Molecular details of ligand selectivity determinants in a promiscuous β-glucan periplasmic binding protein
BMC Structural Biology
Molecular details of ligand selectivity determinants in a promiscuous -glucan periplasmic binding protein
Parthapratim Munshi 0 1 2
Christopher B Stanley 0
Sudipa Ghimire-Rijal 0
Xun Lu 0
Dean A Myles 0
Matthew J Cuneo 0
0 Neutron Sciences Directorate, Oak Ridge National Laboratory , Oak Ridge, TN 37831 , USA
1 Department of Chemistry & Center for Informatics, Shiv Nadar University , Dadri, Uttar Pradesh 203207 , India
2 Department of Chemistry, Middle Tennessee State University , Murfreesboro, TN 37132 , USA
Background: Members of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to specifically bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. The tmCBP binding site is bipartite, comprising a canonical solvent-excluded region (subsite one), adjacent to a solvent-filled cavity (subsite two) where specific and semi-specific ligand recognition occur, respectively. Results: A molecular level understanding of binding pocket adaptation mechanisms that simultaneously allow both ligand specificity at subsite one and promiscuity at subsite two has potentially important implications in ligand binding and drug design studies. We sought to investigate the determinants of ligand binding selectivity in tmCBP through biophysical characterization of tmCBP in the presence of varying -glucan oligosaccharides. Crystal structures show that whilst the amino acids that comprise both the tmCBP subsite one and subsite two binding sites remain fixed in conformation regardless of which ligands are present, the rich hydrogen bonding potential of water molecules may facilitate the ordering and the plasticity of this unique PBP binding site. Conclusions: The identification of the roles these water molecules play in ligand recognition suggests potential mechanisms that can be utilized to adapt a single ligand binding site to recognize multiple distinct ligands.
Periplasmic binding protein; Carbohydrate recognition; Laminarin; ABC transport; Ligand specificity
The periplasmic binding proteins (PBP) are a protein
superfamily that serve as primary receptors for a diverse
group of metabolic solutes in signaling , chemotaxis 
and metabolite transport systems in bacteria ,
eukaryotes and archaea. PBP mediated transmembrane transport
of ligands are coupled to either ATP hydrolysis (ABC
transport)  or H+/M+ motive force (TRAP transport or
tripartite tricarboxylate transport) . In addition, the PBP
module is also found in enzymes , transcriptional
control elements  and eukaryotic neurotransmission
systems . PBPs bind multiple ligands that range in size
from a few Daltons to as large as 1 kDa, including ions ,
amino acids , peptides , monosaccharides ,
oligosaccharides , polyamines , oxidized inorganics
. Many other ligands continue to be discovered through
current genome sequencing technology .
Despite the wide variation in PBP cognate ligand size
and chemical functionality, the three-dimensional
structure is highly conserved across all PBPs. PBPs are
comprised of two / domains connected by a flexible linker
region that serves as a pivot point for the ligand induced
hinge-bending motion that this protein superfamily is
known for [17-20]. PBPs were initially classified into three
distinct sub-groups based upon the topology of -strands
in each domain . Recently, the PBP super-family was
re-categorized into six distinct clusters by combining
known ligand specificities with the wealth of structural
information available in the Protein Data Bank .
PBPs typically bind cognate ligands with exquisite
specificity, discriminating among anomeric/epimeric
carbohydrates or different ions [23,24]. This remarkable
ability to specifically bind their cognate ligands from
pools of similarly related molecules has been attributed
to the localization of the PBP ligand binding site at
the interdomain interface . In the apo form, ligand
binds to a highly adaptable solvent exposed surface
which upon complexation with ligand, and the other
PBP domain, produces an environment similar to a less
adaptable solvent excluded protein core [26,27]. In most
cases PBP function relies on differential recognition of
the apo and ligand bound forms of the protein by
transmembrane-bound proteins . Ligand binding at
the interdomain interface stimulates a conformational
change which is characterized as a rigid body
hingebending/twisting motion about the interdomain linker
region. The relative orientation of the two domains changes
by as much as 60-70 [29,30], although the magnitude of
the hinge-bending motion is variable and can be rather
small in some cases . The conformational coupling
of ligand binding and function is also conserved when
the PBP module is found in larger multidomain
proteins, such as the eukaryotic glutamate receptor  and
the LacI family of transcriptional regulators .
This PBP ligand binding paradigm was recently
altered when the crystal structure of the Thermotoga
maritima cellobiose binding protein (tmCBP) was
solved . Unlike other PBPs, the tmCBP binding site
is bipartite, being composed of a typical PBP solvent
excluded disaccharide binding site (subsite one) that is
adjacent to an atypical large solvent filled cavity (subsite
two) where three additional saccharide rings could be
placed (Figure 1). The structure of tmCBP was solved in
the presence of (1,4) linked sugars, however the size of
the tmCBP binding cavity and molecular modeling
suggested additional glucan sugar linkages could be
accommodated in both the disaccharide binding site and the
solvent filled cavity (Figure 1). tmCBP is found in an
operon that consists of an ABC transport system and an
endoglucanase, the natural substrate of which has been
predicted to be the algae-based storage polysaccharide
laminarin [32-34]. Using a series of laminarin-based
(1,3) linked carbohydrates we sought to further
identify the molecular mechanisms underlying simultaneous
encoding of specificity and promiscuity in tmCBP
subsites. These studies suggests ways that the bound
hydrogen bonding rich water molecules bound in subsite two
can potentially be used to adapt and expand ligand
binding sites beyond the functionality encoded by the
fixed protein scaffold.
Figure 1 Carbohydrates accommodated in the tmCBP binding
site. Previous molecular modeling studies of tmCBP identified that
in addition to (1,4) glucosaccharides (cyan), (1,3) (blue) and mixed
(1,3)/(1,4) (magenta) could be accommodated in the water-filled
(red spheres) non-specific ligand binding subsite. Adapted from .
Results and discussion
Thermal stability and ligand binding specificity of tmCBP
Previous molecular modeling of the tmCBP binding 
site suggested that in addition to (1,4) oligosaccharides,
additional linkages such as (1,3) carbohydrates or mixed
(1,4)/(1,3) could be accommodated in the bipartite
binding site (Figure 1). Thermal denaturation of tmCBP,
monitored by the change in circular dichroism (CD)
signal, was used to assess the binding of the xylan-based
(1,4) linked xylose pentasaccharide, xylopentaose, and
the laminarin-based (1,3) linked glucose disaccharide,
laminaribiose (LR2), and pentasaccharide, laminaripentaose
(LR5). To bring the thermal melting point (Tm) into a
measurable range, experiments were carried out in the
presence of the chemical denaturant guanidine
hydrochloride at a concentration of 2 M [17,35]. Addition of LR2 and
LR5 shifted the Tm of the protein from 94.8C to 99.2C
and 105.2C respectively (Figure 2). These studies indicate
that the tmCBP binding site accommodates (1,3)
glucosaccharides ranging in size from two to five sugar
rings, which is consistent with earlier CD binding studies
for the (1,4) glucosaccharides cellobiose and cellopentaose
. Unexpectedly, the (1,4) linked xylose-based sugar,
xylopentaose, did not induce a change in the Tm indicating
a lack of a stabilizing or binding interaction with tmCBP
(Figure 2). In the previous molecular modeling of
the tmCBP binding site, the first sugar ring of either the
(1,3) or (1,4) glucosaccharides, and in-turn the C6
hydroxyl, is coincident among the two ligand bound forms
(Figure 1). It is likely that the hydrogen bonding
interactions of the carbohydrate ring C6 hydroxyl with the
protein are important for the discrimination of
xyloFigure 2 Thermal denaturation of tmCBP in the presence of
laminarin-based carbohydrates. Circular dichroism was used to
monitor that thermal denaturation of tmCBP in the absence (solid
triangle) and presence of 1 mM laminaribiose (square),
laminaripentaose (circle), or xylopentaose (open triangle). Solid lines
are a fit to a two-state model for thermal denaturation that takes
into account the native and denatured baseline slopes.
and glucosaccharides, as the xylosaccharides lack the
C6 carbon and hydroxyl atoms.
Solution structure of apo and ligand-bound tmCBP
In order to characterize the conformational changes
induced upon addition of ligand, small-angle neutron
scattering (SANS) data was used to characterize both the
apo and ligand bound forms of tmCBP. Cellobiose was
used for these studies, which based on the previous
crystal structures produces a closed state essentially identical
to the laminarin-based carbohydrates. Comparison of
the apo and ligand-bound curves show significant
differences in both the high q and low q data, indicative of a
ligand induced conformational change (Figure 3a). These
raw data can be transformed into a Kratky plot where
geometrical differences, such as compactness and
flexibility, in the scattering particles can be highlighted. In
the case of a multi-domain protein connected by flexible
linkers, the Kratky plot would show a broad peak at
lower q-values, with an upturn at the higher q-values.
The Krakty plots for the apo and cellobiose-bound
protein are similar in shape and indicative of a globular
protein rather than two domains connected by a flexible
linker (Figure 3b). This suggests that the tmCBP hinge
does not allow for significant conformational flexibility
in the absence of ligand which is consistent with the
previous small-angle scattering studies of the group II
maltose binding protein . It is interesting to speculate as
to whether the flexibility of the PBP hinge may be
inherent to a particular PBP group as structures of group I
PBPs in the absence of ligand have been shown to adopt
a series of domain closure angles, perhaps suggesting
flexibility in the absence of ligand .
Upon addition of ligand the protein undergoes large
scale conformational changes as evidenced in the decrease
in the radius of gyration and Dmax (Table 1). The large
differences in these biophysical parameters of the scattering
particles are of a greater magnitude than one would expect
based upon previous small-angle scattering studies of
other group II PBPs . Molecular weight determination,
based upon the intensity at zero scattering angle suggests
that the apo protein forms inter-protein associations that
are alleviated upon addition of cellobiose (Table 1).
Interprotein associations have previously been reported for this
protein superfamily [37,38]. No molecular modeling of
the apo protein was carried out. The SANS data of the
cellobiose-bound protein is well accounted for by the
previously determined cellobiose-bound tmCBP crystal
structure (Figure 3a). This is also observed in comparison of
the cellobiose-bound crystal structure and the ab-initio
model generated from the SANS data in the presence of
5 mM cellobiose (Figure 3c).
Crystal structure of laminaribiose complex
The crystal structure of tmCBP complexed with LR2 was
solved to a resolution of 2.05 by molecular replacement
using the previously determined tmCBP structure 
(Figure 4a). The structure was refined to Rwork and Rfree
values of 18.4% and 20.3%, respectively. The final model
consists of 582 amino acids, a larminaribiose molecule and
293 water molecules. The overall fold and conformation of
the protein is similar to the structure of the cellobiose
complexed protein (all atom RMSD= 0.4 ). Data
collection, stereochemistry and refinement statistics are
summarized in Table 2.
An extensive network of polar and non-polar amino
acids and water molecules bind the LR2 ligand (Figure 4b).
A LigPlot+ representation of the LR2 binding pocket is
shown in Additional file 1: Figure S1a . As in other
periplasmic carbohydrate binding proteins, a network of
aromatic amino acids envelops the ligand between the
Nand C-terminal domains. A total of six tryptophan residues
(Trp15, Trp380, Trp383, Trp426, Trp510 and Trp535)
form van der Waals interactions with the two sugar rings.
In total, ten hydrogen bonds can be formed with the first
sugar ring. Two hydrogen bonds are formed with the C3
and C4 hydroxyl each, whereas the C2 and C6 hydroxyls
form three hydrogen bonds each. All but one hydrogen
bond are directly formed with the protein, with the C6
hydroxyl being ligated by a single specifically bound water
molecule, W20. For second sugar ring of the laminaribiose,
only the C2 hydroxyl forms a direct hydrogen bond with
the main chain carbonyl of Gly12, which also forms a
hydrogen bond with the C2 hydroxyl of the first ring. The
other six hydrogen bonds with the second ring are with
Figure 3 Small-angle neutron scattering of apo and ligand bound tmCBP. (a) I(q) SANS scattering data of tmCBP in the presence (red
squares) and absence (black squares) of 5 mM cellobiose. Solid black line is the CRYSON generated theoretical scattering curve based on the
previously determined tmCBP cellobiose complex. (b) Krakty plot of apo (black) and cellobiose bound (red) tmCBP. Error bars omitted for clarity.
(c) Ab-initio model of cellobiose bound tmCBP (surface representation) superimposed with the crystal structure of the cellobiose complex
water molecules. The C1 hydroxyl of second ring forms
hydrogen bonds with specifically bound water molecules,
W47 and W150, whereas the C4 and C6 hydroxyls form
hydrogen bonds with W39 and W6 water molecules. C2
hydroxyl also forms hydrogen bonds with W47, which
forms another hydrogen bond with the main chain
carbonyl of Ala13. The O5 hemiacetal oxygen of second ring
forms a hydrogen bond with another water molecule,
W179 (Figure 4b).
Unlike the other tmCBP structures that have been
solved thus far, both the LR2 complex and the LR5
complex contain a pentagonal bipyrimidally-bound calcium
ion (Figures 4a and 5). This calcium ion was bound from
the crystallization precipitant solution that contained
Table 1 SANS data analysis
Sat (5 mM Cellobiose)
*No model available for apo form.
Figure 4 The X-ray crystal structure of ligand bound tmCBP. (a) Ribbon representation of the overall structure of the laminaribiose bound
tmCBP. The laminaribiose ligand is shown in ball and stick representation and the calcium ion is represented as a green sphere. (b) Close-up view
of the tmCBP amino acids involved in hydrogen bonding (black dashed lines) and van der Waals interaction. The laminaribiose ligand and the
amino acids interacting with the ligand are shown in ball and stick representation. (c) Ribbon representation of the overall structure of the
laminaripentaose bound tmCBP. The laminaripentaose ligand is shown in ball and stick representation and the calcium ion is represented as a
green sphere. (d) Close-up view of the tmCBP amino acids involved in hydrogen bonding (black dashed lines) and van der Waals interaction. The
laminaripentaose ligand and the amino acids interacting with the ligand are shown in ball and stick representation.
calcium acetate. The carbonyl of Tyr37, the side-chain
Gln142, and the main carbonyl and side chain carboxylate
of Asp33 fill four of the seven coordination sites, while the
remainders are filled by a network of specifically bound
water molecules (Figure 5).
Crystal structure of laminaripentaose complex
The crystal structure of tmCBP complexed with
laminaripentaose (LR5) was solved to 2.07 resolution by
molecular replacement using the previously determined
tmCBP structure  (Figure 4c). This structure was
refined to Rwork and Rfree values of 18.7% and 22.1%,
respectively. The final model consists of 582 amino acids, a
laminaripentaose molecule, and 237 water molecules. A
calcium ion is also bound in an identical manner as the
LR2 complex. The overall fold of the protein is similar to
the structure of the cellopentaose complexed protein (all
atom RMSD= 0.2 ). Data collection, stereochemistry and
refinement statistics are summarized in Table 2.
Like the LR2 complex, an extensive network of polar and
non-polar amino acids and water molecules also bind the
LR5 ligand (Figure 4d). A LigPlot+ representation of the
LR5 binding pocket is shown in Additional file 1: Figure
S1a . The first and second LR5 sugar rings are bound
in an identical manner as the LR2 sugar rings. However,
The O5 hemiacetal oxygen of the second LR5 sugar ring
forms an intra-molecular interaction with the third sugar
ring C4 hydroxyl, which replaces the water molecule
W179 of LR2. All the hydroxyls of ring 3 of LR5 have only
water mediated hydrogen bonds. The C2 hydroxyl and the
Resolution range ()
r.m.s.d.b from ideal
Num. of reflections
(working /test set)
aNumber in parentheses represent values in the highest resolution shell.
br.m.s.d. indicates root mean square deviation.
LR5 ring 3/4 hemiacetal each form two hydrogen bonds
with three water molecules, while C4 and C6 hydroxyls
form single hydrogen bond with W73 and W15,
respectively. Unlike the ring three of LR5, ring four forms direct
hydrogen bonds with the protein. The C6 hydroxyl forms
two hydrogen bonds, while the C4 hydroxyl and O5
oxygen each form a single hydrogen bond with the protein.
An additional water molecule, W129, hydrogen bonds
with the C4 hydroxyl of the LR5 ring 4. The C4 hydroxyl
of the fifth LR5 ring forms two hydrogen bonds, while the
C6 hydroxyl forms a single hydrogen bond with the
protein. Three specifically bound water molecules hydrogen
bond with the C1, C2 and C4 hydroxyls; two of these
waters also separately hydrogen bond with the ring 4/5 O3
hemiacetal oxygen and the O5 oxygen of ring 5. Ring 4 is
Table 2 Data collection and refinement statistics
Figure 5 The tmCBP calcium binding site. Close-up view of the
molecular interactions of tmCBP with an endogenously bound
calcium ion (green sphere). Water molecules are shown as red
spheres and amino acids involved in hydrogen bonding are
represented as ball and stick models. Direct metal hydrogen bonds
are represented as black-dashed lines whereas the remainder of the
hydrogen bonding network is red. The LR5 crystal structure was
used for this analysis.
essentially occupying the positions of W49, W87 and
W171 of LR2 complex while ring 5 replaces the W119
water of the LR2 complex.
The calcium ion of the LR5 complex (Figure 4c), with
slight variation in coordination distances, has identical
coordination geometry as the calcium ion present in the LR2
structure. It is interesting to note that the Asp33, which
primarily coordinates the calcium ion also forms hydrogen
bonds to C6 hydroxyl of LR5 ring 4 and a water molecule
that hydrogen bonds to the C2 hydroxyl of the fifth sugar
ring (Figure 5). Moreover, the side chain of Gln34, of which
the main chain carbonyl interacts with a water molecule
that coordinates with the calcium ion, also forms two
hydrogen bonds with the C4 hydroxyl of the LR5 ring four.
The localization of this calcium ion suggests it may
potentially have a functional role in ligand binding, rather than a
structural role as found in other PBPs .
Comparison of laminarin and cellodextrin
Comparison of the laminarin and cellodextrin bound
tmCBP structures allow for the identification of the
molecular details of ligand selectivity in this semi-specific
periplasmic binding protein. Superposition of the
laminaripentaose (LR5) and cellopentaose (CP5) complexes
demonstrates that the conformation of almost every
amino acid that forms van der Waals interactions or
hydrogen bonds with the LR5 or CP5 in both subsites is
in an identical conformation (Figure 6). A single amino
acid in subsite two, Gln142, adopts an alternate rotamer
between the two structures. As the ligands occupy
Figure 6 The tmCBP cellopentaose and laminaripentaose binding site. Stereo view of the superposition of tmCBP bound with cellopentaose
(magenta) and laminaripentaose (cyan). All amino acids that interact with either ligand are shown in line representations.
distinct regions of the binding pocket, which remain
fixed in position regardless of which ligand is bound, we
postulate that the identical conformation of the
hydrogen bonding and van der Waals ligand interaction
network suggests the protein binding pocket is pre-ordered
for binding of either ligand.
Role of water molecules in organizing the tmCBP
The tmCBP binding site accommodates both (1,3) and
(1,4) carbohydrates by utilizing the non-specific, subsite
two, binding site . Although the ligands occupy
different regions of the non-specific subsite, there is significant
overlap of the amino acids involved in the recognition of
either ligand. We sought to understand how this occurs
when the protein amino acid network remains in a fixed
conformation. Superposition of the LR5 and CP5 bound
structures suggest the bound water network may play an
important role in the plasticity of the tmCBP binding site
(Figure 7). Five distinct classes of water molecules that are
specifically bound to either the N or C-terminal domain
are found potentially mediating and modulating ligand
selectivity in the tmCBP ligand binding site.
1. Waters in identical positions coordinating identical
atoms on different ligands (Figure 7, red water
molecule): The first LR5 and CP5 sugar rings are
in identical positions and the water molecule
coordinating the C6 hydroxyl is also found in an
identical position. This water simultaneously forms
hydrogen bonds with both the N and C-terminal
domains of the protein, the C6 hydroxyl of the ligand,
and another bound water molecule that interacts with
the second sugar ring. As five membered -xylan
carbohydrates do not bind to tmCBP it is possible that
the positioning and the bonding network of this water
molecule make it important in transducing to the
protein that a six membered ring is bound in subsite
one. The combination of the lack of this water
molecule, and the lacking of the molecular interactions
with the C6 carbon and hydroxyl group, potentially
impede formation of the closed ligand bound state of
the protein in the presence of xylans. These types of
bridging, ligand-binding-induced interdomain contacts
have been postulated to be important in stimulating
the PBP conformational change .
2. Waters in identical positions coordinating different
ligand atoms (Figure 7, green water molecules): Beyond
the first sugar ring, the conformation or localization of
the sugars in the binding pocket significantly differ.
Although coordinating distinct ligand atoms which are
located in different regions of subsite two, several
water molecules are found conserved in the LR5 and
CP5 bound structures. Water molecules that hydrogen
bond with ring 2, 3, or 5 of the LR5 ligand are also
found in the CP5 bound structure. The conservation
of water molecules is also observed in the water
molecules interacting with ring 2, 3, or 5 of the CP5
ligand. The rich hydrogen bonding potential of water
molecules allows for conservation of water position in
Figure 7 The network of water molecules modulating the ligand selectivity of tmCBP. (a) View of the LR5 water network. Water molecules
found interacting with the LR5 are shown as blue spheres. The different classes of conserved water molecules also found in the CP5 binding site
are colored as follows: red, waters in identical positions coordinating identical atoms; green, waters identical positions coordinating different
ligand atoms; black, waters in identical positions forming ligand contacts in one form and not the other; orange, waters mimicking hydroxyl
atoms of ligand. (b) View of the CP5 water network. Water molecules found interacting with the CP5 are shown as magenta spheres. The
different classes of conserved water molecules also found in the LR5 binding site are colored as in (a).
both the CP5 and LR5 forms and thereby potentially
permits the preordering of the hydrogen bonding
potential of subsite one for either type of ligand.
3. Waters in identical positions forming ligand contacts
in one form and not the other (Figure 7b, black water
molecules): Beyond the first two sugar rings found
in subsite one, the localization of the LR5 and the
CP5 in the tmCBP subsite two differ significantly.
However, several water molecules that are involved
in forming hydrogen bonds with LR5 are still
present when CP5 is bound in subsite two. The
same is also true for subsite two waters that
hydrogen bond with CP5 and not LR5. This class of
water molecule is involved in pre-ordering the
rotameric state of subsite two hydrogen bonding
residues. Although crystal structure of apo form of
tmCBP protein could not be obtained, it is possible
the same water-mediated hydrogen bonding network
pre-orders subsite 2 for binding of (1,3) or (1,4)
ligands in the apo state.
4. Waters that mimic hydroxyls/hemiacetals of other
ligand (Figure 7, orange water molecules): Several
water molecules are present in either the LR5 or
CP5 bound structure that mimic the localization in
subsite two of ligand hydroxyl groups of the other
ligand. Similar to the class 3 water molecules, this
class of water molecules preforms both the water
and protein hydrogen bonding interactions for either
ligand, the role of which is potentially important for
promiscuous ligand recognition of the apo protein.
5. Secondary shell waters involved in coordinating
primary shell waters (and/or involved coordinating
preordering of binding pocket): Beyond the primary
shell water molecules that directly interact with the
ligands, a conserved network of at least twelve water
molecules is found ordering either the primary shell
waters or the amino acids that are involved in
interacting with the ligands.
Depending on biological function, PBPs ligand selectivity is
modulated through a combination of binding pocket
adaptations that mediate ligand positioning, alter ligand size
selection, or alter the free energy of ligand binding in such a
manner that excludes incorrect ligands [35,40]. The novel
bipartite tmCBP binding site represents an additional,
interesting alteration of PBP ligand recognition, exemplifying
how both specificity and promiscuity are encoded in a
single binding site. Comparison of the tmCBP structures
bound to laminarin-based and cellodextrin-based
carbohydrates allows for identification of the novel binding
selectivity determinants found in this binding site.
The structural changes accompanying ligand binding in
PBPs typically involves a re-organization of side-chain
rotamers or the protein backbone. Although in some cases
one of the two binding half sites in each domain undergoes
conformational changes upon ligand binding and this has
been suggested as a mechanism of ordering ligand binding
. Although lacking the apo crystal structure, analysis of
the tmCBP ligand bound state suggests the binding site
may be pre-ordered for binding of either laminarins or
cellodextrins. Essentially no structural differences are
observed among the amino acids in the ligand recognition
sphere of either class of ligands. It should be pointed out that
no side-chain movement is observed even when the same
residue forms direct or indirect interactions with either ligand.
The conservation and positioning of water molecules
trapped in the tmCBP binding pocket suggest they
potentially play a role in tuning tmCBP ligand selectivity. Several
classes of water molecules, playing distinct functional roles,
are found in the tmCBP binding site. This network of
water molecules preforms the ligand hydrogen bonding
network and side chain conformations of ligand interacting
amino acids, and thereby reduces the entropic penalty of
ligand binding. Additionally, these water molecules are rich
in hydrogen bonding potential, allowing for conservation
of water placement while facilitating the plasticity of this
bipartite binding site.
The mode of ligand binding found in tmCBP represents
an interesting adaptation mechanism not previously
observed in other PBPs. The downstream carbohydrate
transport systems have a narrow, predefined limit to the size and
type of carbohydrate that can be processed. The tmCBP
binding cavity pre-filters this pool of ligands, potentially
optimizing the transport process and in-turn eliminating the
energetic penalty of presentation of incorrect carbohydrates
to the transport machinery. In E. coli maltose binding
protein ligands that do not fit within the binding site are still
bound and presented to the transport machinery .
However, with tmCBP a single protein is used to
promiscuously select a molecular class of ligands while at the
same time sterically restricting the number of rings that
can be placed within the constraints of the tmCBP binding
cavity. Larger ligands are likely not bound as they would
impede the hinge bending motion and in-turn specific
Seven additional oligosaccharide binding proteins with
varying substrate specificities are found in the T. maritima
genome . It remains to be observed whether the mode
of ligand recognition in tmCBP is unique or found across
this subset of periplasmic carbohydrate binding proteins.
The adaptation mechanisms observed in tmCBP allow for
expansion of binding site selectivity while maintaining
specificity for a molecular class of ligands. These types of
adaptation mechanisms could potentially be recapitulated in
drug design studies where the rich hydrogen bonding
potential of water molecules can be utilized to expand binding
sites, or enable multiple drugs to bind to a single target site.
Protein expression and purification
The tmCBP plasmid was transformed into BL21-RIL
cells for heterologous expression in either terrific broth
media, M9 minimal media or Enfors minimal media
supplemented with carbennicillin and chloramphenicol.
Growth on terrific broth or M9 minimal media produced
protein that was bound with a disaccharide ligand as
determined by circular dichroism (CD) and X-ray
crystallography (data not shown). Ligand-free protein was produced
by growth on the glycerol based medium, Enfors minimal
media. In all cases, tmCBP was purified as previously
described [13,32], with slight modifications. Cell pellets were
lysed by sonication. The resulting lysate was clarified by
centrifugation (34,000 g) for 20 minutes. Following
nickel chelation chromatography purification of the lysate,
the protein was loaded on to a Superdex S75 26/60
(Amersham) gel filtration column that was equilibrated with
20 mM Tris, pH 8.0, 150 mM NaCl. This purified material
was used for all other experiments.
CD experiments were performed on a Jasco CD
spectrophotometer. Thermal denaturations were determined by
measuring the CD signal at 225 nm as a function of
temperature using 0.5 M protein in 10mM TrisHCl pH
8.0 and 40 mM NaCl. In the absence of guanidinium
chloride, tmCBP is too stable to exhibit temperature-induced
denaturation and all measurements were carried out in the
presence of 2 M guanidine hydrochloride and 1.0 mM
ligand. CD measurements were fit to a two-state model that
takes into account the slope of the native and denatured
Small angle neutron scattering data collection
Small-angle neutron scattering (SANS) experiments were
performed on the extended Q-range small-angle neutron
scattering (EQ-SANS, BL-6) beam line at the Spallation
Neutron Source (SNS) located at Oak Ridge National
Laboratory (ORNL) . Protein was concentrated to 16.7
mg/mL and dialyzed in to 20 mM Tris pH 8.0, 40 mM
NaCl in 100% D2O for SANS measurements that were
performed at 20C. Cellobiose was added to the apo protein
at a concentration of 5 mM for measurements of the ligand
bound form. Data reduction followed standard procedures
using MantidPlot (http://www.mantidproject.org/) .
Upon verifying a Guinier regime  in the SANS
profiles, the pair distance distribution function, P(r), was
calculated from the scattering intensity using the indirect
Fourier transform method implemented in the GNOM
program  (Table 1). The real-space radius of gyration,
Rg, and scattering intensity at zero angle, I(0), were
determined from the P(r) solution to the scattering data.
The molecular mass, M, was calculated by I 0
22=N A , where = contrast in scattering length
density between protein and D2O buffer solution (= prot
buf ), protein partial specific volume 0:73 ml=g,
and NA = Avogadros number. The GASBOR program 
was used to generate ab initio shape reconstructions.
Crystallization and X-ray data collection
tmCBP was concentrated to 20 mgmL and dialyzed into 10
mM Tris, 40 mM NaCl 0.5 mM TCEP for crystallization.
Laminaribiose (LR2) or laminaripentaose (LR5) was added
to a final concentration of 1 mM prior to crystallization
trials. Crystals were grown by hanging drop vapor diffusion
in drops containing 2 L of the protein solution mixed
with 2 L of 0.20.3 M magnesium acetate or calcium
acetate, 2030% (wt/vol) PEG 3350 equilibrated against 900 L
of the same solution. Crystals were transferred to 35%
(wt/vol) PEG 3350 for cryoprotection, mounted in a nylon
loop, and flash frozen in liquid nitrogen. All X-ray diffraction
data were collected at 100 K on a Rigaku 007HFmicromax
X-ray generator with a Raxis IV++ detector. The
diffraction data were scaled and indexed using HKL3000 .
The data collection statistics are listed in Table 2.
Structure determination, model building and refinement
The LR2 bound and LR5 bound tmCBP structures were
solved by molecular replacement using the Phaser
program . The crystal structure of the previously
determined tmCBP was used as the initial model for fitting
the X-ray data (, PDB code 3I5O). Manual model
building was carried out in COOT  and refined using
REFMAC5  and PHENIX . The models exhibit
good stereochemistry as determined by MolProbity ;
final refinement statistics are listed in Table 2.
Atomic coordinates and structure factors have been
deposited in the Protein Data Bank  under the accession
codes 4JSD and 4JSO for the LR2 complex and LR5
Additional file 1: Figure S1. Interaction network of tmCBP laminarin
ligands. The polar and non-polar contacts of the LR2 (a) and LR5 (b)
ligands as generated by LigPlot . The interaction network that is
coincident among the LR2 and LR5 structures are highlighted in red,
hydrogen bonding interactions are represented as black dashed lines and
water molecules as red spheres.
The authors declare that they have no competing interests.
MJC, PM and DAM designed the research and drafted the manuscript. MJC
and CBC performed small-angle scattering experiments. MJC, PM, SG, and XL
performed CD and X-ray crystallography experiments. All authors read and
approved the final manuscript.
A portion of this research was performed at Oak Ridge National Laboratory's
Spallation Neutron Source, sponsored by the U.S. Department of Energy,
Office of Basic Energy Sciences. PM was funded in part through a research
grant from the National Science Foundation (Award 0922719).
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