Consequently, the overall arrangement of sugar-binding sites in MLI differs from those in monomeric type-II RIPs: two N-terminal sugar-binding sites are 15 A˚ apart on one side of the dim
Trang 1Crystal structure at 3 A˚ of mistletoe lectin I, a dimeric type-II
ribosome-inactivating protein, complexed with galactose
Hideaki Niwa1, Alexander G Tonevitsky2, Igor I Agapov3, Steve Saward1, Uwe Pfu¨ller4and Rex A Palmer1 1
School of Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK;2Institute of Transplantology and Artificial Organs, Moscow, Russia;3Institute of Genetics and Selection of Microorganisms, Moscow, Russia;
4
Institut fu¨r Phytochemie, Universita¨t Witten/Herdecke, Witten, Germany
The X-ray structure of mistletoe lectin I (MLI), a type-II
ribosome-inactivatingprotein (RIP), cocrystallized with
galactose is described The model was refined at 3.0 A˚
resolution to an R-factor of 19.9% using21 899 reflections,
with Rfree24.0% MLI forms a homodimer (A–B)2in the
crystal, as it does in solution at high concentration The
dimer is formed through contacts between the N-terminal
domains of two B-chains involvingweak polar and
non-polar interactions Consequently, the overall arrangement of
sugar-binding sites in MLI differs from those in monomeric
type-II RIPs: two N-terminal sugar-binding sites are 15 A˚
apart on one side of the dimer, and two C-terminal
sugar-bindingsites are 87 A˚ apart on the other side Galactose
bindingis achieved by common hydrogen bonds for the
two bindingsites via hydroxy groups 3-OH and 4-OH and
hydrophobic contact by an aromatic ring In addition, at the
N-terminal site 2-OH forms hydrogen bonds with Asp27 and Lys41, and at the C-terminal site 3-OH and 6-OH undergo water-mediated interactions and C5 has a hydro-phobic contact MLI is a galactose-specific lectin and shows little affinity for N-acetylgalactosamine The reason for this
is discussed Structural differences amongthe RIPs investi-gated in this study (their quaternary structures, location of sugar-binding sites, and fine sugar specificities of their B-chains, which could have diverged through evolution from
a two-domain protein) may affect the bindingsites, and consequently the cellular transport processes and biological responses of these toxins
Keywords: lectin; mistletoe (Viscum album); ribosome-inac-tivatingprotein; b)trefoil
The use of the mistletoe plant (Viscum album), well known
in ancient religious ceremonies, is thought to have extended
to more medicinal purposes since ancient times [1] More
recently, particularly since the beginning of the 20th century,
extracts of the plant have been applied in the treatment of
cancer, especially in continental Europe, although its
efficacy in this respect is not fully understood (http://
www.cancer.gov/cancerinfo/pdq/cam/mistletoe) It is now
considered that mistletoe lectin I (MLI) is the most
important component in this respect MLI is a type-II
ribosome-inactivatingprotein (RIP) which consists of two
chains [1]: a catalytic A-chain, which inactivates protein
synthesis, and a lectin B-chain, which binds to carbohydrate
moieties of cell surfaces, triggering the internalization of
MLI into the cell The two chains are linked by a disulfide
bond Other type-II RIPs include the plant toxins ricin from
Ricinus communis and abrin from Abrus precatorius
In type-I RIPs there is a single polypeptide chain which shares sequence and structural homology with the A-chains
of type-II RIPs The low cytotoxicity of type-I RIPs is considered to be due to the lack of the cell internalizing facility exerted by the B-chains of type-II RIPs [2–4] After endocytosis of a type-II RIP, it is transported to the Golgi network and then to the endoplasmic reticulum [5] The toxin is finally translocated across the endoplasmic reticulum membrane into the cytosol to exert its catalytic action on ribosomes Studies usingMLI [6,7] indicate that the disulfide bond that links the two chains is reduced and the A-chain is unfolded before translocation into the cytosol
The A-chain inhibits protein synthesis by cleavingthe N-glycosidic bond in adenosine A4324 in 28S eukaryotic rRNA by hydrolysis (EC 3.2.2.22) [8] The structure of the rRNA loop where this adenine exists has been studied using synthetic nucleotides [9], and the location of the loop in a ribosome was identified in a 5.0-A˚ electron density map for the crystal structure of a bacterial 50S ribosome subunit [10] How the specific adenine is recognized by an RIP, the mechanism of the catalytic action itself, and why the depurination of the single adenine arrests protein synthesis are not properly understood It has also been demonstrated that RIPs not only release adenine from rRNA, but in vitro, both type-I and type-II RIPs release adenine from DNA, and many type-I RIPs can release adenine from poly(A) [11] Three type-II RIPs or mistletoe lectins, MLI, MLII and MLIII, have been isolated from mistletoe extract [12], MLI
Correspondence to H Niwa, School of Crystallography, Birkbeck
College, University of London, Malet Street, London WC1E 7HX,
UK Fax: + 44 20 76316803, Tel.: + 44 20 76316800,
E-mail: h.niwa@mail.cryst.bbk.ac.uk
Abbreviations: MLI, MLII, MLIII, mistletoe lectin I, II, III;
RIP, ribosome-inactivatingprotein; MLA, MLI A-chain; RTA,
ricin toxin A-chain; ABA, abrin-a A-chain; MLB, MLI B-chain;
RTB, ricin toxin B-chain; ABB, abrin-a B-chain.
(Received 11 March 2003, revised 27 April 2003,
accepted 30 April 2003)
Trang 2beingthe most abundant MLI exists as a noncovalently
associated dimer (A–B)2in high concentration [13,14] while
ricin, abrin, MLII and MLIII are monomeric toxins The
importance of the fact that MLI is cytotoxic as a dimeric
type-II RIP has been emphasized, as it is known that some
type-II RIPs exist as dimers or tetramers and are not
cytotoxic to a whole cell system, even though their A-chains
can inhibit protein synthesis in a cell-free system [15] These
include dimeric R communis agglutinin [16], an isolectin of
ricin, and Sambucus nigra agglutinin V [17] from the elder
plant, and also a tetrameric S nigra agglutinin I [18]
Type-II RIPs are usually Gal/GalNAc-specific lectins,
S nigra agglutinin I alone having specific affinity for
terminal sialic acid sequences [19] MLI also has some
affinity for terminal sialic acid sequences, which is another
major difference from ricin [20] AmongGal/GalNAc
lectins, affinity for Gal and GalNAc differ MLI is Gal
specific, showinglittle affinity for GalNAc [21], whereas
MLIII is GalNAc specific and MLII has similar affinity
for both [12] Whereas ricin shows similar affinity for both
Gal and GalNAc, R communis agglutinin exhibits little
affinity for GalNAc [22] S nigra agglutinin V has higher
affinity for GalNAc than Gal [17] Why these differences
occur in homologous proteins or lectins is a subject for
further study
Previous papers have described the crystallization of
MLI [23] and subsequently the structure of MLI at 3.7 A˚,
showingit to be a dimer in the solid state [24] In this
paper, we present the structure of MLI with noncovalently
bound galactose The present structure, including bound
galactose molecules, was refined at 3.0 A˚ The
sugar-bindingsites are clearly defined and are discussed in detail
The biological importance of MLI as a dimeric cytotoxic
type-II RIP is also discussed and details of the dimer
interface are analysed The availability of the known
structure of MLI without a specific sugar [25] (pdb 1ce7,
2mll) has enabled us to make a comparison with the
present structure
Weak diffraction is often associated with high solvent
content and a large unit cell However, Krauspenhaar et al
[26] recently reported the structure of MLI with adenine
bound in the A-chain active site These crystals, grown in
a microgravity environment, exhibited improved diffraction
quality over other MLI complexes The method of
crystal-lization may have contributed to the improvement in
diffractingpower
Materials and methods
Extraction and purification of MLI has been described
previously [7] MLI crystals were obtained by the hanging
drop method The protein solution at 18 mgÆmL)1
concen-tration contained 0.1Mgalactose and 0.01Macetate buffer
at pH 4.0 The reservoir solution contained 0.9M
ammo-nium sulfate and 0.1Mglycine buffer at pH 3.4 The droplet
consisted of 1 lL of the protein solution and 1 lL of the
reservoir solution Hexagonal crystals grew to about
0.2 mm in a few weeks Sequence data were obtained as
described previously [24] and in [27]
The X-ray data were collected at the Synchrotron
Radiation Source (SRS) at the Central Laboratory of the
Research Councils (CLRC) Daresbury Laboratory, UK,
on station 7.2, with wavelength 1.488 A˚ and a 30-cm Mar Research image plate detector Before being mounted on a loop, the crystal was soaked in a cryoprotectant solution containingabout 30% glycerol and was flash cooled to
100 K for data collection Each image was collected with a 1.5 step and a 900-s exposure time A total of 16 images for the sweep range 24.0 were collected
Data processingwas carried out withDENZOand SCALE-PACK[28], and subsequently withSCALAin the CCP4 suite [29] The space group was P6522 as previously determined [24], with unit cell parameters of a¼ b ¼ 107.65 A˚,
c¼ 311.92 A˚ The solvent content was calculated to be 71% for one molecule of MLI with molecular mass 62 kDa per asymmetric unit [30] The data were processed to 2.9 A˚, providing68 126 measured reflections, which reduced to
24 175 unique reflections The overall completeness and multiplicity (redundancy) to 2.9 A˚ were 98.0% and 2.8, respectively, and the overall R-merge was 6.9%
Molecular replacement was performed withAMORE [31] usingthe previous partial 3.7-A˚ model [24], and the more complete ricin model [32] (pdb 2aai) was used independ-ently for checkingpurposes The structure was refined usingX-PLOR[33] and in the later stages theCNSpackages [34], with manual intervention on graphics using O [35] Refinement was carried out usingdata up to 3.0 A˚ (The R-merge between 3.12 A˚ and 3.0 A˚ was 28.1%.) There was a total of 21 899 reflections, of which 1117 (5%) were kept separate to calculate Rfree All reflections were used for the refinement with a bulk-solvent correction proce-dure Individual isotropic B-factors were subsequently employed This reduced the R-factor and Rfree by 2.1% and 1.9%, respectively, without introducingany unrea-sonable B values An anisotropic overall B-factor was also refined
The C-terminal residues after Gly248 of the A-chain could not be located because the electron density was too weak, presumably because of disorder A total of six glycosylating sugar units was included in the final model
as follows For the four putative glycosylation sites of type Asn-X-Thr/Ser in MLI (Asn112 in the A-chain, Asn61, Asn96 and Asn136 in the B-chain), it was possible to model the first glycosylating sugar as GlcNAc to all four sites and in addition a second GlcNAc at Asn96 and Asn136 in the B-chain A model includingfour water molecules was refined to an R-factor 20.6% with Rfree 25.8% [36] This model was further refined to include 47 water molecules, which were selected from the peaks above 3.5 r in the Fo–Fcmap by examiningthe geometry and the electron density The final R-factor and Rfreewere 19.9% and 24.0%, respectively Ramachandran plots were calculated using PROCHECK [37] For nonglycine and nonproline residues, 87.6% were in the most favoured regions, and 12.4% were in additional or generously allowed regions
Other important refinement statistics are summarized in Table 1 Hydrogen bonds and hydrophobic contacts between sugar molecules and protein, and between dimer molecules were analysed usingHBPLUS[38] Figures 1 and 6–8 were drawn withMOLSCRIPT[39], and Figs 4 and 5 were drawn with SETOR [40] The refined coordinates and structure factors have been deposited at PDB with the accession code 1OQL
Trang 3Overview and the A-chain
Figure 1 shows a ribbon diagram of the structure of MLI
The overall structure of the MLI monomer is similar to two
other type-II RIPs, ricin [32] (pdb 2aai) and abrin-a [41]
(pdb 1abr), and when the current structure was super-imposed on to the other type-II RIPs, the number of matched Ca atoms for MLI and ricin was 469 (91.8% out of 511) with rmsd 0.92 A˚, and 479 (93.7%) with rmsd 1.05 A˚ for MLI and abrin-a, usinga 3.0-A˚ cut-off distance As differences exist between the current and already published sugar-free MLI [25] in terms of sequence and detailed structure as described below, a structure-based sequence alignment of the A-chains of MLI (MLA), ricin (RTA) and abrin-a (ABA), together with a type-I RIP momordin [42] (pdb 1mom) is shown in Fig 2
Although the structure of MLA determined here clearly has some features in common with other RIPs, important differences do occur, for example in solvent-exposed regions, particularly in the region running from strand e
to helix B (residues 91–100 of MLA) In this region MLA and ABA are structurally similar, whereas in RTA and momordin this region is more extensive, with additional amino-acid residues and consequent differences in the structures This region has been proposed [43] as being an antigenic epitope site in RIPs, and, in fact, this is the site where a monoclonal antibody against MLA has been found
to bind [7] Another notably labile region is the sheet g–h Structural lability in this region is commonly observed in both type-I RIPs and the A-chains of type-II RIPs, and is the region in the present structure with the most consistently high B-factors
The active site is located in a prominent, centrally located cleft of the A-chain Six residues conserved among all RIPs (Fig 2; Tyr76, Tyr115, Glu165, Arg168, Trp199 and Ser203) are located here in a structurally highly conserved hydrogen-bond network Tyr76 alone in this region exhibits various conformations in known RIP structures [42], and its conformational change is the main difference in active-site geometry compared with the A-chain of ricin, as reported in sugar-free MLI [25] In MLA, there is a glycosylation site at Asn112 at the edge
of the active-site cleft, which is discussed further in the Discussion section
B-chain The B-chain of MLI (MLB) consists of two homologous globular domains Each domain has a diameter of 30 A˚ and consists of three repetitive subdomains, which form
a pseudo-threefold symmetry around a hydrophobic core The fold of one such domain has been classified as the b-trefoil fold [44] Sequence alignment of the six subdomains with those of ricin (RTB) and abrin-a (ABB) is shown in Fig 3 Three B-chains can be aligned without insertion or deletion except for the N-terminal region It should be noted that the assignment of subdomains here differs from that described for RTB [45]: in the RTB description, 1a1 (the suffix indicates a strand number in a subdomain shown in Fig 3) was not included in the repetitive subdomain, and one unit consists of, for the first one for example, 1a2, 1a3, 1a4 and 1b1, if the strand designation in Fig 3 is used, resultingin 1c and 2c units beingone strand short Three disulfide bonds are conserved in MLB, but one in the 1a subdomain is lost as the result of a mutation from Cys to Ser39 There are two glycosylation sites in RTB and ABB, which are conserved in MLB In addition, MLB has
Table 1 Refinement statistics.
No of reflections (total, test set) 21899, 1117
No of atoms
Total, proteins 4132, 3977 (511 residues)
Covalently bound sugars 84 (6 GlcNAc)
Ligands (galactose) 24 (2 Gal)
Average B-factors (A˚ 2 )
Covalently bound sugars 60.3
Ligands (galactose) 65.0
Rmsd from ideal values
Fig 1 Ribbon representation of the structure of MLI The A-chain is
located above and the B-chain is below The disulfide bond between
the two chains is shown in yellow The glycosylating sugars included in
the final structure are shown in brown Galactose molecules are
depicted in ball-and-stick Tyr76 and Tyr115 in the active site of the
A-chain, and Asp23 in the N-terminal sugar-binding site of the B-chain
are shown in red.
Trang 4another glycosylation site at Asn61 The structure of one
subdomain may be summarized as: strand 1 fi turn fi
b-sheet (strands 2 + 3) fi W-loop (includinga 310-helical
region at the end) fi strand 4 Strands 1 and 4 also form a
b-sheet An interestingfeature of the fold is provided by the
repetitive Gln-X-Trp (QxW) sequences (Fig 3) [46]
Sugar-binding sites
Sugar-binding sites in RTB exist in the subdomains 1a and
2c [45], and the residues involved in protein–sugar
inter-actions in RTB are also retained in these (and only these) two subdomains in MLB (Fig 3) Clear electron density for binding sugar molecules was observed at each site (Fig 4) b-Galactose was modelled into both sites, the electron density beingcomplete for the C-terminal site, but lacking for 1-OH at the N-terminal site The electron density for the side chain of Lys254 in the C-terminal site was also weak and tenuous, indicatingdisorder Water molecules bound to galactose, three in the C-terminal site, were included in an attempt to complete all possible sugar-binding interactions Each sugar-binding site exists in a shallow cleft formed by
Fig 2 Sequence alignment of MLA, RTA, ABA and momordin (MOM) The secondary-structure designation follows that of ricin [32] The C-terminal residues not included in the refined structure are indicated in lower case Identically conserved residues amongthese four proteins are shown in bold: those that are also identically conserved amongall RIPs are darkly shaded, and those highly conserved are lightly shaded Conserved residues in RIPs may be obtained from the Pfam database Possible glycosylation sites are underlined.
Trang 5contiguous stretches in the protein chain with some 20
residues in one subdomain, from strand 2, a 310-helical kink,
strand 3 and anW-loop with a 310-helical kink at the end,
as indicated in Fig s 3 and 5
Possible interactions between MLB and galactose are
summarized in Fig 5 together with those between RTB and
lactose usingthe same criteria for comparison, and Fig 6
provides a stereoview of the N-terminal sugar-binding site in MLB Protein–galactose interactions that are common in both the N-terminal and C-terminal sugar-binding sites of RTB [45] are also common to the two sites of MLB These are: (a) via hydroxy groups 3-OH and 4-OH of galactose, donatinghydrogen bonds to Od2 and Od1 of aspartate (Asp23 in the N-terminal site, Asp235 in the C-terminal
Fig 3 Sequence alignment of six subdomains in MLB, RTB and ABB The strand regions are indicated by horizontally striped rectangles with the strand number in the top row, and the common 3 10 -helical (kink) regions are indicated by obliquely striped rectangles Identically conserved Ile and Trp are shown in dark shading and the conserved hydrophobic residues in light shading Cysteines that make disulfide bonds are in yellow, the key residues involved in sugar binding are in magenta and possible glycosylation sites are marked in cyan Repetitive QxW sequences are underlined.
Fig 4 Stereoview of an electron density map of the C-terminal MLB sugar-binding site Cyan: 2F o –F c + 1.25 r; blue: F o –F c + 3.0 r; red:
F o –F c ) 3.0 r Purple, F o –F c + 3.0 r, was calculated by deletingcoordinates of galactose and water molecules.
Trang 6Fig 5 Schematic drawings of the sugar-binding sites of MLB and RTB (A) MLB N-terminal site (B) MLB C-terminal site (C) RTB N-terminal site (D) RTB C-terminal site Key residues, hydrogen bonds that are formed with bound sugar, and secondary-structure elements are shown.
Fig 6 Stereoview of the N-terminal MLB sugar-binding site showing a hydrogen-bond network.
Trang 7site); (b) Nd2 of asparagine (Asn47, Asn256) donating a
hydrogen bond to 3-OH of galactose; (c) an aromatic ring
(Trp38, Tyr249) makinghydrophobic contact by stacking
its ringapproximately parallel to the C3–C4–C5 plane of
galactose These aspartates, asparagines and aromatic rings
are identically conserved residues in all B-chain
sugar-bindingsites
In addition to these common interactions, specific
contacts are made in each site, as described below in
comparison with RTB In the N-terminal site, Nf of Lys41
interacts with 2-OH and 3-OH of galactose, and 2-OH
donates a hydrogen bond to Od2 of Asp27 In RTB,
although the lysine is conserved (Lys40), because of the
distance between the probable hydrogen of Nf and 2-OH or
3-OH being slightly greater than the criterion used (2.5 A˚),
it was not selected as a hydrogen bond here Asp27 in MLB
corresponds to Gly26 in RTB, which consequently does not
form a hydrogen bond In RTB, 6-OH forms hydrogen
bonds with Ne2 of Gln35 and a main-chain N, whereas in
MLB 6-OH is not involved in interactions with the protein
The glutamine is conserved in the two proteins, however,
and differences in orientation of the CH2OH groups in
galactose are responsible for the changes in these
interac-tions The main-chain N of Asp26 in MLB donates a
hydrogen bond to 4-OH, but in RTB a bond between N of
the correspondingresidue (Asp25) and 6-OH is probable, as
mentioned above In the C-terminal site of MLB, a
water-bridged hydrogen bond is made between 3-OH of galactose
and Oc1 of Thr252, which is equivalent to Lys41 at the
N-terminal site In RTB, Thr252 is mutated to His251, of
which Ne2 directly donates a hydrogen bond to 3-OH In
MLB, positive electron density was observed extending
from 6-OH of galactose to Arg245, where two
hydrogen-bondingwater molecules were located (Fig 4) Ile247,
which is equivalent in position to Gln36 of the N-terminal
site, makes hydrophobic contact with C6 of galactose, as is
also observed in RTB The main-chain N of Gln238 donates
a hydrogen bond to 4-OH, as equivalent interactions are
observed in the N-terminal site of MLB (Asp26–4-OH, as
described above) and in the C-terminal site of RTB
(Ala237–4-OH) Although specific interactions with bound
sugar are not established, Asp26 in MLB with galactose
undergoes conformational change (v1¼)164) as
com-pared with Asp25 in RTB with lactose (v1¼)65)
Hydrogen-bond networks of the sugar-binding sites
Oc2 of the identically conserved aspartate (Asp23, Asp235),
which accepts a hydrogen bond from 3-OH of galactose,
accepts another hydrogen bond from Ne2 of g lutamine
(Gln48, Gln257) (Fig 6) The Ne2 and Oe2 of this
glutamine form hydrogen bonds with the main-chain atoms
in the W-loop Nd2 of identically conserved asparagine
(Asn47, Asn256), which donates a hydrogen bond to 3-OH
of galactose, also donates a hydrogen bond to the
main-chain O of valine (Val24, Val236), while the main-main-chain N
of this valine donates a hydrogen bond to the main-chain O
of the asparagine, thus forming a bridge between the region
from strand 2 to the helical kink (left-hand side of a
sugar-bindingsite in Figs 5 and 6) and the helical kink at the end
of the bindingsite (right-hand side) The above valine and
glutamine are identically conserved in all B-chain
sugar-binding sites (Fig 3) A hydrogen bond that corresponds to the one from the valine N to asparagine O is correspond-ingly observed in any subdomain of the RIP B-chain Dimer structure
In the MLI dimer, shown in Fig 7, two A–B monomers are related by crystallographic twofold symmetry and face each other at the N-terminal domain of the B-chain, where the three hairpin loops in the b-trefoil fold make major contacts with those of the other in the followingway: a« c¢, b « b¢,
c« a¢ [24] When viewed perpendicularly to the twofold axis, two oblong-shaped MLB molecules are seen to make
an angle of 160 The dimensions of the dimer are
157· 63 · 48 A˚3, and the contact area is 755 A˚2 per monomer The existence of a dimer structure is consistent with an earlier electron microscope study of MLI [14], which corroborates the idea that this is a real dimer form, not an artefact of the crystal packing The distances between sugar-bindingsites in one dimer, calculated as straight distances between correspondingO4 atoms of galactose, are shown in Fig 7
A number of both polar and nonpolar contacts are observed at the dimer interface, as shown in Fig 8 Residues
in the 1b subdomain (the side chain of Tyr68, the main-chain atoms of Ala72 and Gly73, and Val74) make hydrophobic contact with the equivalent region in the other molecule In addition, Og of Tyr68 makes a hydrogen bond with the side chain of Gln122 in the 1c¢ subdomain Several hydrogen bonds are formed between residues in the 1a and 1c¢ subdomains, some of which are water mediated In addition, Ile114 makes hydrophobic contacts with carbon atoms in the side chains of Arg25 and Asn26 Among the residues that make polar interactions, Gln34, which makes hydrogen bonds with the main-chain atoms of Thr118, and Ser111, which makes water-mediated hydrogen bonds with the O of Gly32, are not conserved in RTB and ABB However, Oc of Ser111 is located in a similar position to Oc1 of Thr110 of RTB Amongthe hydrophobic contacts, Ilel14 of MLB is mutated to an asparagine in RTB and
a serine in ABB
Comparison of MLI structures Two MLI structures are currently available in the PDB, namely 1ce7 and 2mll [25] The coordinate sets for these are identical and will be referred to only as 2MLL, which
is MLI without lectin-bound sugar 2MLL was refined at 2.7 A˚ to an R-factor¼ 25.1% and Rfree¼ 31.9%, accord-ingto the PDB file The published model comprises 241 residues in the A-chain, 255 residues in the B-chain, 3 glycosylating GlcNAc sugars, and 215 water molecules Excludingthe C-terminus of the A-chain, where residues could not be located in either the current structure or 2MLL, and the N-terminus of the B-chain, where the structure is not defined well in the current structure and residues were not located in 2MLL, the numbers of differences in sequence between the two structures are 39
in the A-chain and 31 in the B-chain These differences arise from: (a) differences in original sequences determined by two methods [27,47]; (b) the fact that 2MLL contains truncated residues in solvent-exposed loops and also a total
Trang 8of 11 deleted residues compared with its original sequence
[47]
The A-chains of the present structure and 2MLL can be
superimposed, with rmsd 0.56 A˚ for 232 matched residues,
and the B-chains with 0.45 A˚ for 253 matched residues with
3.0-A˚ cut-off distance In the A-chains, structural
differ-ences exist in solvent-exposed regions, and there is a shift by
one residue in the strand a at the N-terminus However,
there is no significant discrepancy in the active site, including the conformation of Tyr76 (Tyr75 in 2MLL), which differs from that of ricin
In the B-chains, three points relatingto sugar bindingare: (a) in the N-terminal sugar-binding site, the locations of Oe1 and Ne2 of a glutamine (Gln36 in the current structure and Gln32 in 2MLL) are interchanged; (b) in the C-terminal site, 2MLL has deletions at Gln238 and Ala239 of the current
Fig 8 Stereoview of the MLI dimer interface Three hairpin loops (strand 2 and 3) of the N-terminal domains of the B-chains are shown The dimer molecules are related by twofold symmetry and the view is alongthe twofold axis Residues that make hydrophobic contacts are in darker grey.
Fig 7 Ribbon representations of MLI dimer (A) and (B) show orthog onal views of the whole dimer with bound galactose molecules (C) is the top view of (A), depictingonly B-chains, where in the N-terminal domains only the hairpin loops (strand 2 and 3) of three subdomains are depicted and their design-ations are shown in the same colour Distances between galactose molecules (straight distan-ces between two O4 atoms) are also shown.
Trang 9structure and, as a result, the position of Ala239 in the
current structure is occupied by Asn232 in 2MLL, which
corresponds to Asn240 of MLI; (c) the conformation of
a lysine (Lys254 in the current structure and Lys246 in
2MLL) is different in the two structures; however, as
mentioned previously the electron density of this side chain
is weak in the current structure (and possibly also in 2MLL)
and this point does not justify further discussion
Discussion
The refined structure of MLI complexed with galactose is
presented The molecular structure shares common features
with other RIPs without extra or shortened main-chain
loops MLA has a glycosylation site at the rim of the
active-site cleft, which is unique amongRIPs On superimposition
of the structure of the RTA–ApG complex [48] on to MLA,
a glycosylating MLA sugar at Asn112 was seen to occupy
(at least partially) the guanine-binding site of the RTA
substrate analogue However, glycosylation appears not to
affect the catalytic activity because it is known that
recombinant MLA shows similar activity to that of
plant-derived MLA [27] As molecular dynamics studies also
suggest [48,49], it is possible that, when the RNA substrate
loop binds to an A-chain of a type-II RIP, it adopts a
different conformation from that of the dinucleotide
substrate analogue In fact, the conformation of the bound
ApG in ricin differs from that in the structure of the
ribosomal loop determined by NMR [50] (pdb 1scl) or by
X-ray crystallography [9] (pdb 430d, 483d)
The two sugar-binding sites of MLB exhibit common
features sometimes observed in sugar-binding sites in
proteins other than RIP B-chains: an extensive
hydrogen-bond network and hydrophobic stacking[51] Sugar-affinity
studies have shown that 4-OH and then 3-OH are the
hydroxy groups of galactose that strongly affect sugar
bindingfor MLI [21] and ricin [52], and they are involved in
the common bindingmode for the two sugar-bindingsites
of both proteins
It is known that GalNAc binds only to the C-terminal
sugar-binding site of ricin, which is the high-affinity site [53]
However, experimental results on the difference in sugar
specificity between the two bindingsites are not available for
MLB In interpretingsugar-bindingspecificity assays of
lectins, consideration should be paid to the actual number of
bindingsites in each protein The two sugar-bindingsites
in type-II RIP B-chains are structurally and chemically very
similar, but not identical As a dimer, MLI in fact possesses
four sugar-binding sites, and this property, in view of the
novel interbinding-site distances resulting from this
dimeri-zation, is likely to affect the toxin’s ability to bind to cell
surface sugars Ambiguity exists when the sugar specificity
of a single bindingsite is examined usingbiochemical data
that are unavoidably from all of the available binding
sites With respect to the specificity of MLI for GalNAc,
however, evidence derived from solid-phase assay [54]
indicates that each bindingsite in fact does not have high
GalNAc specificity
There is insufficient space for the N-acetyl group of
GalNAc to be accommodated in the N-terminal site of
MLB, because it is blocked by the two residues that form
hydrogen bonds with 2-OH, namely Asp27 and Lys41
As to the C-terminal site, there is a possibility that the disordered side chain of Lys254 causes steric hindrance with the N-acetyl group of GalNAc However, because of the disorder, whether it completely hinders sugar binding is not conclusive When a GalNAc is located at the C-terminal sugar-binding site of RTB so that its pyranose ring is superimposed on that of the galactose of bound lactose, it is found that the oxygen in the acetyl group could form a hydrogen bond with the hydroxy group of Ser238 In MLB Ser238 in RTB is mutated to Ala239 and cannot make a hydrogen bond with a sugar It is proposed that this serine
in RTB may contribute to GalNAc binding
In the present MLI structure, the electron density of galactose in the N-terminal site of MLI was less pronounced than that in the C-terminal site This may suggest that, of the two sites, the N-terminal site has the lower affinity for galactose, as is also the case for ricin
MLI crystallizes readily from ammonium sulfate in acidic conditions (pH 3.4 for the actual crystal used for the current structure analysis) Sugar affinity decreases under acidic conditions for MLI [55] and ricin [56], therefore the electron density of galactose shown in Fig 4 may be regarded as correspondingto a partially bound sugar The cause of this decrease in affinity is considered to be either due to protonation of the aspartate in the sugar-binding site, which can be a hydrogen-bond acceptor, or conformational change As there is little conformational change among the crystal structures of MLI (pH 3.4), ricin (pH 4.75) [32] and abrin-a (pH 8.0) [41], the decrease in sugar affinity in acidic conditions is probably associated with protonation of the identically conserved aspartate, which interacts with 3-OH and 4-OH of bound galactose in these sugar-binding sites The MLI dimer involves several polar and hydrophobic contacts through the N-terminal domains of two B-chains
As the formation of the dimer is concentration dependent [13,14], the overall interaction is weak In the 1a subdomain
of the B-chains of some type-II RIPs, the hairpin loop is stabilized by a disulfide bond, as in RTB and ABB However, this S-S bond does not exist in MLB because of the mutation from a cysteine to Ser40, and the loop is more flexible Therefore, it may be speculated that the increased flexibility caused by the loss of the disulfide bond in MLB may play a crucial role in dimer formation [24] As little shift of the 1a loop is observed in the superimposition of MLB, RTB and ABB, it appears that at least permanent dislocation of the loop is not a requisite for dimer formation It is not possible
to conclude, however, from this study alone how important each of the various interactions described in the Results section is individually for the dimer formation Although hydrophobic 1b hairpin loops do in fact contact each other, this region is also hydrophobic in monomeric RTB and ABB,
as seen in the sequence in Fig 3, therefore this hydrophobic contact alone is not enough to cause MLI dimer formation Amongthe residues that make interactions, Gln34 and Ile114 are unique to MLB Mutation studies of these residues may reveal their role in the dimer formation
In contrast with some type-II RIPs mentioned in the Introduction, the dimeric structure of MLI does not interfere with its toxic activity It may be, however, that the quaternary structure does affect other aspects of the biological processes One factor that may be involved
is the relative disposition of the sugar-binding positions
Trang 10The distance between two sugar-binding sites in a monomer
is 47 A˚, whereas in the MLI dimer two N-terminal
sugar-bindingsites are 15 A˚ apart on one side of the dimer and
two C-terminal sites are 87 A˚ apart on the other side of the
dimer (Fig 7) All sugar-binding sites are centrally located
in the MLI dimer Unless some quaternary structural
change occurs, the dimer must bind to the cell surface by
layingthe whole (A–B)(B–A) structure alongthe cell surface
so that the bindingsites are close enough to cell-surface
oligosaccharides
As there is no significant difference in catalytic activity
between recombinant MLA and plant-origin MLA, it
may be argued that differences in cytotoxicity between
MLI and ricin are mainly influenced by the B-chains [27]
The actual bindingsites of the toxins on cells, subsequent
transport processes, and biological responses are probably
affected by differences in fine sugar specificity and/or
quaternary structure with associated differences in the
arrangement of the sugar-binding sites Recent studies
highlight differences in the membrane-binding sites of
MLI and ricin [57] It has been sug g ested [58] that ricin
may in fact dimerize on bindingto cell surface receptors
However, the study was carried out on ricin with
chemically blocked sugar-binding sites, and the actual
dimer form was not specified
There is evidence to suggest that RIP B-chains evolved
from a primordial peptide of about 40 residues: (a) by gene
duplications and fusions into a three-subdomain protein
(a b-trefoil domain); and (b) by further duplication into a
two-domain protein [59] The fact that some bacteria have
proteins with a b-trefoil domain sharingsequence and
structural homology with RIP B-chains [60] suggests that
b-trefoil fold proteins existed in early evolutionary
proces-ses When the six subdomains of MLB are aligned, it is seen
that the sequence identity between subdomains and also
between two domains is about 20% However, overall
sequence identity between MLB, RTB and ABB is more
than 50% [27] Hence, it is reasonable to speculate that RIP
B-chains diverged evolutionarily from a precursor protein
that already had a two-domain structure
Acknowledgements
We thank members of the School of Crystallography at Birkbeck
College for their valuable discussions, and colleagues at Daresbury
Laboratory, UK for their help and support duringthe course of this
work.
References
1 Franz, H (1985) Inhaltsstoffe der Mistel (Viscum album L.) als
potentielle Arzneimittel Pharmazie 40, 97–104.
2 Barbieri, L., Battelli, M.G & Stirpe, F (1993)
Ribosome-inactivatingprotein from plants Biochim Biophys Acta 1154,
237–282.
3 Van Damme, E.J.M., Hao, Q., Chen, Y., Barre, A.,
Vandenbus-sche, F., Desmyter, S., Rouge´, P & Peumans, W.J (2001)
Ribo-some-inactivatingproteins: a family of plant proteins that do more
than inactivatingribosomes Crit Rev Plant Sci 20, 395–465.
4 Peumans, W.J., Hao, Q & Van Damme, E.J.M (2001)
Ribo-some-inactivatingproteins from plants: more than RNA
N-gly-cosidases? FASEB J 15, 1493–1506.
5 Sandvig, K & van Deurs, B (1996) Endocytosis, intracellular transport, and cytotoxic action of Shiga toxin and ricin Physiol Rev 76, 949–966.
6 Agapov, I.I., Tonevitsky, A.G., Moysenovich, M.M., Mal-uchenko, N.V., Weyhenmeyer, R & Kirpichnikov, M.P (1999) Mistletoe lectin dissociates into catalytic and bindingsubunits before translocation across the membrane to the cytoplasm FEBS Lett 452, 211–214.
7 Agapov, I.I., Tonevitsky, A.G., Maluchenko, N.V., Moysenovich, M.M., Bulah, Y.S & Kirpichnikov, M.P (1999) Mistletoe lectin A-chain unfolds duringthe intracellular transport FEBS Lett.
464, 63–66.
8 Endo, Y & Tsurugi, K (1987) RNA N-glycosidase activity of ricin A-chain J Biol Chem 262, 8128–8130.
9 Correll, C.C., Wool, I.G & Munishkin, A (1999) The two faces of the Escherichia coli 23S rRNA sarcin/ricin domain: the structure at 1.11 A˚ resolution J Mol Biol 292, 275–287.
10 Ban, N., Nissen, P., Hansen, J., Capel, M., Moore, P.B & Steitz, T.A (1999) Placement of protein and RNA structures into a 5 A˚-resolution map of the 50S ribosomal subunit Nature (London)
400, 841–847.
11 Barbieri, L., Valbonesi, P., Bonora, E., Gorini, P., Bolognesi, A & Stirpe, F (1997) Polynucleotide: adenosine glycosidase activity of ribosome-inactivatingproteins: effect on DNA, RNA and poly (A) Nucleic Acids Res 25, 518–522.
12 Franz, H., Ziska, P & Kindt, A (1981) Isolation and properties of three lectins from mistletoe (Viscum album L.) Biochem J 195, 481–484.
13 Olsnes, S., Stirpe, F., Sandvig, K & Pilh, A (1982) Isolation and characterization of viscumin, a toxic lectin from Viscum album L (mistletoe) J Biol Chem 257, 13263–13270.
14 Lutsch, G., Noll, F., Ziska, P., Kindt, A & Franz, H (1984) Electron microscopic investigations on the structure of lectin I from Viscum album L FEBS Lett 170, 335–338.
15 Citores, L., Ferreras, J.M., Iglesias, R., Carbajales, M.L., Arias, F.J., Jime´nez, P., Rojo, M.A & Girbe´s, T (1993) Molecular mechanism of inhibition of mammalian protein synthesis by some four-chain agglutinins: proposal of an extended classification of plant ribosome-inactivatingproteins (rRNA N-glycosidases) FEBS Lett 329, 59–62.
16 Sweeney, E.C., Tonevitsky, A.G., Temiakov, D.E., Agapov, I.I., Saward, S & Palmer, R.A (1997) Preliminary crystallographic characterization of ricin agglutinin Proteins 28, 586–589.
17 Van Damme, E.J.M., Barre, A., Rouge´, P., Van Leuven, F & Peumans, W.J (1996) Characterization and molecular cloning
of Sambucus nigra agglutinin V (nigrin b), a GalNAc-specific type-2 ribosome-inactivatingprotein from the bark of elderberry (Sambucus nigra) Eur J Biochem 237, 505–513.
18 Van Damme, E.J.M., Barre, A., Rouge´, P., Van Leuven, F & Peumans, W.J (1996) The NeuAc (a2,6) Gal/GalNAc-binding lectin from elderberry (Sambucus nigra) bark, a type-2 ribosome-inactivatingprotein with an unusual specificity and structure Eur J Biochem 235, 128–137.
19 Shibuya, N., Goldstein, I.J., Broekaert, W.F., Nsimba-Lubaki, M., Peeters, B & Peumans, W.J (1987) The elderberry (Sambucus nigra L.) bark lectin recognizes the Neu5Ac (a2–6) Gal/GalNAc sequence J Biol Chem 262, 1596–1601.
20 Wu, A.M., Song , S.-C., Hwang , P.-Y., Wu, J.H & Pfu¨ller, U (1995) Interaction of mistletoe toxic lectin-I with sialoglycopro-teins Biochem Biophys Res Commun 214, 396–402.
21 Lee, R.T., Gabius, H.-J & Lee, Y.C (1994) The sugar-combiningarea of the g alactose-specific toxic lectin of mistletoe extends beyond the terminal sugar residue: comparison with a homologous toxic lectins, ricin Carbohydr Res 254, 269–276.