b-galacto-sidase, the lactose transporter and thiogalactoside Keywords galactose uptake; glucose ⁄ galactose-binding protein; glyceryl galactoside; lactose uptake; Salmonella enterica se
Trang 1Salmonella typhimurium in complex with the physiological
Sanjeewani Sooriyaarachchi1, Wimal Ubhayasekera1, Winfried Boos2and Sherry L Mowbray1
1 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden
2 Department of Biology, University of Konstanz, Germany
Glucose⁄ galactose-binding protein (GBP) was the first
sugar-binding protein for which roles in active
trans-port [1] and chemotaxis [2] were demonstrated The
transport occurs via a typical ABC system [3]
consist-ing of three components: the periplasmic bindconsist-ing
protein (GBP, or alternatively, MglB) that acts as the
primary recognition site; a membrane-bound permease
(MglC); and a cytoplasmic module (MglA) that
cou-ples the binding⁄ hydrolysis of ATP to transmembrane
transport of the cognate substrates In Escherichia coli
and Salmonella enterica serovar Typhimurium (S
ty-phimurium), both galactose and glucose are
physiologi-cally important ligands [4,5] As well as having affinity
for the nonphysiological b-methyl-galactoside, from
which the name Mgl is derived, it was recognized early that the GBP from E coli also binds glyceryl-b-d-ga-lactopyranoside [6] Further work showed that only the (2R) diastereomer was bound [7], consistent with the fact that only this stereoisomer (hereafter referred
to as GGal) is found naturally as the polar head group
of plant glycolipids An estimated 16.6% of the total lipids in runner bean leaves represents GGal [8], and a similar abundance has been found in other plants, such
as red clover [9] Conjugated forms are common in both plants and animals
Interestingly, GGal is also a good substrate for all three components of the lac operon, i.e b-galacto-sidase, the lactose transporter and thiogalactoside
Keywords
galactose uptake; glucose ⁄ galactose-binding
protein; glyceryl galactoside; lactose uptake;
Salmonella enterica serovar Typhimurium
Correspondence
S L Mowbray, Department of Molecular
Biology, Swedish University of Agricultural
Sciences, Box 590, Biomedical Center,
SE-751 24, Uppsala, Sweden
Fax: +46 18 53 6971
Tel: +46 18 471 4990
E-mail: mowbray@xray.bmc.uu.se
Website: http://xray.bmc.uu.se/
(Received 13 December 2008, revised 31
January 2009, accepted 2 February 2009)
doi:10.1111/j.1742-4658.2009.06945.x
Periplasmic binding proteins are abundant in bacteria by virtue of their essential roles as high-affinity receptors in ABC transport systems and chemotaxis One of the best studied of these receptors is the so-called glucose⁄ galactose-binding protein Here, we report the X-ray structure of the Salmonella typhimurium protein bound to the physiologically relevant ligand, (2R)-glyceryl-b-d-galactopyranoside, solved by molecular replace-ment, and refined to 1.87 A˚ resolution with R and R-free values of 17% and 22% The structure identifies three amino acid residues that are diag-nostic of (2R)-glyceryl-b-d-galactopyranoside binding (Thr110, Asp154 and Gln261), as opposed to binding to the monosaccharides glucose and galac-tose These three residues are conserved in essentially all available glucose⁄ galactose-binding protein sequences, indicating that the binding of (2R)-glyceryl-b-d-galactopyranoside is the rule rather than the exception for receptors of this type The role of (2R)-glyceryl-b-d-galactopyranoside in bacterial biology is discussed Further, comparison of the available struc-tures provides the most complete description of the conformational changes
of glucose⁄ galactose-binding protein to date The structures follow a smooth and continuous path from the most closed structure [that bound to (2R)-glyceryl-b-d-galactopyranoside] to the most open (an apo structure)
Abbreviations
GBP, glucose ⁄ galactose-binding protein; GGal, [2R]-glyceryl-b- D -galactopyranoside; PDB, Protein Data Bank (http://www.rcsb.org).
Trang 2transacetylase [10] The (2R), and not the (2S),
diaste-reomer is formed by E coli b-galactosidase during
transfer of the galactosyl residue from any galactosyl
donor (including lactose) to glycerol [7,11,12] Further,
unlike lactose itself, GGal is an excellent inducer for
LacI, the repressor of the operon [13,14] Considering
these properties, one may be inclined to regard the
name ‘lactose operon’ as a misnomer, as it seems likely
that GGal, and not lactose, is the natural substrate of
the system Thus, GGal taken up by the Mgl
trans-porter will induce expression of the lac operon, and so
promote further uptake and utilization of the
com-pound Enterobacteriaceae, found in the gut of
ani-mals, encounter GGal in large quantities via the
ingestion of plant leaves (indeed, much more
fre-quently than an adult mammal is exposed to the
lac-tose contained in milk) In contrast to E coli and most
other Enterobacteriaceae, Salmonella has a deletion of
the entire lac operon However, because GGal can still
be transported quite effectively by the Mgl transport
system, it is expected that some other
b-galactosi-dase(s) in Salmonella can be used to metabolize it
The Km of the Mgl transporter for GGal is 2.8 lm,
comparable with the reported Kdof GBP for this
com-pound (3.2 lm) [6] Measured using the same methods,
the Kmand Kdvalues for galactose are similar, 0.5 and
1 lm, respectively; values for glucose are almost
identi-cal to those of galactose, its C4 epimer [15] Earlier
crystal structures of GBPs from E coli [16–18] and
Salmonella[19–21] showed the basis of recognition for
the monosaccharides Here, we report the crystal
struc-ture of Salmonella GBP in complex with GGal We
find that the protein provides a specific binding pocket
for the d-glyceryl moiety, and that the amino acids
lin-ing this pocket are highly conserved, reflectlin-ing the
widespread importance of GGal as a bacterial carbon
source
Results and Discussion
Overall structure
The structure of GBP in complex with GGal was
determined by molecular replacement using the
Salmo-nella GBP–Gal structure (PDB entry 1GCA) [21] as
the search model, and refined to 1.87 A˚ resolution with
final R and R-free values of 17% and 22% (Table 1)
Electron density was observed for all except residues
1–2 and 308–309 of the complete sequence in both
molecules of the asymmetric unit The structure is
composed of two similar domains, each representing a
b sheet sandwiched between two layers of a helices
(Fig 1A) Domain 1 is composed of residues 1–110
and 257–293; domain 2 includes residues 111–256 and 295–307
In each molecule, structural sodium and calcium ions are observed, bound in the loops following the first helices of domains 1 and 2, respectively The EF-hand-like calcium site of domain 2 was described earlier, and tight binding of the ion was shown to con-tribute to the integrity of the protein structure [17,22] The sodium site involves close interactions ( 2.3 A˚) with Gly28-O, Ala31-O and Val34-O, as well as with well-ordered water molecules (2.3–2.6 A˚) Although the concentration of sodium in the crystallization experiment (150 mm) falls within the generally accepted physiological range, no structural sodium ion was noted at the same position in earlier GBP struc-tures, from either Salmonella or E coli However, our inspection of the previous structures suggests that, in some cases, electron density modeled as a water mole-cule could actually be a sodium ion One thiocyanate ion is also located in the asymmetric unit, based on the characteristic linear shape of the electron density, and the presence of 0.2 m NaSCN in the crystallization
Table 1 Data collection and refinement statistics.
Data collection a
Environment ESRF ID14:4 Wavelength (A ˚ ) 0.955 Cell dimensions (A ˚ ) a = 36.4, b = 109.3,
c = 150.7 Space group P212121 Resolution (A ˚ ) 30.0–1.87 (1.97–1.87) Unique reflections 49 021
Average multiplicity 5 (5) Completeness (%) 96.4 (98.4)
<(I) ⁄ r (I)> 9.9 (3.4) Refinement
No reflections (completeness, %) 46 530 (96%) Resolution range (A ˚ ) 30.0–1.87 R-factor, R-free (%) 17.0, 22.2
No protein atoms (average B, A˚2 ) c
A molecule 2327 (9.6)
B molecule 2329 (9.9)
No water molecules (average B, A˚2 ) c 710 (21.3)
No ligand atoms (average B, A˚2)c 34 (5.4)
No ions (average B, A ˚ 2 ) c
Rms bond length (A ˚ ) 0.008 Rms bond angle () 1.052 Ramachandran plot outliers (n, %)d 4 (0.7%)
a
Values in parentheses are for the highest resolution shell.
b Rmerge= P
h
P
l jI hl ) ÆI h æ| ⁄ P
h
P
l < Ih> c Calculated using MOLE-MAN [48] d A stringent-boundary Ramachandran plot was used [49].
Trang 3solutions; this site appears to have no links with struc-ture or function
The rms difference when all Ca atoms of the two molecules in the asymmetric unit are compared is 0.3 A˚, slightly greater than the expected coordinate error in the structures ( 0.1 A˚) When the two domains are compared individually with a tightened cut-off of 0.5 A˚, it is seen that there is a very small (1.5) difference in their relative orientations A nearly perfect twofold axis (179) relates the two molecules, with 750 A˚2 on domain 1 of each molecule buried at the interface Dimers have been reported previously for the E coli protein under some conditions [23], however, inspection of a number of other GBP structures does not reveal any similar example, resulting from either non-crystallographic or crystallographic symmetry
GGal binding Electron density for the GGal ligand is clearly observed in the cleft between the two domains (Fig 1)
As illustrated in Fig 2, 15 hydrogen bonds directly link protein and ligand, six of which arise from domain 1, and nine from domain 2 Two water mole-cules also make hydrogen bonds with the ligand; several other residues contribute hydrophobic inter-actions (Fig 2)
Most of these interactions have been identified previ-ously in complexes with glucose or galactose [20,21,24,25] Asn91 is now shown to have an addi-tional role, forming a hydrogen bond to O2¢ of the glyceryl moiety Asn256 was known to interact with O1 of the preferred b-sugars [26], and this role is preserved for the glycoside oxygen of GGal Three other residues are exclusively linked to binding of the glyceryl moiety (marked with red ovals in Fig 2B):
N
A
B
C
Na+
Ca+2
GGal
Fig 1 Structure of the GBP–GGal complex (A) Overall structure of
GBP, color-coded using a scheme going from blue at the
N-termi-nus, through the rainbow to red at the C-terminus The GGal ligand
is shown in royal blue Structural sodium and calcium ions are
shown in red and blue, respectively (B) Electron density of GGal
in the final SIGMAA-weighted 2m|Fo| – d|Fc| map [50] contoured at
1 r = 0.49 e ⁄ A˚ 3
Fig 2 Interactions in the binding site (A) Stereoview of bound GGal showing GBP residues making hydrogen-bonding and aromatic inter-actions (B) Schematic diagram of the hydrogen bonds between GBP and GGal Interactions specific to the glyceryl moiety are marked with red ovals.
Trang 4Thr110 and Asp154 interact with O3¢, and Gln261
interacts with O2¢ These interactions increase the
number of hydrogen bonds between the protein and
ligand by five compared with the monosaccharides
The glyceryl moiety of GBP lies near the hinge of
the protein, in a pocket that is otherwise filled only
with water molecules (Fig 3) Indeed, this pocket,
which is lined by polar side chains, extends to the
sur-face of the protein, suggesting that even longer
com-pounds could be accommodated by GBP However, it
is not known what such compounds might be, or
whether they could be accepted by the transport
sys-tem It is probably significant that the sugar unit of
GGal lies closest to the portions of GBP that will
make first contact with the permease, as deduced from
mutagenesis studies summarized previously [27] By
presenting the sugar first, recognition by the permease
can be largely independent of the presence or absence
of the glyceryl moiety
Comparison with available sequences
The presence of the equivalents to residues Thr110,
Asp154 and Gln261 in a given GBP sequence would
thus be expected to indicate GGal binding, as opposed
to simply glucose⁄ galactose binding These residues
are, in fact, well conserved in the sequences of proteins
annotated as GBPs, some examples of which are given
in Fig 4 Asp154 and Gln261 are most tightly
con-served, whereas Thr110 may be conservatively replaced
by a serine residue; in more distant relatives, an alanine
is sometimes observed in this position We conclude
that GBP’s role in the binding and transport of GGal
is widespread in nature By contrast, the residues lining
the ‘extension’ of the glyceryl pocket that reaches the
surface are not conserved (Fig 4)
It should also be noted that a large number of
sequences are annotated incorrectly, as periplasmic
binding proteins of unknown specificity, lacI-type
repressors or even enzymes (Fig 4) Although designa-tion of a particular binding protein’s specificity should ultimately rely on a complete biochemical characteriza-tion, the patterns of conservation indicate that it is rather simple to distinguish GBPs from even their nearest relatives, the ribose-binding proteins Examples
of such features include residues Tyr10, His152 and Asp154, which are clearly present in the YP_087835.1 sequence (annotated as a RbsB), but replaced by other residues in the authentic ribose-binding proteins In addition, the repressor sequences include a DNA-bind-ing headpiece, and so are consistently longer than those of the binding proteins, even if one includes their signal sequences; for example, the sequence of E coli LacI is 363 residues, whereas the longest binding pro-tein of this type is typically 350 residues or fewer, and lacks the characteristic DNA-binding domain Thus, modest improvements to the existing methods of anal-ysis⁄ annotation would provide significant benefits, given that such proteins account for a large proportion
of the bacterial genome
An unrelated type of glucose-binding protein has been identified in some bacteria; its fold is not similar
to GBP, but rather to that of the larger maltose-bind-ing protein This kind of protein is exemplified by the Thermus thermophilus protein, PDB entry 2B3B [28] The mode of binding the monosaccharide is completely different in terms of orientation of the sugar, and inter-actions between protein and sugar, from that observed for GBP Further, there appears to be no room within the structure to accommodate the additional glyceryl moiety Thus, GGal binding is not expected to be a characteristic of this family of proteins
Conformational changes
As described above, the two molecules in the asymmet-ric unit of our structure differ only slightly ( 1.5)
in their degree of opening The similarity between the
Fig 3 Extension of the GGal site
Stereo-view of the residues lining the water-filled
tunnel that extends from the glyceryl moiety
to the surface of GBP are shown.
Trang 5Fig 4 Sequence alignments Representative sequences were identified by a BLAST search, and aligned using INDONESIA [45] after removal of the signal sequences using the SIGNAL P program [51] Residues interacting directly (via either van der Waals interactions or hydrogen bonds) with the monosaccharide unit in the current complex are marked with cyan, and those specifically related to the glyceryl moiety with red Residues lining the tunnel extending from the glyceryl site are marked in gray The sequences were annotated as follows (number of resi-dues given in each case in parentheses): YP_001783460, periplasmic binding protein ⁄ LacI transcriptional regulator Haemophilus somnus
2336 (328); YP_087835.1, RbsB protein from Mannheimia succiniciproducens MBEL55E (330); ZP_01786351, galactose-1-phosphate uridylyl-transferase from Haemophilus influenzae 22.4-21 (331); ZP_01169389.1, probable galactoside ABC transporter from Bacillus sp NRRL B-14911 (353); ZP_00134897.2, periplasmic component of ABC-type sugar transport system, Actinobacillus pleuropneumoniae serovar 1 str.
4074 (323); YP_720691.1, putative galactoside ABC transporter from Trichodesmium erythraeum IMS101 (342); ZP_02849935.1, periplasmic binding protein ⁄ LacI transcriptional regulator from Paenibacillus sp JDR-2 (338); YP_001311499.1, periplasmic binding protein ⁄ LacI transcrip-tional regulator Clostridium beijerinckii NCIMB 8052 (356); ZP_02035313.1, hypothetical protein BACCAP_00909 from Bacteroides capillosus ATCC 29799 (333) 2GX6 and 2IOY are authentic ribose-binding protein sequences for which structures are known [52] (M J Cuneo and
H W Hellinga, unpublished results).
Trang 6two molecules indicates that their conformation is
affected very little by differences in crystal packing
Comparison with the structures of Salmonella GBP in
complex with galactose (1GCA) [21] and glucose
(3GBP) [20] indicates that both are more open by
5, as illustrated in Fig 5A The structure of the
same protein, closed but without bound sugar (1GCG)
[25], is even more open ( 7 compared with the new
structures)
A number of structures are also available for E coli
GBP (1GLG, 2GBP, 2HPH, 2FVY, 2FW0, 2IPN,
2IPM, 2IPL, 2GX6) [18,24–26] (M J Cueno and
H W Hellinga, unpublished results), which given the
94% amino acid sequence identity, can be compared
with Salmonella GBP with confidence Least-squares
superimposition of domain 1 of all of the GBP
struc-tures is shown in Fig 5B, illustrating the ‘fan’ of
related conformations observed The GGal complex is the most closed structure found to date, perhaps because of the significantly larger number of hydrogen bonds compared with the structures with simple sug-ars The other structures represent a series of confor-mations that ‘link’ the GGal complex to the most open (apo, 2FW0) structure (by 37) through similar motions at the hinge As shown in Table 2, the three hinge strands do not contribute equally Changes in relatively few main-chain dihedral angles (primarily ones in the first hinge segment, that near residue 110) account for most of the motion observed Interestingly, Gly109 is a Ramachandran outlier in the closed struc-tures, but not in the most open one We conclude that, like the ribose- and allose-binding proteins of the same structural class [29,30], GBP has a preferred conforma-tional pathway in its motions However, inspection of Table 2 quickly shows that the motions are not of the same character in the three proteins, and that the three hinge segments contribute to different degrees The
A
B
Fig 5 Conformational changes (A) Stereo representation showing
the different domain relationships seen when binding galactose
(PDB entry 1GCA, gold) compared with GGal (A molecule, blue).
Domain 1 of the two structures is superimposed (B) Superposition
of domain 1 in the available GBP structures from Salmonella and
E coli The structures are colored progressing from blue (most
closed) to green (most open) in the series: GGal, GGal molecule B
(1.5), 2GBP (1.7), 1GLG (1.8), 2IPN (2.0), 2HPH (2.0), 2IPM
(2.0), 2IPL (3.4), 1GCA (5.1), 3GBP (5.4), 1GCG (7.0), 2FVY
(9.8) 2FW0 (opened by 36.8) was not shown for reasons of
clarity.
Table 2 Comparison of conformational changes Structures of GBP (GBP–GGal versus PDB entry 2FW0), ribose-binding protein (2DRI versus 1URP) and allose-binding protein (1RPJ versus 1GUD) were compared with the delta-dihedral command of the program
LSQMAN [44,48], which calculates Ca-Ca-Ca-Ca torsion angles Only differences > 10 are shown for residues in the three hinge seg-ments of each protein; equivalent residues of the various structures are aligned Where more than one molecule was present in the respective asymmetric unit, the A molecule was used for the calculation Both open ribose- and allose-binding protein structures differ by 43 from their closed forms The two proteins have 34% amino acid sequence identity to each other, and 28% and 25%, respectively, to Salmonella GBP.
Protein GBP
Ribose-binding protein
Allose-binding protein Segment 1
Val108 10.3 Ile101 )14.4 Gly109 39.7 Ala102 )24.2 Thr110 18.4 Thr112 12.7 Asp111 )25.7
Set112 )18.9 Glu114 )10.9 Segment 2
Val254 21.4 Ile233 13.4 Val245 35.9
Ala234 )30.5 Ala246 )54.0 Gln235 12.9 Gln247 )10.1
Asn248 )12.9 Segment 3
Val291 )12.8 Pro262 )21.0 Val293 16.5 Val281 )11.5 Pro294 )15.0 Asp264 16.5 Asp282 )25.3 Tyr295 )13.9 Leu265 )44.0 Ser283 )26.8 Val296 10.5 Ile284 12.9
Trang 7changes observed must be relevant both to the closing
that traps bound sugars, and the opening required for
a ligand’s release into the membrane-bound
compo-nents of the ABC transport systems Differences in the
direction of the motion could provide an additional
level of specificity in the action of such systems
Experimental procedures
Protein purification
E colistrain LA5709 [31], transformed with plasmid pBD10
[32], was used to overexpress GBP in Luria–Bertani medium
containing 50 lgÆmL)1 ampicillin, as described previously
[20,33] Following expression, the osmotic (chloroform)
shock fluid was removed and precipitated overnight using
60% (w⁄ v) ammonium sulfate The pellet was resuspended
in 10 mm Tris⁄ HCl buffer (pH 8.0), then dialyzed against
the same buffer The resulting sample was centrifuged at
5000 g at 4C for 15 min, passed through a membrane filter
(0.22 lm) and concentrated (Vivaspin concentrator, 10 kDa
cut-off, from Vivascience, Littleton, MA, USA) The
con-centrated samples were purified using cation-exchange
chro-matography, followed by anion exchange and gel filtration
on a Superdex 75 16⁄ 60 column The eluted fractions were
analyzed by SDS⁄ PAGE
To remove endogenously bound sugar, the purified
pro-tein sample was treated with 8 m urea and incubated at
room temperature for 30 min, then dialyzed in steps against
6, 4, 2, 1 and 0 m urea in 10 mm Tris⁄ HCl buffer (pH 7.4)
containing 1 mm CaCl2 at 4C The final concentrated
protein sample was analyzed by SDS and native PAGE to
confirm its homogeneity Protein was stored in 10 mm
Hepes (pH 7.0), 150 mm NaCl at)20 C
Crystallization
GBP was crystallized using the hanging-drop vapor
diffu-sion method at room temperature Drops were composed
of 1.0 lL mother liquor [20% w⁄ v poly(ethylene
gly-col) 3350, 0.2 m NaSCN] and 1.0 lL of a solution
com-posed of 0.29 mm (10 mgÆmL)1) protein and 0.60 mm GGal
(synthesized as described earlier [11]) Crystal formation
was facilitated by streak-seeding immediately after set-up
Prior to data collection, the thin plate-like crystals were
stabilized by a cryoprotectant solution [35% w⁄ v
poly(eth-ylene glycol) 3350 in the same buffer] and then flash-cooled
directly in liquid nitrogen
Data collection, structure solution, refinement
and model building
X-Ray data were collected at 100 K at beamline ID14:4 of
the European Synchrotron Radiation Facility (ESRF,
Grenoble, France) Data were processed with mosflm [34] and scaled with scala [35] Analysis of the unit-cell content
of GBP suggested that there would be two molecules in the asymmetric unit, consistent with a solvent content of 46% and a Vm of 2.3 [36] A relatively high Rmerge arose from some anisotropy in the data attributable to the thin, plate-like shape of the crystals Molecular replacement with molrep [37], as implemented in the ccp4 interface [38,39], utilized the protein only of the unliganded form of GBP (PDB entry 1GCA [21] as the search model The clear solution was improved with rigid-body and restrained refinement in refmac5 [40] The protein was rebuilt as needed in o [41] and refined in a cyclical fashion Waters were placed using the ARP⁄ warp-solvent command in ccp4 [38] Statistics for the data processing and final refined model are presented in Table 1 Structure factors and coordinates have been deposited at the PDB with the accession code 3GA5
Structural analysis, comparisons and figure preparation
Similar proteins were located using blast [42] Structures were obtained from the PDB [43] and compared using o and lsqman[44] Similar sequences were aligned using indonesia [45] Figures were prepared with the programs o, molscript [46], molray [47] and isis⁄ draw (http://www.mdli.com)
Acknowledgements
This work was supported by grants from the Swedish Research Council (VR) We thank ESRF staff mem-bers for their support during the data collection
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