91405 Orsay
Cedex, France
Received for publication, November 2, 2000, and in revised form, February 9, 2001
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ABSTRACT |
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The yeast enzymes involved in UDP-GlcNAc
biosynthesis are potential targets for antifungal agents. GNA1, a novel
member of the Gcn5-related N-acetyltransferase (GNAT)
superfamily, participates in UDP-GlcNAc biosynthesis by catalyzing the
formation of GlcNAc6P from AcCoA and GlcN6P. We have solved three
crystal structures corresponding to the apo Saccharomyces
cerevisiae GNA1, the GNA1-AcCoA, and the GNA1-CoA-GlcNAc6P
complexes and have refined them to 2.4, 1.3, and 1.8 Å resolution,
respectively. These structures not only reveal a stable,
The Gcn5-related N-acetyltransferases
(GNATs)1 represent a large
superfamily of functionally diverse enzymes that catalyze the transfer
of an acetyl group from AcCoA to the primary amine of a wide range of
acceptor substrates (for a review, see Ref. 1). Recently,
three-dimensional structural information has become available with the
structures of two aminoglycoside N-acetyltransferases from
Serratia marcescens (SmAAT) (2) and Enterococcus
faecium (EfAAT) (3); five histone acetyltransferases (HATs): PCAF
(4), HAT1 (5), Tetrahymena GCN5 (tGCN5) (6, 7), yeast GCN5 (yGCN5) (8), and Hpa2 (9); and one arylalkylamine
N-acetyltransferase, the serotonin
N-acetyltransferase (AANAT) (10). These structures have
revealed a structurally conserved GNAT core that is largely involved in
AcCoA binding and incorporates elements from the four conserved
sequence motifs (A to D) initially identified by sequence analysis (11,
12).
Although the GNAT AcCoA binding site is well documented, the binding
site of the acceptor substrate has been characterized in only two
cases: the histone binding site in the tGCN5-CoA-H3 peptide complex
structure (7) and the serotonin binding site in the AANAT-bisubstrate
analog complex structure (10). These two structures also provide the
first glances into the catalytic machinery of GNAT. Yet, a better
understanding of the diverse modes of acceptor substrate recognition
and the catalytic mechanism of the GNATs as well as further insights
into the evolution of this superfamily still await additional
structural studies of GNAT-substrate complexes.
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) is a
novel amino-sugar N-acetyltransferase member of the GNAT
superfamily. GNA1 holds a key position in the pathway toward de
novo synthesis of the essential metabolite UDP-GlcNAc and is
present in various eukaryotic organisms (Fig. 1A). The GNA1
murine homologue, EMeg32, which possesses an extra 31-residue
NH2-terminal region compared with the yeast
homologues, has recently been characterized (13). EMeg32 is essential
for embryonic development, and its inactivation in mouse embryonic
fibroblasts generates resistance to apoptotic stimuli and defects in
cell proliferation (14). GNA1 has also been characterized in yeast (15)
and shown to be essential to the survival of Saccharomyces
cerevisiae in which it controls multiple cell cycle steps (16). In
addition, Candida albicans GNA1 null mutants exhibit reduced
virulence when injected into mice (17), making GNA1 a potential target
for the development of new antifungal agents.
We present here three high resolution crystal structures of GNA1. The
apo GNA1 and the binary GNA1-AcCoA complex structures were solved
independently using MAD techniques and refined to 2.4 and 1.3 Å resolution, respectively (Table I). The structure of the
GNA1-CoA-GlcNAc6P ternary complex was solved by molecular replacement
and refined to 1.8 Å resolution (Table
I). These three structures reveal GNA1
catalytic features and provide the first complete picture of an
amino-sugar GNAT active site as a first step toward the development of
specific inhibitors.
Expression and Purification of the Native and Selenomethionyl
Proteins--
Full-length GNA1 was amplified from genomic S. cerevisiae DNA, cloned into the pQE30 expression vector, and
transformed into M15pREP4 (Qiagen) Escherichia coli cells.
Protein expression was induced with 0.1 mM
isopropyl-1-thio- Crystallization--
Crystals of the native and selenomethionyl
protein were grown at 20 °C using the hanging-drop vapor diffusion
method by mixing equal volumes of the protein solution and the
reservoir, which contained 17-22% polyethylene glycol 600 and 0.2/0.4
M imidazole/malate, pH 5.1. Crystals obtained from the apo
protein belong to the orthorhombic space group
P212121 and contain 6 molecules/asymmetric unit. Those obtained from GNA1 preincubated
overnight with 1 mM AcCoA or CoA belong to the monoclinic
space group C2 and contain 4 molecules/asymmetric unit. For data
collection, the crystals were transferred to a reservoir solution
containing 32% polyethylene glycol 600 and flash-frozen at 100 K in
gaseous nitrogen.
Data Collection--
A three-wavelength MAD experiment was
performed at the ESRF beamline ID14-4 (European Synchroton
Radiation Facility, Grenoble, France) on both the AcCoA-complexed and
apo selenomethionyl protein crystals at 2.8 and 3.2 Å resolution,
respectively. High resolution data sets were collected at 2.4 and 1.3 Å resolution on an apo GNA1 crystal and a GNA1-AcCoA complex crystal,
respectively, on beamline ID14-2 (ESRF, Grenoble). A 1.8 Å resolution data set was collected at EMBL-X31 (DESY, Hamburg)
from a GNA1-CoA complex crystal soaked in the reservoir solution
supplemented with 1 mM GlcNAc6P for 6 days.
Structure Solution, Model Building, and Refinement--
All data
were processed and reduced using DENZO (19) and the CCP4 program suite
(20). The apo and AcCoA-complexed GNA1 structures were solved
independently using SOLVE (21). The experimental MAD electron density
maps were improved by solvent flattening, non-crystallographic
symmetry averaging, and phase extension with the program DM (20). The
apo GNA1 model was built manually using TURBO-FRODO (22). The
GNA1-AcCoA complex model was built automatically using ARP/wARP (23).
These two models were refined against their respective high resolution
data set using the program CNS (24), including bulk solvent and
anisotropic B-factor corrections. NCS restraints were used only for the
apo GNA1 model. High temperature factors and weak electron density maps
are associated with residues Gln-52 to Lys-57 in the two models. The 3 intertwined dimers of the apo GNA1 model have an average root mean
square deviation of 0.7 Å for all C
Figs. 1, 2, 4, and 5 were generated with SPOCK (26) and Raster3D (27)
except for Fig. 1A, which was computed with Alscript (28).
Overall Structure--
The three-dimensional structures of GNA1 in
its apo state and complexed forms with AcCoA or with CoA and GlcNAc6P
have been solved and refined at 2.4, 1.3, and 1.8 Å resolution,
respectively. Overall, the electron density is well defined for these
structures (Fig. 1B) except
for a surface loop comprising residues Gln-52 to Lys-57 (cf.
"Experimental Procedures"). As predicted from sequence analysis,
GNA1 shares structural similarities with other GNAT superfamily members
(1, 11). The GNA1 fold consists of a central core, composed of a mixed
5-stranded
The GNA1 structure is dimeric in the crystal as well as in solution, as
attested from gel filtration data (not shown). The crystalline dimer is
made of two intertwined GNA1 monomers in which strand The Cofactor Binding Site--
In each subunit of the GNA1 dimer,
AcCoA is positioned in a large hydrophobic cleft located at the site
where the two parallel strands,
AcCoA adopts a conformation similar to that described in other
AcCoA-complexed GNAT structures (1). The acetyl group of AcCoA, which
marks the active site, is located between strand
Superimposition of the apo and AcCoA-complexed GNA1 structures
shows that AcCoA binding induces subtle structural rearrangements that
are confined to the edges of the cleft and result in a slightly narrower cleft. Residues 102-109 in the The Acceptor Substrate Binding Site--
The GNA1 amino-sugar
binding site exhibits an atypical architecture, as it is built at the
dimer interface and involves residues from the exchanged Catalytic Mechanism--
Several reports on GlcN6P
N-acetyltransferases suggest that catalysis requires
sulfhydryl group-containing residues, such as a cysteine that could act
as a nucleophile in a two-step mechanism involving the formation of a
covalent acetyl-cysteine enzyme intermediate (35, 36). In contrast,
structural and kinetic data available for GNATs support a mechanism
proceeding through a direct nucleophilic reaction of acceptor substrate
on AcCoA (1, 32, 33).
In the GNA1-AcCoA complex structure, the cysteine residue closest to
the AcCoA acetyl group lies 6.5 Å apart, too far to play a role in
acetyl transfer. Furthermore, no other appropriate nucleophile residue
is found in the proximity of the acetyl group, which makes the
formation of an acetyl-enzyme intermediate very unlikely and supports
the hypothesis of a single-step mechanism as suggested for GNATs.
Consistent with this hypothesis is the position of the amino group of
the product GlcNAc6P (similar to that of the substrate GlcN6P;
cf. "Experimental Procedures"), which is ideal to
allow a direct nucleophilic attack at the AcCoA carbonyl (Fig. 2B). In addition, the nucleophilic character of the amine is
enhanced by the hydrogen bond it establishes with the backbone carbonyl of Asp-134 (Fig. 2C). The AcCoA carbonyl is polarized via
hydrogen bonds to the backbone amides of Asp-99 and Ile-100, located in the oxyanion hole, a feature that facilitates the nucleophilic attack
and stabilizes the negative charge building up on the oxygen atom of
the tetrahedral reaction intermediate (Fig.
3). Finally, the Tyr-143 hydroxyl group,
which lies within hydrogen bond distance of the AcCoA sulfur atom (Fig.
2B), could serve to stabilize the thiolate anion of the
departing CoA molecule. Tyr-143 also establishes hydrophobic contacts
with the acetyl group, probably playing a role in correctly positioning
the acetyl group for the reaction. A critical role of Tyr-143 in
catalysis is supported by mutagenesis data (15). A close inspection of
the active site of GNA1 also pinpoints two significant structural and
functional differences with others members of the GNAT superfamily.
The first striking difference resides within the GNA1
The second important difference concerns the deprotonation of the
acceptor substrate amino group prior to the reaction. In the case of
tGCN5 and AANAT, a chain of well ordered water molecules, or
"proton wire," connecting the acceptor substrate amino group to the
proposed catalytic bases tGCN5 Glu-122 or AANAT His-120 was suggested
to be involved in this proton removal (7, 10). In the GNA1-CoA-GlcNAc6P
complex, a similar proton wire is observed, leading to Glu-98 in which
the side chain occupies a similar position as that of tGCN5 Glu-122 and
AANAT His-120. However, the E98A mutation does not abolish the
GNA1 activity (15), suggesting that Glu-98 might not function as the
general base. Nevertheless, deprotonation prior to the reaction might
not be necessary in the case of GNA1, because the
pKa of GlcN6P (~7.75) is lower than that of other
GNAT acceptor substrates such as lysine (8.95) or serotonin (~10).
This hypothesis is also supported by the fact that the optimum pH of
purified mammalian GlcN6P N-acetyltransferases lies in the
alkaline range (35, 36) and by the lower Km value of
S. cerevisiae GNA1 for GlcN6P at pH 8 than at pH 7.5 (13),
suggesting that GNA1 may preferentially bind the basic/deprotonated form of GlcN6P.
Substrate Specificity among GNATs--
Although the GNAT enzymes
share structural similarities, they have distinct acceptor
specificities, consistent with their implication in various biological
processes. A comparative analysis of the complexes of
GNA1-CoA-GlcNAc6P, tGCN5-CoA-H3 peptide (7), and AANAT-bisubstrate
analog (7, 10) highlights the structural determinants responsible for
the substrate specificities among GNATs. Importantly, this knowledge is
essential for the design of specific inhibitors for medical applications.
The structural comparison of these three complexes reveals that
both the NH2- and COOH-terminal regions diverge between the different GNAT structures and are important for substrate specificity. The NH2-terminal structural differences concern the
In GNA1, the shorter Role in the Cell Cycle--
GNA1 was shown to control multiple
cell cycle steps in S. cerevisiae (16). It is still unclear
whether this role is related to the N-acetyltransferase
activity of GNA1 in UDP-GlcNAc biosynthesis (which implies a
physiological link between UDP-GlcNAc and cell cycle progression) or if
it is the consequence of an additional function of GNA1.
The hypothesis of an additional HAT activity for GNA1 was
addressed, but no HAT activity could be detected in vitro
(15). Interestingly, a comparison of GNA1 with the related GNAT
structures reveals that the closest structural homologue of GNA1 is the
HAT Hpa2, which also adopts an intertwined dimeric structure (9). Superimposition of the two structures reveals differences in the relative arrangement of the two subunits, resulting in different acceptor substrate binding sites. A narrow open-ended channel, in which
the histone tail could insert, is found in the Hpa2 structure instead
of the rounded pocket of GNA1, which seems unlikely to accommodate an
extended and bulky histone tail.
Could GNA1 fulfill an additional function via the noncovalent
association with a particular cell compartment or with a protein partner? An association with the cytoplasmic face of organelle membranes has been described for EMeg32 (the GNA1 murine homologue) which also co-purifies with the cdc48 homologue protein (p97/VCP) (13). Double-hybrid systematic experiments performed in S. cerevisiae revealed interactions between GNA1 and a
priori unrelated or unknown proteins (38). Further biochemical
experiments are needed to determine the biological relevance of these
protein/membrane interactions.
UDP-GlcNAc is a key precursor of chitin (a component of the yeast and
fungal cell wall) as well as of the glycosylphosphatidylinositol anchor
of membrane-bound proteins and is essential to N-linked glycosylation and O-GlcNAc modification of proteins.
Glycosyltransferases involved in N-glycosylation, such as
the yeast GPT/alg7, which uses UDP-GlcNAc as a substrate,
have been suggested to play a role in the cell cycle (39). In addition,
a recent report shows that EMeg32-dependent UDP-GlcNAc
levels influence cell cycle progression and apoptosis signaling (14).
Hence, the role of GNA1 in cell cycle progression appears to be linked
to its key GlcN6P N-acetyltransferase activity in de
novo UDP-GlcNAc biosynthesis. The structural data presented here
have allowed us to propose a catalytic mechanism for GNA1, as well as
providing a structural template for GNA1 homologues and related
aminoglycosides GNATs. Finally, these results further exemplify the
remarkable diversity of the GNAT superfamily and represent a critical
step toward the development of specific inhibitors.
-intertwined, dimeric assembly with the GlcNAc6P binding site
located at the dimer interface but also shed light on the catalytic
machinery of GNA1 at an atomic level. Hence, they broaden our
understanding of structural features required for GNAT activity,
provide structural details for related aminoglycoside N-acetyltransferases, and highlight the adaptability of the
GNAT superfamily members to acquire various specificities.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Structural statistics
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside for 20 h at 37 °C. Selenomethionyl GNA1 was produced as previously published (18). The recombinant His-tagged native and selenomethionyl enzymes were purified via nickel affinity and anion exchange
chromatographies, dialyzed against 10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM dithiothreitol, and
concentrated to 10 mg/ml. The activity of these enzymes was verified.
atoms. In the GNA1-AcCoA
complex model, the root mean square deviation value between the 2 dimers is 0.4 Å for all C
atoms. The GNA1-CoA-GlcNAc6P complex
structure was obtained from a rigid body refinement using the
GNA1-AcCoA complex as a starting model. Fourier difference maps clearly
revealed the location of the bound CoA and GlcNAc6P in two of the four molecules. The structure of the GNA1-CoA-GlcN6P complex was also solved
at 2.5 Å; superimposition of the two ternary complexes (GNA1-CoA-GlcNAc6P and GNA1-CoA-GlcN6P) revealed that GlcN6P (the substrate) and GlcNAc6P (the reaction product) were positioned similarly. Because the GNA1-CoA-GlcNAc6P structure was obtained at a
higher resolution than that of GNA1-CoA-GlcN6P, we only considered in
the analysis the GNA1-CoA-GlcNAc6P complex structure. The
stereochemistry of the refined models was analyzed by PROCHECK (25); no
residue was found in the disallowed regions of the Ramachandran plot. The coordinates of apo, AcCoA-, and CoA-GlcNAc6P-complexed GNA1 have
been deposited in the Protein Data Bank (accession codes 1I21, 1I12,
and 1I1D).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sheet flanked by 4
-helices, and a COOH-terminal
strand
6, which is projected away from the central core (Fig.
2A).
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Fig. 1.
Sequence conservation and quality of the GNA1
structure. A, sequence alignment of GNA1 homologues.
Conserved and similar residues are highlighted with
black and gray backgrounds, respectively. GNA1
secondary structure elements forming the structurally conserved GNAT
core are shown in black. The sequence alignment for SmAAT
and Hpa2 is based on a structural comparison with GNA1. Subunit 1 residues involved in the GlcNAc6P and AcCoA binding sites are
identified by filled circles and filled
triangles, respectively, and are shown in black for the
residues making GNAT conserved interactions. The unfilled
circles indicate subunit 2 residues that complete the GlcNAc6P
binding site of subunit 1. The four GNAT sequence motifs are
boxed. B, stereoview of the 1.3 Å resolution
experimental, solvent-flattened, averaged electron density map
contoured at 1.25 around an AcCoA molecule.
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Fig. 2.
Structure of GNA1 and AcCoA, GlcNAc6P
binding sites. A, ribbon representations of the GNA1
fold (left) and the intertwined GNA1 dimer
(right). In subunit 1, the GNA1 secondary structure elements
forming the structurally conserved GNAT core are shown in
green, the exchanged -strand in yellow, and
the remaining structural elements in cyan. Subunit 2 is shown in magenta with its exchanged strand
6 in
red. The molecular surface of AcCoA-(B) and
CoA-GlcNAc6P-(C) complexed GNA1, oriented as in Fig.
1B (left view) and color-coded (B) as
in Fig. 1B, with the regions undergoing small structural
rearrangements upon AcCoA binding displayed under a transparent surface
(the cyan and yellow bonds refer to the apo and
AcCoA-complexed GNA1 models, respectively). AcCoA is shown with carbon
(white), nitrogen (blue), sulfur
(green), oxygen (red), and phosphorous
(purple) atoms. C, the color code is according to
the electrostatic potential with positive and negative charges shown in
blue and red, respectively. The essential
catalytic Tyr-143 is displayed through a transparent surface. CoA and
GlcNAc6P are shown with yellow carbon atoms. D,
stereoview of the GlcNAc6P binding site with residues from subunit 1 and 2 shown in cyan and magenta, respectively.
The dotted lines indicate hydrogen bonds. Residues within
the GNAT conserved
-bulge are displayed in green.
6 of one
subunit exchanges with the identical strand from the other subunit
(Fig. 2A). A
-strand exchange between subunits in a dimer
is an unusual feature among GNATs and has been observed only in the HAT
Hpa2 structure (9). In all other structurally characterized GNAT,
except Hat1 that lacks a
6 strand (5), the hinge loop preceding
strand
6 folds back onto its own subunit. This difference is
reminiscent of three-dimensional domain-swapped proteins in which the
loop that precedes the exchanged domain can switch from a closed to an
opened conformation thereby leading to either a monomeric or a dimeric
form (29). In the case of GNA1 or Hpa2, the
4-
6 loop is too
small to undergo such a conformational switch, and the dimeric assembly
is further stabilized by a hydrophobic interface, two features
that make three-dimensional domain swapping unlikely. Nonetheless, the
monomeric GNATs and the intertwined dimers of GNA1 and Hpa2 are most
probably related by divergent evolution from a common ancestor, and the
evolutionary mechanisms that have led to dimer formation may have
included three-dimensional domain swapping.
4 and
5, diverge because of a
-bulge in strand
4 that positions the side chains of Glu-98 and
Asp-99 on the same face of the
-sheet. The presence of this
-bulge is remarkably well conserved among GNATs, which suggests a
critical role for this structural element in the formation of the AcCoA
binding site.
5 and the
-bulge
and is largely stabilized by contacts with the protein; the two carbon
atoms contract hydrophobic interactions with residues Ile-100, Leu-133,
and Tyr-143, and the carbonyl oxygen inserts into an oxyanion hole
formed by the backbone amides of residues Asp-99 and Ile-100. Such an
oxyanion hole has been observed in the structurally related
N-myristoyltransferase (30) but is a unique feature within
the structurally characterized GNATs.
3-
5 loop and 134-143 in the
5-
4 loop plus the N-cap of
4 move by ~1.3 and 1.1 Å, respectively, toward the center of the cleft (Fig. 2A).
Whether these conformational changes, induced upon cofactor binding,
are a prerequisite for acceptor substrate binding as shown for other GNATs (31-33) needs to be ascertained by kinetic studies. A detailed comparison with other GNATs reveals that these rearrangements differ
from those reported for (i) tGCN5, in which the cofactor-binding cleft
opens slightly upon AcCoA binding to accommodate the histone tail (7);
and (ii) AANAT, in which a major rearrangement of the
1-loop-
2
region occurs upon AcCoA binding to complete the serotonin binding site
(10). Therefore, although the binding of AcCoA is similar among GNATs,
it induces different conformational changes that can contribute to the
specific binding of the acceptor substrate.
-strand,
two features found only in a few intertwined oligomeric structures such
as that of bovine seminal ribonuclease (34). GlcNAc6P binds at the base
of the AcCoA cleft within a small pocket that is lined mostly with
electronegative residues except for a patch of positively charged
residues that specifically accommodate the 6-phosphate group (Fig.
2B). Remarkably, the GlcNAc6P acetyl group is positioned
similarly to the cofactor acetyl group in the GNA1-AcCoA complex
structure (Fig. 2B). The sugar-6-phosphate establishes
numerous hydrogen bonds, mainly via side chain atoms, together with a
few hydrophobic contacts such as that found between Leu-27 and the
-face of the sugar ring (Fig. 2C). Superimposition of the
structures of the cofactor-complexed SmAAT (2) or EfAAT (3) (the two
structurally characterized aminoglycoside GNATs) on that of the
GNA1-CoA-GlcNAc6P complex shows that SmAAT Phe-51, EfAAT Trp-25, and
GNA1 Leu-27 are identically positioned, an observation that supports a
common functional role for these residues, thereby identifying an
aminoglycoside recognition feature of GNATs.
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Fig. 3.
Schematic representation of the proposed GNA1
catalytic mechanism.
-bulge which,
when compared with the
-bulges in other GNAT structures, shows a
markedly different hydrogen bonding pattern. The GNAT
-bulge is an
irregularity of the antiparallel
structure in which two residues on
one strand are facing a single residue on the other strand (37). Such a
-bulge is formed when two consecutive residues in a
-strand
direct their backbone carbonyl or amide toward the same side of the
strand, thereby breaking the typical alternated pattern of a
-structure. In GNA1, the backbone amides of Asp-99 and Ile-100 are
projected toward the active site and form the oxyanion hole, whereas
the carbonyls of Glu-98 and Asp-99 point toward
3. In all other
members of the GNAT superfamily (except EfAAT in which a proline
perturbs the bulge conformation (3)), the situation is reversed; the
oxyanion hole is absent because the two consecutive backbone amides are
now directed toward strand
3 (establishing an hydrogen bond with the
backbone carbonyl of the facing residue), whereas two consecutive main
chain carbonyls are found pointing into the active site (Fig.
4). These two carbonyls have been
suggested to play a role in acceptor substrate binding in tGCN5 (7) and
in the stabilization of catalytic water molecules in AANAT (10); this
indicates that, in addition to a common role in structuring the
cofactor binding site, the
-bulge could also fulfill other
nonconserved catalytic functions.
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Fig. 4.
The -bulge structure
in GNA1 and comparison with another GNAT superfamily member. The
structure of GNA1-CoA-GlcNAc6P (thick sticks with
light gray carbons) is superimposed with that of
AANAT (thin sticks with dark gray carbons) in the
vicinity of the
-bulge. Nitrogen and oxygen atoms from the protein
main chain or the acetylated amine of GlcNAc6P are shown in
black. Light and dark gray dotted
lines indicate hydrogen bonds in the structures of GNA1 and AANAT,
respectively.
1-loop-
2 region and are relatively minor, whereas more dramatic
changes occur in the COOH-terminal end. In AANAT, the
4-
6 loop
orients toward the active site as it folds back on its own subunit.
This loop, along with the
1-
2 loop, almost covers the active
site, thus facilitating the binding of a hydrophobic substrate (Fig. 5A). In tGCN5, the
20-residue segment inserted between
4 and
6 contributes to one
side of the substrate binding canal, providing specific binding
residues for the histone tail (Fig. 5B).
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Fig. 5.
Structural comparison of substrate binding
sites of AANAT, tGCN5, and GNA1. The molecular surfaces
around the active site of the AANAT-bisubstrate analog (A),
the tGCN5-CoA-H3peptide (B), and the GNA1-CoA-GlcNAc6P
complex structures (C) are viewed with a similar
orientation. From top to bottom, the
serotonin-like moiety and the acetyl group are shown in red,
the H3 peptide backbone in yellow, with its reactive Lys-14
side chain in orange, and GlcNAc6P in purple.
Structural divergences within the NH2- and COOH-terminal
regions are highlighted in dark blue and
blue, respectively. The 3-
4 insertion loop unique to
AANAT is shown in brown.
4-
6 loop does not participate directly in
acceptor substrate binding, but it forces strand
6 to extend and
exchange with the identical strand of the other subunit in the dimer.
Instead of using this loop, GNA1 exploits its intertwined oligomeric
state to achieve specific binding, because an important part of the
GNA1 active site consists of residues from the other subunit in the
dimer (Fig. 5C). In contrast, this region is partly replaced
in monomeric AANAT by a long loop inserted between strands
3 and
4, which contributes largely to one wall of the serotonin binding
site (Fig. 5A). For the monomeric tGCN5, the short
3-
4 turn contributes to the canal-shaped active site designed to
accommodate a long peptidic chain (Fig. 5B).
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ACKNOWLEDGEMENTS |
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We thank Véronique Charrier, Bernard Henrissat, Pascale Marchot, Florence Fassy, and Gerlind Sulzenbacher for helpful discussions, Frédérique Pompéo for enzymatic assays, and the ESRF staff for technical support in data collection.
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FOOTNOTES |
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* This work was supported by grants from the CNRS to UMR 6098 (Marseille, Y. B.) and to UMR 8619 (Orsay, D. M.-L.).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 1I21, 1I12, and 1I1D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Holder of a CNRS Ph.D. fellowship.
To whom correspondence should be addressed. Tel.:
+33-4-91-16-45-08; Fax: +33-4-91-16-45-36; E-mail:
yves@afmb.cnrs-mrs.fr.
Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M009988200
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ABBREVIATIONS |
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The abbreviations used are: GNAT, Gcn5-related N-acetyltransferase; HAT, histone acetyltransferase; AANAT, serotonin N-acetyltransferase; MAD, multi-wavelength anomalous dispersion.
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REFERENCES |
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