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§1§ The ι-Carrageenase of Alteromonas fortis §1§
§2§ A β-HELIX FOLD-CONTAINING ENZYME FOR THE DEGRADATION OF A HIGHLY
POLYANIONIC POLYSACCHARIDE[15]* §2§
1. [16]Gurvan Michel[17]‡[18]§,
2. [19]Laurent Chantalat[20]‡,
3. [21]Eric Fanchon[22]‡,
4. [23]Bernard Henrissat[24]¶,
5. [25]Bernard Kloareg[26]§ and
6. [27]Otto Dideberg[28]‡[29]‖
1.
From the ^‡Laboratoire de Cristallographie Macromoléculaire, Institut
de Biologie Structurale Jean-Pierre Ebel, CNRS/Commissariat Å
l'Energie Atomique, 41, rue Jules Horowitz, 38027 Grenoble Cedex 1,
France, the ^§Station Biologique de Roscoff, UMR 1931 (CNRS and
Laboratoires Goëmar), Place Georges Teissier, BP 74, 29682 Roscoff
Cedex, France, and the ^¶Architecture et Fonction des Macromolécules
Biologiques, UMR 6098 (CNRS, Universités d'Aix-Marseille I et II), 31
Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
[30]Next Section
§2§ Abstract §2§
Carrageenans are gel-forming hydrocolloids extracted from the cell
walls of marine red algae. They consist ofd-galactose residues bound by
alternate α(1→3) and β(1→4) linkages and substituted by one
(κ-carrageenan), two (ι-carrageenan), or three (λ-carrageenan)
sulfate-ester groups per disaccharide repeating unit. Both the κ- and
ι-carrageenan chains adopt ordered conformations leading to the
formation of highly ordered aggregates of double-stranded helices.
Several κ-carrageenases and ι-carrageenases have been cloned from
marine bacteria. κ-Carrageenases belong to family 16 of the glycoside
hydrolases, which essentially encompasses polysaccharidases specialized
in the hydrolysis of the neutral polysaccharides such as agarose,
laminarin, lichenan, and xyloglucan. In contrast, ι-carrageenases
constitute a novel glycoside hydrolase structural family. We report
here the crystal structure of Alteromonas fortisι-carrageenase at 1.6 Å
resolution. The enzyme folds into a right-handed parallel β-helix of 10
complete turns with two additional C-terminal domains. Glu^245,
Asp^247, or Glu^310, in the cleft of the enzyme, are proposed as
candidate catalytic residues. The protein contains one sodium and one
chloride binding site and three calcium binding sites shown to be
involved in stabilizing the enzyme structure.
Carrageenans are the main components of the cell walls of various
marine red algae (Rhodophyta) where they play a variety of structural
(cell-cell cohesion and exchange boundary) and signaling (cell-cell
recognition) roles ([31]1, [32]2). They consist of linear chains of
galactopyranose residues in the d-configuration linked by alternating
α(1→3) and β(1→4) linkages. This regular structure is modified by
3,6-anhydro bridges and substitution with sulfate-ester groups. On the
basis of the level and position of sulfate substitution, carrageenans
are classified into four types, namely furcellaran and κ-, ι-, and
λ-carrageenans. κ-Carrageenan consists of repeated units of the
disaccharide
4-sulfate-O-1,3-β-d-galactopyranosyl-1,4-α-3,6-anhydro-d-galactose,
also known as neocarrabiose sulfate. At the primary structure level,
ι-carrageenan differs from κ-carrageenan in the presence at C-2 on the
α-linked galactose residues of one additional sulfate substituent per
repeating disaccharide (Fig.[33]1). ι-Carrageenans therefore contain
two sulfate groups per repeat unit, i.e. one anionic group per
monosaccharide. Such a high linear charge density is reminiscent of
that seen in alginic acid, the main cell wall polysaccharide of brown
algae, and in polygalacturonic acid, the nonmethylated component of
higher plant pectins, which both contain one carboxyl group per
monomeric unit ([34]1).
[35]Figure 1
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Figure 1
Schematic diagram showing two disaccharide repeating units of
ι-carrageenan. The two different glycosidic bonds in ι-carrageenan are
labeled, and further extensions of the polymer at the nonreducing and
reducing ends are indicated, respectively, by R′ and R". As
ι-carrageenase cleaves the β(1→4) glycosidic bond, its subsites for
substrate binding are labeled in accordance with the established
nomenclature ([39]49).
In aqueous solution, κ- and ι-carrageenans form thermoreversible gels
([40]3) and are used in a variety of industrial applications as gelling
or thickening agents. The mechanism of gelation involves the formation
of double helices of carrageenan chains followed by the association of
the double helices into a macromolecular three-dimensional network
([41]4, [42]5). Gelation is promoted by the presence of cations,
potassium in κ-carrageenan and calcium in ι-carrageenan. By binding to
the helices, either electrostatically or through specific binding
sites, these cations considerably reduce the charge density of the
helices and enhance their tendency to aggregate ([43]6). The fine
structure of ι-carrageenan in the solid state has been studied in
detail by x-ray diffraction of calcium carrageenate fibers ([44]7). The
polysaccharide chains were shown to adopt a twisted-ribbon conformation
with a 3[1] symmetry, stabilized by the formation of double helices,
themselves aggregated into larger clusters through the coordinated
binding of calcium ions ([45]7). In the cell walls of red algae,
carrageenans are laid out as highly ordered molecules associated with
the cellulose microfibrils or in the microfibril-less intercellular
matrix ([46]8). ι-Carrageenans are mainly located in the outer cortical
tissues, whereas κ-carrageenans constitute the walls of the inner
cortical and medullary cells ([47]9).
Enzymes that degrade carrageenans, namely κ-, ι-, and λ-carrageenases,
have been isolated from various marine bacteria. They all are
endohydrolases that cleave the internal β(1→4) linkages of carrageenans
yielding products of the neocarrabiose series ([48]10-13). Since these
galactan hydrolases display a strict substrate specificity, they
obviously recognize the sulfation pattern on the digalactose repeating
unit and thus provide an opportunity for investigating the
structure-function relationships of hydrolases that degrade sulfated
polysaccharides. With this aim, we have undertaken the structural
analysis of a representative set of carrageenases. Recently we reported
the deduced amino acid sequences of ι-carrageenase fromAlteromonas
fortis and Zobellia galactanovorans. They share no sequence similarity
with κ-carrageenases, and unlike β-agarases and κ-carrageenases, they
are inverting hydrolases. They represent the first members of a new
family of glycoside hydrolases ([49]14), family 82 (13). In this
context, a high-resolution three-dimensional structure for a
ι-carrageenase would be pivotal in determining the fold prevailing in
family 82 and in delineating the molecular bases for the recognition of
ι-carrageenan. To this end, we have recently overexpressed and
crystallized A. fortisι-carrageenase ([50]15), and we report here its
structure at 1.6 Å resolution.
[51]Previous Section[52]Next Section
§2§ EXPERIMENTAL PROCEDURES §2§
§5§ Expression, Purification, and Crystallization of Native and
Selenomethionyl-ι-carrageenase §5§
Full details of the expression and purification of native
ι-carrageenase have been described previously ([53]15). Briefly,
ι-carrageenase was expressed using the pET20b vector ([54]Novagen) as a
His-tagged fusion protein in the periplasm of Escherichia coli
BL21(DE3) strain and purified by metal affinity chromatography on a
column of Chelating Fast Flow Sepharose (Amersham Pharmacia Biotech)
loaded with NiSO[4]. The yield was 7–8 mg/liter of culture medium, and
the purified protein was concentrated to 6 mg/ml using a dialyzing
concentrator (Amicon Co.). Single crystals were obtained with
polyethylene glycol, and the presence of calcium ions appeared to be
crucial for crystallization. High quality crystals, typically 0.25 ×
0.25 × 0.15 mm in dimensions, were grown with 0.1 m sodium cacodylate
at pH 6.5, 15–17% polyethylene glycol (M [r] = 6,000), and 200 mm
calcium acetate.
The seleno-l-methionine labeling procedure was identical to that
described for the expression of seleno-l-methionine
(Se-Met)^1-κ-carrageenase ([55]16). As the expression yield was only
800 μg/liter of culture medium, the volume had to be scaled up to
obtain a sufficient amount of protein. During the concentration step,
the Se-Met-ι-carrageenase appeared to be less soluble than the native
protein and could only be concentrated to 2 mg/ml. Crystallization of
Se-Met-ι-carrageenase was performed under similar conditions to those
already reported ([56]15) except for the addition of 1 mm
dithiothreitol and the replacement of sodium cacodylate by imidazole to
avoid the reaction between cacodylate and dithiothreitol ([57]17).
Under these conditions, small crystals with a maximal size of 100 × 100
× 50 μm were obtained in 1 month of equilibration, whereas large native
crystals appeared in a few days.
§5§ Data Collection and Processing §5§
Crystals were successively soaked for 30 s in crystallization solutions
in which the glycerol concentration was increased by steps of 5% to a
final concentration of 20%. A single crystal was mounted on a loop,
transferred to the goniometer head, and kept at 100 K in a nitrogen
stream. The multiple anomalous diffraction data were collected on a
Se-Met-ι-carrageenase crystal at three wavelengths around the K (λ =
0.9t95 Å) absorption edge of selenium (beamline FIP/BM 30, European
Synchrotron Radiation Facility (ESRF), Grenoble, France). A 345-mm
Marresearch image plate detector was used. All intensity data were
integrated and reduced using DENZO/SCALEPACK ([58]18). Based on unit
cell dimensions two molecules of protein are present in the asymmetric
unit. Using the Patterson method and SHELX 97 (19), the positions for 7
of the 16 selenium atoms expected were determined. This partial
structure was input into SHARP ([59]20) for a first approximation of
the phases. Five other selenium atoms were located in the Fourier
difference electron density map. The final partial structure therefore
contained 12 selenium atoms, and the final phases from SHARP resulted
in a figure of merit of 0.43–2.3 Å resolution. Low (up to 2.2 Å) and
high (up to 1.6 Å) resolution data were also collected on a native
ι-carrageenase crystal using a Marresearch charge-coupled device
detector (beamline ID14 EH2, ESRF). All intensity data were integrated
using DENZO ([60]18). At low temperature, the unit cell parameters were
slightly different: a = 55.87, b = 90.07, c = 124.12 Å, α = γ = 90°, β
= 93.53°. The low and high resolution data were merged and reduced with
SCALA ([61]21).
§5§ Structure Determination and Refinement §5§
Taking advantage of the noncrystallographic symmetry, the electron
density was improved by molecular averaging, histogram matching, and
solvent flattening (50% solvent) with DM ([62]21). Using the native
data, the phases were then extended to 1.6 Å resolution yielding a
figure of merit of 0.75. Due to the high quality of the electron
density map at 1.6 Å, wARP ([63]22) was used to build the model
automatically resulting in the assignment of 85 and 75% of the main and
side chain atoms, respectively. The rest of the model was built
manually using O ([64]23). However, no clear electronic density was
observed for residues 314–333 and 341–350, which are not included in
the final model. The refinement was performed with CNS ([65]24) using
the native data set to 1.6 Å with the maximum likelihood target
function. The program was set up to automatically compute a
cross-validated ς[a] estimate and the weighting scheme between the
x-ray refinement target and the geometric energy function. Corrections
for a flat bulk solvent and for anisotropy in the data were also
applied. The ς[a] weighted maps obtained from the subsequent refinement
models were used for further model building. The first group of water
molecules was added when peaks in the 2F [o] −F [c] density were >2 ς
and had a stereochemistry compatible with at least one hydrogen bond
with a protein atom or another water molecule. In the final stages, the
ς cut-off was reduced to 1.0 ς, and water molecules with a B factor >60
Å^2 were removed from the model. The final model refined at 1.6 Å has
an R[work] of 20.7% and an R[free] of 22.3% ([66]25) and consists of
6,840 protein atoms, 1,110 water molecules, six glycerol molecules, and
six calcium, six sodium, and two chloride ions. All these ions have an
occupancy of 1, and their B factors refine to a value close to
neighboring protein atom B factors. The stereochemistry of the final
structure was evaluated using the PROCHECK program ([67]26).
§5§ Calcium-dependent Activity Test and Proteolytic Digestion §5§
ι-Carrageenase activity was assayed as follows. Three enzyme solutions
were prepared by diluting a stock solution of the purified enzyme (6
mg/ml) in 100 mm Tris-HCl, pH 7.2, 200 mm NaCl and adding either 5 mm
EGTA or 5 mm CaCl[2]. Aliquots (100 μl) were incubated for 15 min at
40 °C with 2 ml of substrate solution consisting of 0.125% (w/v)
ι-carrageenan, 50 mm Tris-HCl, pH 7.2, 100 mm NaCl with or without 5 mm
CaCl[2], and the reaction mixture (200 μl) was assayed for reducing
sugars ([68]27) using boiled enzyme blanks. One unit of enzyme activity
is defined as the amount of enzyme that produces an increase of 0.1 A
[237 nm]/min in the reducing sugar assay.
Purified bovine pancreas trypsin (T1426, 10,000N-benzoyl-l-arginine
ethyl ester units/mg of protein) was purchased from Sigma. Limited
proteolysis of ι-carrageenase (6 mg/ml in 100 mm Tris-HCl, pH 7.2, 200
mm NaCl) was performed using trypsin/ι-carrageenase ratios of 1:100 and
1:20 (w/w) in the presence or absence of 5 mm EGTA or 5 mm CaCl[2]. The
samples were incubated for 1 h at 20 °C, and then the reaction was
stopped by adding SDS loading buffer and boiling the samples for 5 min
at 100 °C. The samples were then loaded onto a 15% SDS-polyacrylamide
gel for electrophoresis, and the gels were stained with Coomassie Blue.
[69]Previous Section[70]Next Section
§2§ RESULTS §2§
§5§ The Overall Structure of A. fortis ι-Carrageenase §5§
The three-dimensional crystal structure of ι-carrageenase lacking the
signal peptide (residues 1–27) was determined at 2.3 Å resolution by
the multiple anomalous diffraction method using a crystal of a
Se-Met-substituted form of the enzyme ([71]28). After phase extension
with a native data set at higher resolution, a high quality electron
density map was obtained (Fig. [72]2) allowing building and refinement
of the model at 1.6 Å resolution. The crystallographic statistics are
shown in Table [73]I. The asymmetric unit contains two mature
ι-carrageenase molecules, each containing amino acids 28–491. Residues
314–334 and 341–350 for molecule A and residues 313–334 and 341–351 for
molecule B are not visible in the 2F [o] − F [c] electron density map
and are presumed to exist in disordered or highly flexible
conformations. Superposition of molecules A and B reveals that the Cα
atoms overlay with a root mean square deviation of 0.21 Å.
Approximately 10 residues in each molecule presented clear alternate
conformations. The need to refine the occupancy for terminal atoms of
several residues, such as aspartate, glutamate, or methionine, suggests
that a fraction of the protein population in the crystal has been
subjected to radiation damage ([74]29).
[75]Figure 2
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Figure 2
Solvent-flattened multiple anomalous diffraction electron density map
at 1.6 Å resolution. Map contoured at 2.0 ς of the N-terminal
calcium-binding hairpin loop. Calcium ion and water molecules are
indicated as yellow andred spheres, respectively. The oxygen, nitrogen,
and carbon atoms in the protein are shown in red, blue, andyellow,
respectively. This figure was created using O ([79]23).
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Table I
Data reduction, phasing, and refinement statistics
The core of ι-carrageenase is folded into a right-handed parallel
β-helix of 10 complete turns (Fig. [82]3). This fold was first
encountered in pectate lyase C from Erwinia chrysanthemi ([83]30). The
lyase structure consists of three parallel β-sheets, PB1, PB2, and PB3.
PB2 and PB3 form planar surfaces almost perpendicular to each other,
while PB1 is in the form of a groove. In the lyase structure, the turns
or loops (depending on the number of amino acids inserted between
consecutive β-helical strands) are referred to as T1 (PB1-PB2), T2
(PB2-PB3), and T3 (PB3-PB1). In β-helix proteins, the assignment of
secondary structure elements is based on the DSSP algorithm ([84]31)
with the additional criterion that any residues with (Φ, ψ) angles in
the left-handed α-helix region are not included in the β-strand. Based
on these rules, PB2 can be divided into two parts, and the
ι-carrageenase β-helix consists of four parallel β-sheets, PB1, PB2a,
PB2b, and PB3, composed respectively of 10, 5, 11, and 10 β-strands.
These strands are relatively short with an average number of 4.0, 2.4,
4.1, and 4.0 residues, respectively. Interestingly, like almost all
β-helix proteins, ι-carrageenase contains in its N-terminal region an
amphipathic α-helix (residues 66–77) that shields the hydrophobic core
of the β-helix from the solvent.
[85]Figure 3
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Figure 3
Folding of A. fortisι-carrageenase. A, stereo view of the Cα trace of
the protein. The N terminus, C terminus, and every 20th residue are
labeled, while every 10th residue is marked with ablack dot. The
secondary structures listed in the text are composed of the following
residues: β15, 187–189; β18, 200–204; β25, 240–245; β26, 258–260; β28,
267–270; β32, 302–304; β35, 378–381; β36, 383–386; β39, 434–436. B,
ribbon representation of the structure. The β-helix core, domain A, and
domain B are shown, respectively, in blue, gold, and red. The small T1
extension, containing an antiparallel sheet (β16-β17) and an α-helix
(α2), is shown ingreen. The red, yellow, andgreen spheres represent
sodium, calcium, and chloride ions, respectively. Figs. [89]3 and [90]4
were prepared using Molscript ([91]50).
The most striking difference between ι-carrageenase (Fig. [92]3) and
the 11 other β-helix proteins of known structure is the presence, in
the C-terminal region, of two large additional domains (both 67
residues long). Domain A (residues 307–373) replaces the T1 turn
between strands β32 and β35 (see legend of Fig. [93]3 A for
definition). Half of this domain (residues 314–333 and 341–350) could
not be built as no clear electron density was observed. The visible
part of domain A features a sheet of two short antiparallel β-strands,
β33 and β34, edged by one α-helix (residues 358–367) and has an average
B value (37.8 Å^2) twice that of the β-helix core (17.8 Å^2). At the
border of the visible part of domain A is a hydrophobic surface,
suggesting that the nonvisible residues complete a globular-shaped
domain. This domain is weakly bound to the β-helix by only four
hydrogen bonds (Glu^310 O-Lys^252 NZ, Asp^358OD1-Ser^442 OG, Asp^358
OD2-Lys^443 N, and Asp^362 OD1-Tyr^444 OH). Moreover, the visible part
of domain A makes no contact with neighboring molecules and is located
in a large solvent channel in the crystal. Domain B consists of
residues 387–430, located on a T3 turn that connect strands β36 and
β39, and the C-terminal extension (residues 469–491). Mainly composed
of long loops, it also features an antiparallel β-sheet (β37-β38), a
3[10] helix (residues 395–402), and a one-turn α-helix at the C
terminus. This domain, also globular in shape, is folded around an
independent hydrophobic core. In contrast to domain A, this bulky
structure is characterized by many side chain-side chain and side
chain-main chain hydrogen bonds, both within the domain and with the
β-helix. These hydrogen bonds have a clear stabilizing effect on domain
B since its average B value (22.0 Å^2) is close to that of the β-helix
core (17.8 Å^2).
Each strand of PB2a is connected to PB2b by an asparagine residue in a
left-handed α-helix conformation. Residues in the α[L]conformation are
also seen in the T2 turns in the structure. The presence of these
residues results in sharp bends of about 100° in the polypeptide chain
without disrupting the hydrogen bond pattern of the parallel β-strands.
PB1 also presents a striking repetition of a structural irregularity:
in each strand, one residue is in a right-handed helix conformation,
thus creating an alignment of nine β-bulges in the groove. With the
exceptions of Ser^186 and the Lys^464-Thr^465 β-bulge in the C
terminus, which are accessible to the solvent, all side chains in these
β-bulges are aliphatic amino acid residues and point toward the
hydrophobic core of the β-helix.
The turns and loops between the β-strands are of different sizes and
have different conformations. Whereas almost all of the T2 turns
consist of a single residue in the α[L] conformation, the T1 and T3
turns are longer and more irregular, their sizes ranging from 1 to 12
and from 2 to 8 residues, respectively. At the N-terminal edge of the
β-helix, the T3 loops form a bulky protrusion above PB1. On the
opposite side of this β-sheet, the T1 turn between strands β15 and β18
folds into a β-sheet of two antiparallel strands (β16 and β17), and the
T1 turn between β25 and β26 folds into a short α-helix. Strands β16 and
β17 and this short α-helix form a domain that is stabilized by a
hydrophobic core (Val^191, Leu^198, Leu^250, Met^251, and Tyr^254) and
hydrogen bonds between Glu^193OE2-Tyr^254 OH and Leu^198 O-Gln^256OE1.
At the surface, two hydrophilic networks (Arg^136-Asp^166-Arg^194 and
Arg^202-Trp^200-Asp^227-Arg^260-Gly^257-Gly^258) firmly bind this
domain to the β-helix core; these amino acids are also present in Z.
galactanovorans ι-carrageenase. On each edge of β-sheet PB1, the
N-terminal T1 and T3 extensions and the protruding domains A and B in
the C-terminal region create a long deep cleft. This large channel,
about 50 Å long and 10 Å wide, is probably the cleft that binds
ι-carrageenan chains.
A. fortis ι-carrageenase contains four disulfide bridges. The first,
Cys^269-Cys^298, is located within the β-helix core, connecting strand
β28 to the T3 turn between strands β31 and β32. The Cys^336-Cys^360
bridge, which links the 334–340 loop to helix α3, probably stabilizes
domain A. This disulfide bridge was opened by radiation damage, but its
existence was confirmed by the refinement of the structure (data not
shown) from the data at 2.0 Å resolution obtained with a less powerful
beamline ([94]15). However, even in this latter electron density map
with an intact disulfide bridge, the above-mentioned nonvisible part of
domain A could not be seen either. The two other disulfide bridges,
Cys^408-Cys^476 and Cys^412-Cys^484, which are strictly conserved in
the two ι-carrageenase sequences, bring the C-terminal extension of the
protein into close proximity with the main part of domain B. Finally,
the ι-carrageenase structure contains two chloride, six sodium, and six
calcium binding sites (see below).
A major characteristic of β-helix proteins is their internal stability
(mean B factor of 15.6 Å^2), which results from numerous intrahydrogen
bonds and side chain stacking of identical or similar residues
([95]32). Consistent with these observations, the ι-carrageenase
structure is dominated by aliphatic stacking in the β-helix interior.
Four major stacks (Ile^209-Leu^234-Ile^267-Val^293-Val^383-Val^453,
Ile^229-Ile^262-Val^288-Val^378,
Ile^187-Ile^221-Leu^242-Val^276-Val^302-Val^434, and
Ile^113-Val^149-Ile^117) form the hydrophobic core, but short stacks
(Val^290-Val^380, Val^135-Val^167, Ile^187-Ile^221, Ile^172, and
Ile^206) also strengthen the β-helix cohesion. In the aligned β-bulges,
stacking of alanine residues (Ala^241-Ala^275-Ala^301-Ala^433), all in
the α[R] conformation, is also seen. In contrast to pectate lyases,
however, clusters of aromatic residues are of minor importance,
involving only short clusters (Phe^381-Phe^451, Phe^147-Phe^175,
Phe^127-Phe^162, Phe^184-Tyr^218). On the surface of the protein, two
rows of asparagine residues in the α[L] conformation
(Asn^102-Asn^137-Asn^169-Asn^203-Asn^228-Asn^261and
Asn^233-Asn^266-Asn^293) flank β-sheet PB2, but few of these amino
acids are engaged in successive hydrogen bonds, and only a short
portion of asparagine ladder (Asn^102-Asn^137-Asn^163) can be
identified. In contrast, as in Flavobactrium heparinumchondroitin lyase
B ([96]33), the ι-carrageenase β-helix features external basic stacks,
which consist of a short lysine pair (Lys^373-Lys^449) and a cluster of
arginines (Arg^136-Arg^168-Arg^202-Arg^260).
§5§ ι-Carrageenase Contains Binding Sites for Sodium, Chloride, and
Calcium §5§
The structure of ι-carrageenase reveals six sodium, two chloride, and
six calcium ion binding sites. Four of the sodium ions are located at
the interface between molecules A and B in the asymmetric unit and form
two clusters related by the noncrystallographic symmetry. In one
cluster, sodium ions are bound to a carboxyl group, Asp^54 or Glu^108,
and to four water molecules, three of which are shared by the ions.
Since ι-carrageenase elutes as a monomer on gel filtration (data not
shown), the presence of these four sodium binding sites is probably due
to crystal packing. The last two sodium binding sites are located in
the same region of molecules A and B in the interior of the β-helix
core, 14–15 Å from the catalytic residues. In one molecule, the sodium
ion is coordinated by five ligands with a trigonal bipyramidal geometry
and at an average distance of 2.35 Å; these ligands are the oxygens of
Leu^159, Ile^183, and Phe^184, the hydroxyl of Thr^182, and one water
molecule. Thr^182, Ile^183, and Phe^184 are conserved in Z.
galactanovoransι-carrageenase ([97]13). Interestingly, Phe^184 adopts a
disallowed conformation (φ = 78, ψ = 139). Each protein molecule binds
one chloride ion, which is located on the surface of the groove,
slightly buried in a hydrophobic pocket and 7 Å from both the
above-mentioned sodium ion and the catalytic residues. This hydrophobic
cavity is composed of the conserved residues Phe^184, Ala^185, Leu^188,
and Tyr^284. The chloride ion is also in contact with the amide group
of the conserved Gln^222, the backbone amino group of Ala^185, and one
water molecule. The activity of ι-carrageenase is known to depend on
the presence of sodium chloride and to decrease rapidly on either side
of the optimum concentration of 100 mm NaCl; complete salt removal or
NaCl concentrations higher than 500 mmresult in complete loss of
activity ([98]34). However, how the presence of sodium and chloride
binding sites in ι-carrageenase accounts for its activation by sodium
chloride remains an open question.
Interestingly, each ι-carrageenase molecule also has three calcium ion
binding sites, all remote from the active site. The first is located at
the surface between the second and third T2 turns. The calcium ion
makes hydrogen bonds with the backbone oxygen and hydroxyl group of
Ser^109, the side chain carbonyl of Asn^145, the oxygen of Gly^146, and
four water molecules. As these ligands are not present in Z.
galactanovorans ι-carrageenase ([99]13) this site appears to be
specific to A. fortis ι-carrageenase. The second and third sites, which
display a hairpin topology, are well conserved between the two
ι-carrageenases. The second site precedes the N-terminal α-helical cap
(Fig. [100]2). The calcium ion, buried inside the hairpin loop,
establishes short contacts (average distance 2.37 Å) with Asn^58 O,
Asp^61 OD1, Ser^63 O, Asp^65 OD1 and OD2, and two water molecules.
Asn^58, Asp^61, and Asp^65 are strictly conserved in the two
ι-carrageenases. The hairpin loop is also structured and anchored to
the β-helix by numerous hydrogen bonds (Asn^58 OD1-Asn^60 N,
Gly^59O-Thr^91 N, Asp^61 OD1-Ser^63 N, Asp^64 OD1-Asp^65 N-Ser^66 N and
OH, Asp^65 OD1-Asn^58 N, and Asp^65OD2-His^93 N). The third calcium
binding site is in a more complex and irregular hairpin loop in the
C-terminal region. It is in interaction with Thr^438 OH, Asp^445 OD1,
Asp^445 OD2, Asp^447 OD1, and one water molecule. This site is
strengthened by Asp^436, which makes a hydrogen bond with Thr^438 OH
and the above-mentioned water molecule. Only Asp^436 and Thr^438 are
strictly conserved in the two enzymes, while Asp^445 and Asp^447 are
replaced by asparagine residues in Z. galactanovoraι-carrageenase.
The potential influence of calcium ions on the stability of
ι-carrageenase was tested. Compared with the activity in the
purification buffer (10 mm Tris-HCl, pH 7.2, 200 mm NaCl), the addition
of either 5 mmCaCl[2] or 5 mm EGTA had no effect on ι-carrageenase
activity. In contrast, the trypsin digestion pattern of ι-carrageenase
was clearly affected by the presence of either EGTA or CaCl[2] (Fig.
[101]4). In the control buffer, trypsin digestion of ι-carrageenase
(1:100 w/w) resulted in the release of several peptides with sizes
ranging from 5 to 53 kDa, but most of the protein was undegraded; under
the same conditions, this pattern was not affected by the presence of 5
mm EGTA. A trypsin/ι-carrageenase ratio of 1:20 resulted in greater
protein degradation especially in the presence of EGTA when the protein
was almost completely degraded. In contrast, at either
trypsin/ι-carrageenase ratio, the addition of 5 mmCaCl[2] significantly
protected ι-carrageenase from proteolysis. Although the enzyme was
purified in the absence of calcium, the marked differences seen in the
digestion patterns in the presence or absence of EGTA at the 1:20
trypsin/ι-carrageenase ratio indicate that the calcium binding sites
were at least partly occupied in the protein, suggesting that they have
a high affinity for calcium. Taken together, these findings demonstrate
that calcium is involved in maintaining the structural integrity of the
protein.
[102]Figure 4
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Figure 4
Protection of A. fortisι-carrageenase from trypsin hydrolysis by
calcium binding. Proteins were separated by SDS-polyacrylamide gel
electrophoresis on a 15% polyacrylamide gel and detected by Coomassie
Blue staining. The number on the left indicates the size of the
molecular mass markers in kDa (Amersham Pharmacia Biotech), while the
band corresponding to trypsin is indicated on the right. Lane 1,
ι-carrageenase (4 μg) in the absence of trypsin; lanes 2–4,
trypsin/ι-carrageenase ratio of 1:100 (w/w); lanes 5–7,
trypsin/ι-carrageenase ratio of 1:20 (w/w). Lanes 2 and5, hydrolysis in
control buffer (100 mmTris-HCl, pH 7.2, 200 mm NaCl); lanes 3 and6,
hydrolysis in the presence of 5 mm EGTA;lanes 4 and 7, hydrolysis in
the presence of 5 mm CaCl[2].
[106]Previous Section[107]Next Section
§2§ DISCUSSION §2§
§5§ Comparison of ι-Carrageenases with Other Right-handed Parallel β-Helix
Proteins §5§
Only 11 protein structures are known to contain a right-handed parallel
β-helix fold. Most are pectin-degrading enzymes and use a mechanism
involving either β-elimination, e.g. the pectate and pectin lyases of
family 1 of the polysaccharide lyases ([108]30, [109]35, [110]36), or
acid/base hydrolysis, e.g. Aspergillus nigerrhamnogalacturonase
([111]37) and polygalacturonase ([112]38) as well asErwinia carotovora
polygalacturonase ([113]39), which constitute family 28 of the
glycoside hydrolases. Other right-handed parallel β-helix
fold-containing proteins include chondroitin lyase B fromF. heparinum
([114]33), a virulence factor (P69 pertactin) from the whooping cough
agent, the bacterium Bordetella pertussis([115]40), the tailspike
rhamnosidase from phage P22 ([116]41), and a pectin methylesterase from
E. chrysanthemi ([117]42). It is worth noting thus that most β-helix
proteins (pectate lyases, rhamnogalacturonases, polygalacturonases,
chondroitin lyases, and ι-carrageenases) are enzymes involved in the
depolymerization of acidic polysaccharides with high linear charge
densities from the extracellular matrix of plants or animals. The
exceptions include pectin lyases, which can degrade the methylated form
of polygalacturonic acid, the P22 tailspike protein, an
endorhamnosidase involved in the recognition and degradation of the
neutral, rhamnose-rich oligosaccharides known as O-antigen receptors
([118]43), and P69 pertactin, the specificity of which is unclear.
Although ι-carrageenase does not share sequence similarity with family
28 pectin hydrolases, its overall structure shows similarities with the
family 28 fold. As in these rhamnogalacturonase and polygalacturonases,
the ι-carrageenase β-helix fold consists of four parallel β-sheets. All
three of these glycoside hydrolases proceed with an inversion of the
anomeric configuration of their substrates. However, the
pectin-degrading enzymes cleave α(1→4) or α(1→2) glycosidic bonds,
whereas ι-carrageenase cleaves β(1→4) glycosidic linkages. It also
appears that the family 28 pectinase catalytic residues Asp^180,
Asp^201, and Asp^202 ([119]38, [120]44) are not structurally conserved
in ι-carrageenase. We thus conclude that ι-carrageenases represent a
genuinely new family of glycoside hydrolases referred to as family 82
(13). Hence, although families 28 and 82 display the same protein fold,
they act on different types of glycosidic bonds (α versusβ) and display
different catalytic amino acids, and therefore they do not belong to
the same clan of glycoside hydrolases ([121]14).
§5§ Structure of the Putative Active Site and Recognition of ι-Carrageenan
Chains §5§
ι-Carrageenase hydrolyzes glycosidic bonds with a mechanism leading to
overall inversion of the anomeric configuration at the site of cleavage
([122]13). In such an inverting hydrolytic mechanism, the glycosidic
bond is protonated by the acid/base catalyst to facilitate aglycon
departure. This protonation step occurs concomitantly with the attack
on the anomeric carbon by a water molecule activated by a base residue,
yielding a product with an anomeric stereochemistry opposite to that of
the substrate ([123]45, [124]46). In inverting glycoside hydrolases,
the acid/base and nucleophile catalysts consist of aspartate and/or
glutamate residues, their carboxyl groups being typically 10 Å apart
([125]47). However, family 28 pectinases feature an active site
topology with a short distance (5–6 Å) between the catalytic residues
([126]38, [127]44).
In the structure of E. chrysanthemi pectate lyase C complexed with a
plant cell wall fragment, the oligosaccharide is found on PB1, defining
the groove as the substrate binding site ([128]48). As discussed below,
the groove in ι-carrageenase also appears to be the binding site for
the ι-carrageenan chain. Of the acidic residues located in this cleft,
only three, Glu^245, Asp^247, and Glu^310, are conserved between the
two ι-carrageenase sequences. Glu^245 and Asp^247 are located at the
bottom of the groove between domains A and B, and their carboxyl groups
are separated by a distance of 4–5 Å. Glu^310 belongs to domain A, and
its carboxyl group, which points toward the cleft, is separated by a
distance of about 10 Å from the carboxyl groups of Glu^245 and Asp^247
(Fig. [129]5). According to the DSSP program, Glu^245, Asp^247, and
Glu^310 are all accessible to the solvent with respective accessible
surface values of 31, 46, and 61 Å^2. Interestingly the entire region
near these residues is well conserved in ι-carrageenases, and we
propose that the nucleophile and acid/base catalysts are a yet to be
determined pair among these three acidic amino acids.
[130]Figure 5
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Figure 5
Stereo view of the potential active site ofA. fortis ι-carrageenase.
The conserved residues of ι-carrageenase are shown in red,blue, and
black for oxygen, nitrogen, and carbon atoms, respectively. The other
residues are shown in ribbon representation. The violet sphere
represents a chloride ion. Residues Glu^245, Asp^247, and Glu^310are
the potential catalytic amino acids, whereas His^281 may maintain the
correct pK [a] of Glu^245. The basic residues Arg^243, Arg^303, and
Arg^353 are probably involved in ι-carrageenan recognition.
The catalytic cleft of ι-carrageenase is not large enough to correctly
accommodate the double-stranded helix of ι-carrageenan for productive
binding. Therefore, ι-carrageenase presumably initiates its action on
single-stranded chains of the amorphous zones of ι-carrageenan gels. In
κ-carrageenan, the glycosidic backbone contains alternating negatively
charged units ofd-galactose-4-sulfate and hydrophobic
3,6-anhydro-d-galactose residues. Consistent with these observations,
the binding of κ-carrageenan by Pseudoalteromonas carrageenovora
κ-carrageenase has been shown to involve both ionic interactions with
sulfate-ester substituents and a specific hydrophobic environment for
the 3,6-anhydro-d-galactose units ([134]16). Since ι-carrageenan bears
a sulfate group at C-2 of the 3,6-anhydro-d-galactose moiety, this
α(1→4)-linked residue is less hydrophobic than in the κ-carrageenans,
and substrate binding by ι-carrageenases should mainly involve ionic
interactions. This assumption is confirmed by the paucity, in the
active site cleft of ι-carrageenase, of aromatic residues, which are
known to interact with neutral saccharides through aromatic
ring-pyranose ring stacking. The ι-carrageenase groove contains no
tryptophan residues and only three tyrosine residues, Tyr^218, Tyr^224,
and Tyr^400. In contrast, four arginine residues (Arg^125, Arg^243,
Arg^303, and Arg^353) and one lysine residue (Lys^122), which are also
present in Z. galactanovorans ι-carrageenase, point toward the cleft.
Fig.[135]6 A shows the mapping of these conserved basic residues and of
the catalytic residues on the surface of ι-carrageenase.
[136]Figure 6
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Figure 6
ι-Carrageenan recognition by ι-carrageenase. A, view of the molecular
surface of the ι-carrageenase groove. The potential catalytic residues
and the conserved basic amino acids are shown inred and blue,
respectively. The distances between clusters of basic residues are
shown. Fig. [140]6 was created using Grasp ([141]51). B, ball and stick
representation of a ι-neocarrahexaose-sulfate ([142]7). Oxygen, sulfur,
and carbon atoms are shown in red, yellow, and black, respectively.
Distances between the sulfate substituents are shown.
Based on the three-dimensional structure of the ι-carrageenan double
helix ([143]7), the distribution of sulfate substituents along the
ι-carrageenan chain is not homogeneous. Within the same repeating unit,
the sulfate groups flank the α(1→3) glycosidic bond and are separated
only by 4.65 Å. The distance between two such successive sulfate pairs
is 11.5 Å, leaving the immediate vicinity of the β(1→4) linkages devoid
of negative charges. Finally, two neighboring pairs of sulfate-ester
substituents on the same side of the glycosidic backbone are separated
by 16 Å (Fig. [144]6 B). This topology of the sulfate substituents on
ι-carrageenan shows a striking match with the distribution of the
conserved basic amino acids in the ι-carrageenase groove (Fig. [145]6
A). Residues Arg^243 and Arg^303, separated by 7 Å, could form salt
bridges with one ester-sulfate pair; this would position the uncleaved
α(1→3) linkage between these two arginine residues and place the β(1→4)
linkage to be cleaved next to the potential catalytic residues Glu^245,
Asp^247, and Glu^310. This assumption is supported by the fact that
Arg^243 and Arg^303 are tightly hydrogen-bonded to Glu^245 and Asp^305,
respectively (Fig. [146]5). However, we do not know whether Arg^243
interacts withd-galactose-4-sulfate and Arg^303 interacts with
3,6-anhydro-d-galactose-2-sulfate or vice versa. Thus, Arg^243 and
Arg^303 participate either in subsites −1 and −2 or subsites +1 and +2,
and the directionality of the carbohydrate polymer chain with respect
to the protein structure remains to be identified. Two other basic
amino acids in the groove, Lys^122 and Arg^125, located 16 Å from
Arg^243 (Fig. [147]6 A), are in the correct topology to bind the
sulfate pair. Finally, the last basic residue, Arg^353, is 10 Å from
Arg^303, again in a correct position to establish a salt bridge with
the sulfate-ester substituent located 11.5 Å from the sulfate-ester
bound to Arg^303.
[148]Previous Section[149]Next Section
§2§ ACKNOWLEDGEMENTS §2§
We thank Dr. Ed Mitchell for invaluable help with the beamline ID14EH2
(ESRF), Dr. Thierry Vernet for support with the Se-Met-substituted
protein expression, Dr. Anne-Marie DiGuilmi for precious help with the
proteolysis analysis, and Dr. Tristan Barbeyron for helpful discussion.
[150]Previous Section[151]Next Section
§2§ Footnotes §2§
* [152]↵* This work was supported by grants from the Action Concertée
Coordonnée Sciences du Vivant (No. V) and Groupement de Rechereches
1002 of CNRS “Biology, Biochemistry and Genetics of Marine
Algae.”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 ) have been
deposited in the Protein Data Bank, Research Collaboratory for
Structural Bioinformatics, Rutgers University, New Brunswick, NJ
([153]http://www.rcsb.org/).
* [154]↵‖ To whom correspondence should be addressed. Tel.:
33-4-38-78-56-09; Fax: 33-4-38-78-54-94; E-mail: otto@ibs.fr.
* Published, JBC Papers in Press, August 7, 2001, DOI
10.1074/jbc.M100670200
* Abbreviations:
Se-Met
seleno-l-methionine
*
+ Received January 24, 2001.
+ Revision received June 13, 2001.
* The American Society for Biochemistry and Molecular Biology, Inc.
[155]Previous Section
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