From the Hauptman-Woodward Medical Research
Institute, Buffalo, New York 14203, the § Roswell-Park
Cancer Institute, Buffalo, New York 14263, the ** Pontificia Universidad
Catolica de Chile, Santiago, Chile, the
§§ Karolinska Institutet, Stockholm SE-17177,
Sweden, and the ¶¶ Cornell High Energy Synchrotron Source,
Ithaca, New York 14853
Received for publication, September 27, 2000
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ABSTRACT |
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Acetylxylan esterase (AXEII; 207 amino acids)
from Penicillium purpurogenum has substrate
specificities toward acetate esters of D-xylopyranose
residues in xylan and belongs to a new class of Plant cell wall hemicelluloses are complex mixtures of
heteropolysaccharides. Their main component is xylan, which is composed of a linear chain of Purification and characterization of AXEs from other xylanolytic
microorganisms have been described previously (4, 5). Although
considerable amounts of work have been performed on the enzymology of
xylanases and AXEs (6-10), the structure-function characterization of
this new class of esterases with regard to their catalytic activities
and substrate specificities is yet to be carried out. We reported the
crystallization and structure determination using the room temperature
data of the first member of the family, P. purpurogenum
AXEII (11-13). Recently, crystallization and preliminary diffraction
studies of the catalytic core of Trichoderma reesei
AXE have been published (14). Here we present for the first time
the complete description of the three-dimensional structure of P. purpurogenum AXEII and its active site at 0.90-Å resolution and
85 K, compare it with the 1.10-Å structure (Protein Data Bank Code:
1BS9) determined at 295 K (room temperature), and investigate the
structural basis for its acetyl D-xylopyranose specificity.
Crystallization and Data Collection--
The enzyme was
crystallized from ammonium sulfate solution in 50 mM
citrate buffer at pH 5.3 (11). Diffraction data were collected at the
A-1 station of Cornell High Energy Synchrotron Source using a 2000 × 2000 pixel charge-coupled device detector. The crystal (0.5 mm × 0.6 mm × 0.1 mm) was flash-frozen in a stream of
liquid nitrogen vapor using a mixture of glycerol and polyethylene glycol as the cryoprotectant. The x-ray beam was tuned to the wavelength 0.920 Å. The entire data collection was carried out in two
separate modes, a high (better than 1.6 Å) and a low resolution. A
total of 403 frames of data were collected from two crystals, yielding
420,882 observations and 95,343 unique reflections between 99.0- and
0.90-Å resolution, an 86.6% complete set in the resolution range. The
intensities were measured and processed with DENZO software package
(15). Table I gives a summary of the
results from the diffraction experiment.
Structure Solution and Refinement--
Details of structure
determination were previously described (12). Briefly, the crystal
structure of AXEII was determined by the single isomorphous replacement
and anomalous scattering (SIRAS) method using an iodine derivative. A
complete atomic model of the protein was built into this SIRAS map.
This starting model was refined separately with both the 1.10-Å and
the 0.90-Å data sets collected at 295 K and 85 K, respectively. The
refinements were carried out first with XPLOR (16) and then with
SHELX97 (17), implemented on a Silicon Graphics Indigo2 workstation. We
describe here the procedure carried out with the cryogenic data. The
initial XPLOR refinement was conducted using a 2.0
In contrast to other high resolution structures in which there were
small regions of disorder (19, 20), neither the 295 K nor the 85 K
structure of AXEII shows any weak or poorly defined electron density
usually associated with dynamic disorder. The difference between the
low and high temperature structures appears to be in the number of
amino acid side chains with multiple conformations. The 85 K structure
has more multiply observed side chains. However, this could also result
from better modeling of side-chain conformations owing to better data
quality and higher resolution. The low temperature structure also shows
more ordered solvent molecules, with higher number of tightly bound
water oxygens and sulfate ions, including the sulfate at the active
site, which is absent in the 295 K structure.
Description of the Secondary and Tertiary Structure--
The
crystal structure of AXEII and the catalytic triad
Ser90-His187-Asp175 are shown in
Fig. 1a. About 60% or 125 of
its 207 amino acids are distributed in ten
The N terminus of the polypeptide chain is anchored at the second
residue by a disulfide bond and then flows into a long strand, Comparison with Fusarium solani Cutinase--
The only other known
structure that is similar to AXEII is that of cutinase, an esterase
that hydrolyzes cutin, a polyester component of the waxy layer
of a plant's cuticle. The structure of cutinase has been determined at
1.0-Å resolution (20). Fig. 2 shows a
superposition of backbones of AXEII and cutinase. Table III is an alignment of the AXEII amino
acid sequence (7) with that of cutinase based on the secondary
structures. In comparison with AXEII, cutinase has 29 additional
residues at the N terminus of which the first 16 belong to a signal
peptide. The next 13 are present in the crystal structure, where
residues are numbered from 17 to 213 (197 amino acids; Protein Data
Bank code: 1CEX). Although the overall similarity between the two
tertiary structures is striking (the root mean squared deviation for
170 C
The secondary structure elements of the two proteins are otherwise
quite similar, except for occasional insertion or deletion of few
residues and their spatial displacement. The The Active Site and Multiple Conformations of Catalytic
Residues--
The active site gorge (Fig. 1a) is bordered
by the following segments of the tertiary structure:
Of the catalytic residues, Ser90 and His187
side chains are fairly exposed to the solvent (each generating 7.1 and
5.7 Å2 of solvent-accessible surfaces, respectively). In
contrast, Asp175 is shielded from the solvent by the helix
Conformational states A and B for the catalytic Ser90 and
His187 exist at averages of 40 and 60%, respectively, of
the volume of the crystal. The A state is not significantly different
from the 1.10-Å structure at 295 K (12). A to B transition
necessitates some rearrangement of other residues of the active site as
well. The most notable of them is the movement of Tyr177,
which is at a van der Waal's contact distance from the
His187 side chain in the A state. Tyr177 moves
about 2 Å away to accommodate His187 in the B state (Fig.
4). The transition to the B state
at 85 K could have been initiated by binding of a sulfate ion to the His187 N
An oxyanion binding site is comprised of two backbone amide groups of
Thr13 and Gln91, as well as Ser90
O Other Residues in Alternate Conformations--
In addition to
Ser90, Tyr177, and His187, 10 serine (residues 31, 36, 50, 58, 70, 74, 120, 160, 196, and 204) and
Gln154 side chains possess two alternate conformations.
These were identified from positive and negative difference electron
density maps. The occupancies of the major conformations range between
55 and 75%. All of the alternate conformers have hydrogen bond-forming
partners, either protein or solvent atoms, in both conformations. Side
chains of the rest of the amino acid residues have well characterized electron densities that uniquely define their conformations. Unlike other high resolution structures, no dynamically disordered region of
the polypeptide chain is identifiable. Only the residues at two termini
have significantly higher than average temperature factors; their
average equivalent isotropic temperature factors are 25 and 45 Å2 for main-chain and side-chain atoms, respectively. The
average equivalent isotropic temperature factor for the rest of the
protein C, N, and O atoms is about 12 Å2.
Packing Interactions in the Crystal--
One of the closest
packing interactions between secondary structure elements in the
crystal occurs between
Intermolecular hydrogen bond formation between two alternate
conformations of serine O
A packing interaction that involves nonpolar contacts occurs in the
region where the short helix The Active Site and Possible Binding Modes of the
Substrate--
Coexistence of two different conformational states,
perhaps representing two oxidation states of catalytic Ser and His
residues in the same crystal, is a major finding that was only possible due to extraordinarily high resolution of the study. In the state A,
which is the resting state of the enzyme resembling the geometry of the
side chains in the 295 K structure (12), it is likely that
Ser90 O
In an attempt to comprehend substrate-protein interactions, a 2-acetyl
xylopyranose molecule, a substrate for AXEII (3), was manually docked
at the active site in one of at least two possible orientations of the
pyranose ring (Fig. 6). In this position, Ser O
Other residues that the substrate may have direct contact with are
Tyr57, Gln91, and Phe152 (Fig. 6).
In the active site gorge, which is about 20 Å long to the back wall
containing the anti-parallel loop
It is possible that the loop Accessibility of the Active Site--
As described under
"Results" and in the discussion above, the active site gorge of
AXEII is more exposed than the only other known structure of the same
family, cutinase. With a water molecule as the probe, the accessibility
surface for the catalytic Ser and His is more for AXEII (12.8 Å2) than for cutinase (7.9 Å2). In contrast,
catalytic residues are relatively buried in cutinase, as has been
illustrated in Fig. 8. Not only the Ser90 and
His187 side chains are more exposed, but also the gorge
stretches wider in AXEII than in cutinase. A few side chains, such as
Tyr177, Tyr57, and Phe152 cover the
gorge, as shown in Fig. 8, reducing its accessibility. Furthermore, the
gatekeeper loop limits the access to the gorge for the xylan
chain. However, as has been pointed out, some of these residues and the
loop belong to flexible regions of the molecule, and it is likely that
they undergo movement to accommodate a xylan chain. The openness of the
active site is suggestive that AXEII is a "pure" esterase as
opposed to a lipase that functions as an esterase on esters of
long-chain fatty acids or substrates containing lipid-like side groups.
AXE II, in contrast, uses polar molecules as substrates, as an AXE should.
Molecular Packing--
Despite the overall compact nature of
the AXEII molecule, 82 of 207 residues are located in loops and turns.
Although each of five disulfide bridges involves at least one Cys from
loops and turns, three have both Cys residues belonging to
extended loop regions of the molecule, namely /
hydrolases. The
crystal structure of AXEII has been determined by single isomorphous
replacement and anomalous scattering, and refined at 0.90- and 1.10-Å
resolutions with data collected at 85 K and 295 K, respectively.
The tertiary structure consists of a doubly wound
/
sandwich,
having a central six-stranded parallel
-sheet flanked by two
parallel
-helices on each side. The catalytic residues
Ser90, His187, and Asp175
are located at the C-terminal end of the sheet, an exposed
region of the molecule. The serine and histidine side chains in the 295 K structure show the frequently observed conformations in which Ser90 is trans and the hydroxyl group is in the
plane of the imidazole ring of His187. However, the
structure at 85 K displays an additional conformation in which
Ser90 side-chain hydroxyl is away from the plane of the
imidazole ring of His187. The His187 side chain
forms a hydrogen bond with a sulfate ion and adopts an altered
conformation. The only other known hydrolase that has a similar
tertiary structure is Fusarium solani cutinase. The exposed
nature of the catalytic triad suggests that AXEII is a pure esterase,
i.e. an
/
hydrolase with specificity for nonlipidic polar substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(1
4)glycosidic-linked
D-xylopyranoses, having various substitutions at carbon 2 and 3 positions (1). Biodegradation of xylan is a complex process
catalyzed by several fungal and bacterial enzymes (2). Although the
linear chain is cleaved by endoxylanases and
-xylosidases,
acetylxylan esterases (AXE)1
hydrolyze O-acetyl substitutions of
D-xylopyranose moieties. Penicillium
purpurogenum secretes at least two forms of AXEs, I and II, that
demonstrate substrate specificities toward acetate esters of
D-xylopyranose and belong to a new class of
/
hydrolases (3).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Data collection statistics
cut off on
structure amplitudes between 99.0 and 0.90 Å, which included 94,055 reflections. The refinement process consisted of 500 cycles of
positional refinement followed by simulated annealing starting at 3000 K. Individual atomic temperature factors were refined isotropically
yielding an R factor of 0.333. The refinement was further
continued with SHELX97. Twelve cycles of conjugate gradient least
squares minimization using SHELX97 were carried out leading to a
convergence. A test data set of 5% of the total was used for
calculating Rfree. During first six cycles, the
model was refined isotropically and 290 fully occupied solvent waters and 3 sulfate ions were included in the model. Multiple conformers of
serines 31, 36, 50, 58, 70, 74, 90, 120, 204 and tyrosine 177 were also
included in the model. At this stage of the refinement, the
R factor was 0.154 with an Rfree of
0.169. The seventh cycle of minimization included modeling of the
disorder of Ser160, Ser196, and
Gln154 side chains. The following cycles of anisotropic
refinement of all nonhydrogen atoms reduced the R factor by
0.04 and Rfree by 0.035. Modeling of the
multiple occupancies of His187 and further addition of
solvent led to the convergence of the refinement process at an
R factor of 0.107 for all data. All the computer graphics
work was performed on a Silicon Graphics Indigo2 workstation running
CHAIN (18). A summary of refinement results is provided in Table
II.
Refinement statistics
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-strands (
1 to
10)
and six
-helices (
1 to
6). The remaining 82 residues are
distributed in five type 1 and 2
-turns, one
-hairpin turn, and
five extended loop regions. Fig. 1b is a schematic showing
the topology of the secondary structure of AXEII. The strands, loops,
and helices have been numbered based on their sequence of occurrence in
the polypeptide. All ten cysteine residues are involved in five
intrachain disulfide bridge formation
(Cys2-Cys79,
Cys46-Cys55,
Cys101-Cys161,
Cys147-Cys179, and
Cys171-Cys178). These disulfide bridges involve
at least one cysteine in the loop regions. The tertiary structure
consists of a doubly wound
/
sandwich, having a central parallel
-sheet flanked by two parallel
- helices on each side. The
catalytic cleft is located at the C-terminal end of the
-sheet near
the center, bordered by helical residues 183-193 from one side and the
loop 105-113 containing an anti-parallel pair of strands from the
other. The geometry and relative orientations of side chains of the
catalytic triad are similar to those previously observed in other
members of the
/
hydrolase family, such as in cholesterol
esterase (21).
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Fig. 1.
a, a ribbon diagram of AXEII as
determined at 85 K and 0.90-Å resolution. The secondary
structures are labeled and color-coded.
The catalytic residues and a bound sulfate ion are shown at the active
site. Carbon atoms are shown in gray, nitrogen in
blue, oxygen in red, and sulfur in
yellow. Disulfide bonds are also shown in yellow.
For clarity, His187 side chain is shown only in the A state
(see text for details) (drawn by SETOR (23)). b, a schematic
diagram of the topology of AXEII and distribution of the secondary
structure elements. and
denote helices
and strands while
denotes loops. The N and C termini of
secondary structure elements are numbered. Open
cylinders represent helices below the central
-sheet, and
shaded cylinders represent the ones above.
1.
The C-terminal end of
1 is adjacent to the catalytic serine at the
active site. The polypeptide chain then describes a loop structure,
loop 1 (
1) between residues 13 and 20, before entering the helix
1. After completing the terminal strand
2 of the central
-sheet via a right-handed
-crossover, the polypeptide chain forms a long loop,
2, between residues 41 and 55, the conformation of which is stabilized by an intra-loop disulfide between
Cys46 and Cys52. The helix,
2, the longest
secondary element in AXEII, comprises residues 56 through 79. The
C-terminal Cys79 of
2 is involved in the disulfide
bridge with Cys2, which anchors the N terminus.
3,
having residues 83-89, is the central strand at the C-terminal end of
which resides the catalytic Ser90 residue. The backbone
conformation of Ser90 belongs to the very restrictive
(+,
) quadrant of the Ramachandran plot, but otherwise is normal for a
catalytic serine in esterases and lipases (22).
3 turns sharply into
3, a three-turn helix, which is the only helix in the interior of
the molecule, surrounded completely by protein atoms. The C-terminal
Cys101 of the helix is disulfide-bridged to
Cys161 from loop
5. This is followed by a 17-residue
loop
3 between 101 and 117, containing a short anti-parallel segment
formed by
4 and
5. The loop ends in a two-turn helix
4
oriented nearly perpendicularly to the sheet, unlike other helices. The
next segment of the polypeptide chain between residues 126 and 171 contributes three strands
6,
8, and
10 with terminal residues
127-131, 142-145, and 167-171, respectively, to the central
-sheet, as well as to a short anti-parallel segment,
7 and
9,
consisting of residues 135-138 and 148-151, respectively, away from
the sheet. The intervening loop
4, the longest loop in the structure
between residues 152 and 166, is held in place by a disulfide bridge
between Cys161 and Cys101 from the C-terminal
end of
4. All of the secondary structure crossovers are
right-handed, whereas the
8-loop-
10 crossover is left-handed.
This segment of the polypeptide consisting of peripheral strands and
loops are held together by two additional disulfide bonds, between
Cys147 and Cys179, and between
Cys171 and Cys178, which also anchor the next
loop,
5 consisting of residues 172-183. Asp175 is
contributed by this loop to the catalytic triad. Held by two disulfide
bonds, the polypeptide chain makes a right-handed turn into a short
helix
5 (residues 184-189), which contributes the last of the
catalytic side chains, His187. This helix is separated from
the C-terminal helix
6 (residues 195-207) by five intervening residues.
atoms is ~1.2Å), long insertions and deletions,
substitutions, and altered disulfide bond structure account for a large
number of local conformational changes, especially in loops around the
active site. AXEII has five disulfide bonds, whereas cutinase has only
four of these cysteines bonded into two disulfides. All three of these
additional disulfides (Cys46-Cys55,
Cys101-Cys161, and
Cys147-Cys179) are involved in anchoring loops
surrounding the active site that are associated with large
conformational differences between two polypeptides. In contrast, loop
1 (residues 13-21) between
1 and
1, adjacent to the active
site but away from the substrate-binding cleft, is conformationally
quite similar to the one in cutinase. The conformations of
2
(residues 41-55) in these two structures are such that they project in
opposite directions: in cutinase more toward the interior, in the
direction of the active site, whereas in AXEII it is pulled outward
away from the active site by the disulfide bridge between
Cys46 and Cys52. A conformation of this loop
like the one in cutinase would be in direct steric conflict with
3
absent in cutinase. Also, the region 41-43 of the loop has a deletion
of three residues in comparison with cutinase, which shortens the path
to the disulfide bridge. The loop
3 consists of an insertion of
residues 101-115 that contain a short anti-parallel segment
(
4-
5). This segment of polypeptide is absent in cutinase. Its
strategic positioning at the rear wall of the active site opening
suggests its distinctive role in recognition and processing of acetate
side groups of a chain of D-xylopyranose as opposed to
cutin molecules by cutinase. The next major difference between the two
structures is in loop regions, including
4, which spans between
residues 148 and 166 for cutinase, corresponding to residues 132 and
166, respectively, in AXEII, in spatial positions. This 16-residue
insertion in this segment is distributed mainly between two short
anti-parallel strands
7 (135) and
9 (148), and the
sixth strand of the central
-sheet,
8. Not one of these features
of the region is present in cutinase. The presence of
Cys101-Cys161 disulfide in AXEII also
drastically alters the course of the backbone between residues 154 and
166, although these residues in the two proteins are analogous to each
other in spatial positions and sequence alignment. The loop
5
(residues 172-183 in AXEII and 172-184 in cutinase) contributes
Asp175 to the catalytic triad and has one conserved
disulfide (Cys171-Cys178); however, the
additional disulfide in AXEII (Cys147-Cys179)
alters the course of the main chain by pulling it away from the
catalytic cleft. In addition to one residue insertion between 179 and
184, the backbone of cutinase in this segment adopts a different path
such that the distance between C
positions of 181 in AXEII
and the equivalent 182 in cutinase is 10.4Å. This altered conformation
of the polypeptide chain in cutinase partially shields catalytic
Asp175 and His188 from exposure to the solvent,
in contrast to exposed catalytic His187 in AXEII. Besides,
a cutinase-like path for this segment of
5 in AXEII would put
residues 178 and 179 in steric conflict with the backbone of 150-152
of
4. Residue 184 marks the beginning of a two-turn helix
5,
which contains the catalytic histidine (His187 and
His188, in AXEII and cutinase, respectively) and is quite
similar in both structures. The C-terminal helix
6 in AXEII is
similar to that in cutinase, except for the fact that cutinase has four
additional C-terminal residues in the helix.
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Fig. 2.
Superposition of backbones of AXEII
(green) and cutinase (pink), shown
roughly in the same orientation as in Fig.
1a. Disulfide bonds are shown in
yellow.
Sequence alignment of AXEII and cutinase based on three-dimensional
structures
-sheet core of the two
structures superimposes almost perfectly, except for the terminal
strand
2 and the missing
8 in cutinase. The C
positions and
side-chain atoms of the catalytic residues superimpose with an
average deviation of 0.3 Å from each other.
1 (residues
13-20) and
2 (residues 41-55) to the left, in the view
in Fig. 1a;
3-to-
3 turn at the center of the gorge,
where the three side chains of the catalytic residues are located; and,
helix
5,
5 (residues 175-183),
6-to-
7 turn (residues
132-134), and
4 (residues 152-161) to the right. The
opening of the active site is ~11 Å across, 7 Å thick from the
surface to the catalytic triad, and has a depth of 20 Å to the back
wall delineated by residues of
3 (101). Residues lining the
gorge are Glu12, Tyr89, Ser21,
Thr24, Gly191, Tyr190,
Thr186, Pro176, Tyr177,
Gln188, Thr13, Thr14,
Gln49, Gly47, Tyr57,
Pro134, and Phe152. A sulfate ion, a glycerol,
and about 40 water molecules fill up the entire cleft.
5 and Pro176. Fig. 3
depicts the two conformations of the catalytic residues observed in the
0.90-Å crystal structure determined at 85 K. The gray and
green side chains of Ser90 and
His187 represent conformations A and B, corresponding to 40 and 60% of the molecules in the crystal, respectively. In the A
conformation, the most commonly observed in the resting state of
esterases and lipases, as well as in the 295 K structure of AXEII (12),
Ser90 side chain is trans (
1 =
172°), whereas the Ser90 O
and Asp175
O
1 atoms are in the plane of the imidazole ring of
His187 (deviations of these two atoms from the plane are
0.03 and 0.02 Å, respectively; root mean square deviation from
planarity of the ring is 0.05 Å). In the B conformation, the
Ser90 side chain is gauche (
1 =
65°) and the O
... N
2 His187 distance is
3.09 Å, at an approach angle of 102° to the imidazole ring. The two
His187 conformations have an average deviation of 0.40 Å with a maximum of 0.80 Å between two N
2 atoms. The (
,
) angles
of His187 A and B conformers are (
62°,
30°) and
(
60°,
20°), respectively, and, side-chain (
1,
2) torsion angles are (
164°, 61°) and (
164°, 51°), respectively.
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Fig. 3.
A view of the catalytic triad in A and B
states. Carbon atoms are shown in gray, nitrogen in
blue, oxygen in red, and sulfur in
yellow. Gray bonds depict the A and
green the B states. Bonds that belong to both states are
also shown in gray. The electron density shown is from a
final (2Fobs Fcalc)
map, contoured at 1.8
. The view is oblique to the imidazole ring of
His187.
2 atom through a hydrogen bond formation
(His187 N
2 ... O2 SO4 210: 2.75Å) (Fig.
5a). O2 of SO4 210 is
also hydrogen-bonded (2.75 Å) to water oxygen 473, which, in turn,
forms a hydrogen bond with Tyr57 side-chain hydroxyl (2.89 Å). The sulfate ion makes four additional hydrogen bond-forming
contacts with protein atoms as shown in Fig. 5a: O3 to
Thr13 O
(3.03 Å), to Thr13 backbone NH
(3.03 Å), and to Gln91 backbone NH (2.98 Å), and O4 to
Thr13 O
(2.75 Å). In addition, the O1 oxygen of sulfate
is coordinated to three water oxygens 465, 488, and 489 through strong
hydrogen bonds (average distance ~2.80 Å) in a tetrahedral manner,
all of which are linked to protein atoms by hydrogen bonds (to
Gln188 N
2, Thr13 backbone carbonyl, and
Ser90 O
; average distance ~2.84 Å). In the B state,
binding of the sulfate ion eliminates three water oxygens that are
present only in the A state, namely, 598, 599, and 600. O3 of the
sulfate ion replaces water oxygen 598. However, binding of the sulfate
ion does not affect water oxygens 465, 473, 488, and 489, all of which are strongly coordinated to protein atoms. These water molecules are
also present in the 295 K structure. There is an estimated net gain of
two hydrogen bonds in the transition from the A state to the B
state.
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Fig. 4.
Movement of Tyr177 from A
(gray) to B (green) states. The
electron density is from a final (2Fobs Fcalc) map, contoured at 1.0
.
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Fig. 5.
Hydrogen bonding interactions at the active
site of AXEII in (a) B states and (b)
A state. Side chains of residues Ser90,
His187, Asp175, Thr13, and
Gln91 in both states, as well as SO4 210 and water
598 in B and A states, respectively, are shown. Carbon atoms are shown
in gray, nitrogen in blue, oxygen in
red, and sulfur in yellow. Bonds in the B state
are shown in green.
and Thr13 O
hydroxyls in the A state, as shown in
Fig. 5b. The Thr13 side chain is stabilized by a
hydrogen bond from Gln91 N
2 (2.90 Å). The water oxygen
598 is thus bound at this oxyanion binding site in a roughly
tetrahedral geometry. In the B state, SO4 210 occupies the oxyanion
hole by replacing water 598; the position of O3 of the sulfate nearly
coincides with the oxygen atom of water 598. Furthermore, in the B
state, O2 of SO4 210 occupies what used to be the Ser90
O
position relative to the His187 imidazole ring in the
A state, thus maintaining an orientation to the imidazole ring similar
to that of O
in the A state.
1 of one molecule and
2 of another.
However, very little direct hydrophobic packing surfaces are involved
in these close contacts. Instead, several solvent molecule-mediated
interactions dominate this molecular interface. Notable among these
include a sulfate ion (213)-mediated contacts between Thr24
O
of
1 and Asn73 N
2 and Ser70 O
of
2. Several water molecules line this interface. The closest approach
between the helices is at Ala62 C
of
2, which is at a
van der Waal's contact distance (3.84 Å) from Ser31 C
of
1. In the second conformation, Ser31 O
also makes
a water-mediated (449) contact to OH of Tyr110 of
5 of
the same neighboring molecule. The backbone amide of N-terminal
Gly35 of
1 donates the proton to the backbone carbonyl
oxygen of Gly109 from
5, thereby forming an
intermolecular hydrogen bond. A second intermolecular hydrogen bond is
formed in this region between the backbone carbonyl of
Ser36 from
2 and Thr111 O
. In addition,
the C-terminal end of
2 is involved in two intermolecular hydrogen
bonds: Ser77 backbone carbonyl to Asn180 side
chain and Gln78 side chain to Gly48 backbone
carbonyl. Gln78 N
2 also accepts a proton from an
alternate conformation of Ser50 O
across the molecular
interface. Asn180, Ser50, and Gly48
are all from extended loop regions of the molecule. Gly53
C
from the disulfide stabilized loop
2 packs (3.83 Å) against Ala149 C
, from the adjacent molecule. Other short
contacts in this loop-loop interaction between two molecules include an
approach (3.07 Å) of Asn42 backbone carbonyl oxygen to
Gly139 C
and packing of Pro17 side chain
against Thr146 C
.
atoms is also observed at the
packing contact between the N terminus of helix
6 and the loop
4
region (residues 159-161) of a neighboring molecule. In one
conformation, Ser196 side chain forms a hydrogen bond to an
alternate conformation of Ser160 O
and in its second
conformation, the hydrogen bond is formed between the side chain and
the backbone amide of Ser160. Again, several water
molecules tightly hydrogen bonded to protein atoms are found at this
interface. The other two direct intermolecular contacts between these
two regions involve a hydrogen bond between Lys203 side
chain of
6 and Gly157 backbone carbonyl, and the packing
of Leu199 C
2 against Gly158 C
(4.16 Å).
Although the C terminus approaches the anti-parallel loop
3, there
is no direct contact between the two. Intermolecular contacts in this
region involve interactions between Lys83 side chain and
backbone carbonyl of Asn107, and between Ala81
backbone carbonyl and Asn107 side chain.
4 approaches
4 after
9. Met124 S
from
4 has a van der Waal's contact (3.75 Å) with Phe152 C
of the adjacent molecule. In the same
region, the carbonyl oxygen of Pro80 has a short contact
(3.25 Å) to C
of Phe152. Ser120 side chain
from
4, in its more occupied conformation, approaches the loop
5
that contributes Asp175 to the catalytic triad, by donating
the proton to the Pro176 backbone carbonyl oxygen. In
addition, side chains of residues Asn183 and
Ala185, near the N terminus of
5 packs against side
chains of Thr14 and Ala15 from
1 and
Ser51 from
2, respectively, of a symmetry-related
molecule. The crystal packing of AXEII is, thus, dominated by polar
interactions; there are only a few interactions among hydrophobic side chains.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
H is the proton donor in the Ser90
O
H ... N
2 His187 hydrogen bond. It was shown for
cutinase that N
2 is deprotonated (19). In the presence of a
negatively charged sulfate ion, which is a strong nucleophile and makes
a direct contact (2.75 Å) to His187 N
2, it is likely
that N
2 retains the proton due to increase in
pKa. (Efforts to locate this proton from the
difference electron density maps were unsuccessful, perhaps due to
complications from modeling the side chain in two alternate
conformations.) The B state, therefore, could be a mimic of the binding
of the tetrahedral intermediate to His in which the imidazole ring has an overall positive charge. Although the Ser side chain in the B state
assumes a catalytically "inactive" position, the proximity and
orientation of the Ser O
proton to the N
2 of His187
could contribute to the stability of the B state. The movement of
residues His187 and Tyr177, demonstrated by
multiple conformations of their backbone and side-chain atoms, is
perhaps reflective of the motion within the catalytic cavity during
intermediate steps of catalysis.
is oriented roughly normal to the acetate plane at a distance of 2.6 Å to the carbon atom. In both orientations, however, the pyranose ring stacks against the Tyr177 side chain.
Interestingly, the location of the pyranose ring nearly coincides with
the only glycerol molecule found in the active site. In this binding
mode, the acetate group is at a van der Waal's contact distance from
the backbone and side-chain atoms lining the pocket, namely
Thr13 NH and His187 CO and side-chain atoms of
His187, Glu12, Tyr89, and
Gln188. The acetate group thus fits nicely in this pocket,
which seems to be tailored to accommodate such a moiety. This
observation is consistent with the biochemical data that AXE II has
high specificity for the acetate ester and only weakly hydrolyzes
esters of longer fatty acids (3). Alternatively, a small substrate like
2-acetyl xylopyranose may flip around and bind in a direction opposite to the one shown in Fig. 6. The acetyl group would then face the longer
end of the active site, thus making room for longer fatty acid side
chains to bind and be hydrolyzed. However, in this mode the
xylopyranose moiety binds to the interior of the molecule, precluding
the possibility of a polymeric substrate such as an acetylated xylan
from binding at the active site.
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Fig. 6.
Docking of 2-acetyl xylopyranose in the
active site of AXEII. Side chains shown are Ser90,
His187, Asp175, Glu12,
Thr13, Tyr57, Gln91,
Phe152, Tyr177, and Gln188. Carbon
atoms are shown in gray, nitrogen in blue, and
oxygen in red. Disulfide bonds are shown in
yellow. In this orientation Ser90 O is
roughly normal to the acetate plane at a distance of 2.6 Å to the
carbon atom.
3 (see the description of the
active site in the result section), an estimated four to five xylose
residues of a xylan chain could be accommodated. If a xylan chain binds
at the active site, then xylose residues may additionally make contacts
with residues Ser58, Asp153,
Glu158, and Asn107.
3 containing the anti-parallel strand
4-
5 plays a special role in recognition and binding of a xylan
chain. This loop, absent in cutinase, is nearly 10 Å away from the
substrate atoms when a single molecule of xylose binds at the active
site (Figs. 7 and
8). However, it may serve as a gate to
the active site for binding of a xylan chain. Interestingly, the loop
is loosely anchored to the rest of the molecule by hydrogen bonds near
the turn, Gln108 N
2 ... O
1
Asp153 (2.92 Å), Asn107 N
2 ...
OC Phe152 (2.99 Å), and Asp105 O
1 ...
O
1 Glu94 (2.54 Å). There are several acidic side chains
in this region that are linked to each other by hydrogen bonds, as
shown in Fig. 7. These residues are, therefore, protonated at the
crystallization pH of 5.3. Deprotonation of these residues, coupled
with conformational changes, could trigger breakage of the hydrogen
bonds and release of the loop. This structural flexibility could be
critical to its role as the "gatekeeper" of the active site gorge
and recognition of a xylan chain.
View larger version (40K):
[in a new window]
Fig. 7.
Hydrogen bond formation by the anti-parallel
loop 3. Other interactions among charged
side chains in the area are also shown. Carbon atoms are shown in
gray, nitrogen in blue, and oxygen in
red. Disulfide bonds are shown in yellow.
View larger version (47K):
[in a new window]
Fig. 8.
Space-filling models of (a)
AXEII and (b) cutinase, viewed directly into the
active site gorge from bottom in Fig. 1a
(or Fig. 2). Catalytic Ser and His are shown in
red and blue, respectively. The Asp of the triad
is not visible. In a, Tyr177 and
Tyr57 are shown in pink, Phe152 in
green, and residues of the anti-parallel loop 3 in
yellow.
2,
3,
4, and
5. The internal rigidity of the AXEII molecule is a direct
consequence of well-packed secondary structure elements and
disulfide-stabilized loops and turns. Some of the loop regions are
highly polar and form parts of the outer surface. The central theme in
packing of AXEII molecules in the unit cell is: few hydrophobic and
many polar interactions, the presence of interfacial solvent molecules
and solvent-mediated protein-protein interactions, and strong loop-loop
contacts in several extended loop regions. These are all
characteristics of a tightly folded monomeric, soluble, globular
protein. A high long range order of the crystal could, however, be a
consequence of the molecule's overall shape and its highly polar
surface property that is conducive to close packing. The overall shape
of the molecule resembles a cylinder of an average diameter of 27 (±3)
Å and a height of 38 (±3) Å. These cylindrical molecules are packed
in the unit cell with the cylinder axes along the z axis,
which, interestingly, is also roughly the direction along which major helices and the central
-sheet are aligned.
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ACKNOWLEDGEMENT |
---|
We thank Prof. George M. Sheldrick of Göttingen University for providing us with a copy of the SHELX97 software.
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FOOTNOTES |
---|
* This work was partially supported by the John R. Oishei Foundation (to D. G.) and FONDECYT (Grants 1960241 and IR 7960006) (to J. E.).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 1G66) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Present address: Center for Advanced Research in
Biotechnology, Rockville, MD 20850.
Present Address: Department of Biochemistry, Michigan State
University, E. Lansing, MI 48825.
¶ To whom correspondence should be addressed: Hauptman-Woodward Medical Research Institute, 73 High St., Buffalo, NY 14203-1196. Tel.: 716-856-9600 (ext. 316); Fax: 716-852-6086; E-mail: ghosh@hwi.buffalo.edu.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M008831200
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ABBREVIATIONS |
---|
The abbreviations used are: AXE, acetylxylan esterase; SIRAS, single isomorphous replacement and anomalous scattering.
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REFERENCES |
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