Different modes of barrel opening suggest a complex pathway of ligand binding in human gastrotropin
Different modes of barrel opening suggest a complex pathway of ligand binding in human gastrotropin
Zita HarmatID 0 2
Andra? s L. Szab o? 0 2
Orsolya T?ke 1 2
Zolt a?n G a?sp a?ri 0 2
Pratul K. Agarwal, University of Tennessee,
0 Faculty of Information Technology and Bionics, Pa ?zma ?ny Pe ?ter Catholic University , Budapest , Hungary
1 Research Centre for Natural Sciences, Hungarian Academy of Sciences , Budapest , Hungary
2 Research, Development and Innovation Office - NKFIH , grant no. NF104198
Gastrotropin, the intracellular carrier of bile salts in the small intestine, binds two ligand molecules simultaneously in its internal cavity. The molecular rearrangements required for ligand entry are not yet fully clear. To improve our understanding of the binding process we combined molecular dynamics simulations with previously published structural and dynamic NMR parameters. The resulting ensembles reveal two distinct modes of barrel opening with one corresponding to the transition between the apo and holo states, whereas the other affecting different protein regions in both ligation states. Comparison of the calculated structures with NMR-derived parameters reporting on slow conformational exchange processes suggests that the protein undergoes partial unfolding along a path related to the second mode of the identified barrel opening motion.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Gastrotropin (also known as ileal bile acid-binding protein (I-BABP) or fatty acid-binding
protein 6 (FABP6)) [
] is involved in the enterohepatic circulation of bile salts. Being
synthetsized in the liver from cholesterol, bile salts are secreted into the proximal small intestine via
the gall bladder and then efficiently recycled blood via the hepatic portal circulation [
This recycling process ensures that only a small amount of bile salts needs to be synthesised de
novo. Gastrotropin is thought to play a role in this recyclinc process via binding interactions
occurring within the absorptive epithelial cells of the distal ileum [4?5] and has an important
role in cholesterol homeostasis [
Gastrotropin belongs to the family of intracellular lipid-binding proteins (iLBPs), a group
of small, approximately 15-kDa proteins that bind fatty acids, retinoids, cholesterol, and bile
salts . Additionally, iLBPs have been shown to have a role in the stimulation of the
transcriptional activity of nuclear hormone receptors [
]. Among the four main groups of the
iLBP family, the subfamily of gastrotropin is unique in the sense that it has the capability of
binding two [11?12] or possibly even three [13?14] ligands simultaneously. NMR solution
structure of the apo form of human gastrotropin (PDB ID: 1O1U) was determined along with
the cholyltaurine bound form (PDB ID: 1O1V) . More recently, the structure of the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
heterotypic doubly-ligated complex of human gastrotropin with glycocholate and
glycochenodeoxycholate has been determined . Similarly to other members of the iLBP family, the
structure of human I-BABP is composed of a ?-barrel formed by ten antiparallel ?-strands
(A-J) and two ?-helices (I-II). The binding cavity of ~1000 ?3 is located inside of the ?-barrel
. Ligand binding in human gastrotropin exhibits positive cooperativity , which has
been shown to be governed by the hydroxylation pattern of the bound bile salts .
Accordingly, hydrogen bonding networks have been shown to have a key mediatory role in positive
binding cooperativity . Besides the observed communication between the two binding
sites, di- and trihydroxy bile salts display a site preference upon binding in each other?s
presence . As it is apparent from the comparison of apo and holo human gastrotropin
structures (Fig 1), bile salt binding is accompanied by large conformational changes in the E-F and
G-H protein regions as well as in the C/D-turn and the proximate helical cap [4,13].
Importantly, NMR relaxation measurements suggest that in the apo form, the ground state is in slow
exchange with a low-populated ?invisible? conformer resembling some structural features of
the the ligand-bound form . Intriguingly, residues undergoing a conformational
fluctuation on the ?s-ms time scale can be grouped into a ?slower? and a ?faster? cluster, which appear
to be spatially separated. Specifically, while the ?slower? cluster involves part of the helical
region, the C/D-turn, and the proximate B and D ?-strands in the N-terminal half, the ?faster?
cluster comprises segments of the EFGH protein region in the C-terminal half .
As the binding site of gastrotropin is located in the interior of the protein, the mechanism
of ligand entry is an important issue to be investigated. The most widely accepted scenario for
the protein family is formulated in the ?portal hypothesis?, stating that access of ligands to the
protein interior is governed by the C/D and E/F-turn regions together with the C-terminal
part of helix II [
]. Based on NMR structural and dynamic studies, a conformational
selection mechanism of ligand binding involving an equilibrium between a closed and a more
open protein state has been suggested for both the human ileal  and the chicken liver
BABP analogues [
]. In line with the NMR spectroscopic analysis of internal motions,
molecular dynamics simulations show evidence of correlated motions in human gastrotropin and in
the absence of ligands indicate a partial unfolding of the E-F protein region [
To improve our understanding of the mediatory role of internal motions in human
gastrotropin-bile salt interaction, we generated conformational ensembles consistent with
experimentally obtained NMR structural and dynamic data published earlier  using a
methodology described earlier [
] and performed ligand docking to obtain an atomic-level
insight into the binding mechanism. Our results reveal different conformational
rearrangements in the protein that are suggested to correspond to motions characteristic of different
time scales indicating a complex mechanism of bile salt entry.
Materials and methods
Ensemble molecular dynamics simulations with NMR restraints
Molecular dynamics calculations were performed using GROMACS version 4.5.5. [
modified to handle S2 order parameters as well as pairwise averaging of NOE distance
restraints over replicas , as proposed for the MUMO (Minimal Under-restraining Minimal
Over-restraining) approach [
]. The OPLS-AA force field [
] and the TIP3P water model
] was used for all molecular dynamics simulations described below.
For modeling the apo structure of gastrotropin, we chose model 7 of PDB entry 1O1U 
based on its highest PRIDE-NMR score [
] among the deposited models. As an initial model
of the holo structure we used model 1 of the PDB entry 2MM3. Ligand topologies for
glycocholic acid (GCA, PDB ligand ID: GCH) and glycochenodeoxycholic acid (GCDA, PDB ligand
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Fig 1. Ribbon representation highlighting the differences between the apo (PDB ID: 1O1U model 7) and the holo
(PDB ID: 2MM3 model 1) form of human gastrotropin. The figure was prepared with UCSF Chimera [
ID: CHO) were generated with the TopolGen script and corrected manually for atom types
where necessary as well as with an in-house Perl script to reassign hydrogen atoms to the
charge groups defined by the heavy atoms they are connected to.
NOE restraints were only available for the holo protein (PDB ID: 2MM3). For the apo form,
we used restraints from the 2MM3 list that were unviolated in the deposited 1O1U structure as
checked with the CoNSEnsX server. Restraints were modified by the removal of
stereospecificity and rounding the restrained distance up to the next integer ?, creating 1 ? wide ?bins? from
4 to 10 ?.
Chemical shifts for the apo structure were obtained from BMRB (BMRB ID: 19843) and for
the holo structure directly from the authors. S2 values for the apo and holo structures measured
at 283, 291, 298, and 313 K were taken from .
After generating a topology using the OPLS-AA force field and TIP3P water model, the
molecule was put into a cubic box, followed by energy minimization with conjugate gradient
method for 5000 number of steps with 0.001 ps step length. The maximum force was set to
200. In the next step, the molecule was solvated and then one of the water molecules was
replaced by a Na+ ion to ensure the neutrality of the system. After that, another energy
minimization was performed using the same parameters, but including the water molecules. In the
last step, a short MD simulation was performed using position restraints of 1000 kJ mol-1 nm-2
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on the heavy atoms of the protein for 2500 steps with 0.002 ps step size using the LINCS
For the production runs, eight replicas were simulated in parallel with the OpenMPI
]. Backbone S2 order parameter restraints were applied on the full ensemble and
NOE distance restraints were averaged between neighboring replicas, similar to the MUMO
(Minimal Under-restraining, Minimal Over-restraining) protocol [
]. The simulations were
performed at four temperatures: 283 K, 291 K, 298 K, and 313 K using S2 restraints measured
at the corresponding temperatures. With LINCS constraining on bond lengths, a timestep of 2
fs was used to generate runs of 2 ns and 6 ns, totaling 16 and 48 ns for the 8 replicas combined,
respectively. Control simulations with the same parametrization but without restraints were
also performed. Topology files for the restrained simulations are included in the
supplementary material as (S1 File).
In order to generate a larger pool of possible conformations in order to further explore the
conformational space, molecular dynamics simulations with only one type of restraint, NOE
or S2, or without any restraints were also performed. Accelerated Molecular Dynamics and
short (500 ps) Targeted Molecular Dynamics simulations were also performed on the apo
structure using the chemical shifts of the holo structure and vica versa in order to achieve
transition from one form to the other.
Docking calculations were performed on selected structures with GCA and GCDA using
Schro?dinger Glide [
]. The binding sites were defined using the ternary complex structure
2MM3. After importing the structure, it was split to separate molecules. As a next step, either
GCA or GCDA was merged with the protein and a mesh grid around the ligand was generated
with the ?Receptor Grid Generation Tool? using default settings. Docking of the respective
ligands was performed using the ?Ligand docking? tool with default settings except requiring
the inclusion of per-residue interaction scores in the output. To dock the second ligand into
the binary complex obtained, the docking result most similar to the pose in the initial 2MM3
structure was merged with the second ligand and used to define the second binding site with
the grid generation tool. For each of the four different setups, i.e. GCA, GCDA, GCA+GCDA
and GCDA+GCA docking runs, 32 different poses were generated and evaluated. Total energy
of the docked complexes was estimated using the MacroModel routine with the OPLS3 force
field and water as solvent.
Correspondence to the experimental parameters was analyzed using the CoNSEnsX webserver
] (for S2 order parameters and chemical shift data) as well as in-house Perl scripts (for
NOE distance restraints). NOE restraints were evaluated on a per-ensemble basis using r-6
averaging both for intramolecular ambiguity and between members of the ensembles.
For the S2 order parameter correspondences, MUMO simulations and the original PDB
ensembles, the corrected S2 values are also displayed. In the correction, those points were
excluded from the analysis, which had greater than 0.2 as an absolute value of the difference
between the experimental and back-calculated values. For the MUMO simulations, maximum
5 such values were found. All the experimental and back-calculated values are depicted in S1
Principal Component Analysis was performed using ProDy [
] and visualized with the
NMWIZ module of the program VMD [
]. Structure-based chemical shift calculations were
performed with the program SHIFTX2 [
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The presence or absence of hydrogen bonds in the ensembles was investigated with an
inhouse Perl program using distance-angle based hydrogen bond identification parameters [
Comparing calculated 15N chemical shift differences with experimentally
derived |??| (15N) values
For each structure, backbone 15N chemical shifts were estimated with Shiftx [
]. For each
conformation in the large conformer pool (see above), the absolute value of the difference of
the predicted chemical shifts relative to those in each calculated unliganded structure in the
MUMO ensembles was calculated. These differences were then compared to experimental |
??| (15N) data derived from CPMG relaxation dispersion NMR measurements for each
residue for which it was available . Both correlation and RMSD measures were calculated after
normalization to the 0?1 range. As there are |??| values available for three temperatures and
the conformational pool is of a heterogeneous source with no well-defined temperature, the
correlation and RMSD values were calculated for all three temperatures and then were
averaged for each structure investigated. The structures with highest correlation and lowest RMSD
values were selected for analysis.
Results and discussion
The generated ensembles reflect experimental parameters
According to the expectations, experimental S2 parameters are generally better reflected in the
MUMO generated ensembles than in the PDB ensembles or the unrestrained ensembles
(Table 1). Interestingly, the MUMO ensemble of the apo protein calculated with the S2
parameters of 283 K and the MUMO ensemble of the ternary complex calculated with the S2
parameters of 291 K corresponds only moderately to these data, while all other restrained ensembles
show good correspondence. The reason for this is the presence of some extremely low (< 0.3)
experimental S2 values, located mostly in turn regions, not reflected in the simulations. Plots
for the experimental and the back-calculated S2 values are depicted in S1 Fig.
The number of ensemble-calculated NOE violations are below 0.5 percent in each of the
MUMO ensembles, despite the clearly higher global RMSD values of the MUMO ensembles
than those for the original PDB ensembles. Correspondence to the amide N and H chemical
shifts are in the same range for the MUMO and the original PDB ensembles.
Gastrotropin ensembles reveal two distinct modes of barrel opening
Principal component analysis (PCA) of the ensembles suggests the presence of two kinds of
modes, both corresponding to the opening of the barrel structure, termed ?Type I? and Type II?
openings below. Type I opening clearly separates the apo and holo structures along PC1,
corresponding to the opening of the barrel between strands F and G. Viewing the structure from
the direction of the helices, this apo to holo structural change can be described as a clockwise
rotation of the E/F- and G/H-turns accompanied by a lower amplitude counterclockwise
rotation of the C/D-turn and helix-II, resulting in the appearance of a large aperture between the
E/F- and G/H-turns at the ?top? of the barrel. PC2 or Type II opening, in contrast, primarily
affects helix-I and the CD-turn, most prominently resulting in the widening of the interhelical
gap and the appearance of an opening between strands D and E.
It is notable that the Type II opening motion occurs in both the apo and the holo structures.
At higher temperatures, the ensembles occupy a larger region of the conformational space
along this particular opening mode (S4 Fig). It should also be noted that this kind of opening
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is also compatible with the portal hypothesis as described in detail for human liver fatty acid
binding protein [
The structural changes can be described in more detail by measuring distances between
selected amino acids. In S2A Fig the correlation of C? distances with each of the two motional
modes is plotted for each amino acid pair as a matrix. On the basis of the correlation of these
C?-C? distances, the most mobile regions corresponding to Type I opening are near the
termini and at the D/E-turn region including ? strand E itself. Regarding Type II motion, the
regions around amino acid 50 (? strand C) and 35 (linker between helix II and ? strand B)
appear to be the most flexible together moving segments.
C?-C? distances displaying the best correlation with Type I opening are between residues
47?69, 48?69, 60?69, 61?69, 62?69, 63?69 (corr. -0.96) as well as 66?70, 67?70 (corr. 0.96).
Regarding Type II motion, the best correlated C? distances are between residues 16?58, 17?
58, and 18?58 (corr. -0.92). The listed C? atom-atom distances are mapped on the structure in
S2B and S2C Fig. Our results suggest that the increasing distance between ? strands C and E or
D and E are correlated with Type I opening, and the increasing distance of helix I from the top
of the barrel (around the C/D.turn) correlates with Type II opening. Note that the residues
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displaying the largest displacement relative to the average structure do not necessarily coincide
with the ones exhibiting the largest changes in interatomic distances.
While the experimentally determined apo structure (1O1U) is clustered with the dynamic
ensembles for the apo state, the NMR structure of the ternary complex (2MM3) is located
between the apo and holo ensembles. This indicates that our ensembles of the complex state
exhibit a more pronounced opening along PC1 than the structure obtained by conventional
NMR calculations. The phenomenon that dynamic ensembles, corresponding reasonably well
to experimental data ?magnify? the differences between different states has been observed in
previous works [
]. This magnification is likely a consequence of the ensemble-based
treatment of NOE restraints allowing more conformational freedom than conventional structure
calculations. It is also the consequence of the different balance between the force field and
experimental restraints than in conventional structure calculation methods.
The apo and holo states exhibit characteristic differences in their hydrogen bond pattern as
well (Fig 2B), (Table 2) and (S1 Table). As shown previously, hydrogen bonds form an
extensive network in human I-BABP [19,21]. According to our calculations, the most significant
differences in hydrogen bond occurence include the formation of one and breaking of two
intrastrand hydrogen bonds upon transition from the apo to the holo state, consistent with a
specific mode of barrel opening between strands E and F. Interestingly, hydrogen bonds with
ligands (purple lines on Fig 2B) are present only in a few conformations, which may indicate a
loose ligand binding as a result of dynamically changing hydrogen bonds. Notable are the
hydrogen bonds of Thr73, where the ?1 OH group forms an intraresidue hydrogen bond in
the holo state that is not present in the apo form. This particular residue in the E/F-turn has
been suggested to have a key role in a conformational selection mechanism of ligand binding
together with proximate residues in the EFGH region of human I-BABP .
Residues involved in the two opening modes coincide with different
exchange rates along the sequence as determined by NMR
Comparing the regions affected by the motions with NMR-derived conformational exchange
data, it is apparent that there is a coincidence of the region affected by Type II opening and the
the NMR-reported ?slow? cluster located in the N-terminal half of the protein  (Fig 2E).
Although kex parameters derived from CPMG relaxation dispersion NMR measurements
report on a motion occuring on a much slower ?s-ms time scale than reflected by the S2
restraints used in our simulations, we suggest that the observed Type II barrel opening is
related to the slow conformational exchange revealed by NMR relaxation dispersion analysis.
Specifically, the fast motions could set the stage for slower, larger-amplitude motions in the
protein along a similar opening mode. The structural transition on a different time scale is also
consistent with the temperature-dependence of the observed motions, i.e. a more even
distribution of conformers along the Type II mode at higher temperatures. Importantly, the
presence of fast motion along this mode in both the apo and the holo states suggests that Type II
motions may have a role in both ligand uptake and release.
The hidden ?holo-like? conformation in the apo state is partially unfolded
Previous NMR investigations of human gastrotropin have identified the presence of an
invisible state that is in slow exchange with the observable apo state . Moreover, it was suggested
that this state exhibits holo-like structural features [16, 21]. In order to get a deeper insight into
the nature of this conformer, we generated a pool of conformers and selected structures that
might be representative of the higher energy state based on the differences in chemical shifts
relative to the apo state when compared with the NMR-derived |??| (15N) values between the
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Fig 2. Description of the Type I and Tye II motions. (A) PCA (Principal Component Analysis) scatter plot of the
simulated and experimentally determined conformer pool. (B) Hydrogen bonds with the largest changes between the
apo and holo states according to the MUMO ensembles. Black numbers denote amino acid residues, black letters
denote atoms, secondary structure elements are labeled with green letters. Red lines represent H-bonds characteristic
of the apo form, blue lines represent those formed mainly in the holo form and purple lines indicate H-bonds between
amino acids and the ligands. Note the central role of Thr73 in the hydrogen bond network. (C) Structural movements
along PC1: barrel opening (D) Structural movements along PC2 (E) Square fluctuation of C? atoms in the two PCA
modes: PC1 (Type I motion, purple), PC2 (Type II motion, orange). The previously measured experimental kex values
indicating two distinct clusters of residues involved in slow conformational exchange processes are depicted as
different gray areas corresponding to the three different temperatures (283 K, 287 K, 291 K) of the measurements. As
only about 30?40 amino acids have displayed ms timescale motion with measurable kex values , a continuous
depiction is used to guide the eye to highlight the regional differences.
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ground and higher energy states of IBABP. We note that with the availability of only backbone
15N chemical shift differences the structural information on the invisible state remains to be
Apparently, the identified conformers with best correspondence to the experimental data
are scattered around a large conformational space (Fig 3). Their common characteristics is
that they are closer to the holo than to the apo state, which is in agreement with the previously
proposed holo-like characteristics of the sparsely populated excited state indicated by NMR
dynamic measurements . Importantly, some of the conformers show a more pronounced
Type II-like opening than the MUMO ensembles. The principal components in Fig 3. are not
direclty corresponding to those in Fig 2. The HD3 and HD5 structures, being close in the PCA
plot, show different degree of Type II-like opening in their helical region. Nevertheless, we
consider this aspect to be the most relevant as Type II opening is clearly identifable in the
MUMO ensembles calculated with a substantial amount of experimental data, in contrast to
other motions identified in unrestrained simulations only.
As shown in Fig 4, secondary structure of the simulated conformers is diverse around the
boundary of the ?-helical and ?-strand elements. In some structures almost all of the ?- helical
and ?-strand elements are partially unfolded. The structures assumed to be the ?holo-like? apo
conformations have low helical and ?-strand content. The E-F region is the most susceptible to
unfolding, in accordance with recent reports [
]. Taken together, these observations suggest
that the transition from the apo to the holo state, instead of being a simple physical opening
along the shortest route, is rather a complex succession of conformational rearrangements
proceeding through a partially unfolded intermediate involving a loosened helical and
C/Dturn regions, resembling in part the observed ?Type II? mode of motions.
Docking simulations support cooperativity of ligand binding
In order to further characterize the mechanism of ligand binding, we performed docking
simulations into selected structures obtained in our calculations.
Based on the PCA analysis, four structures were selected representing extreme states along
Type I and Type II opening, respectively. Additional three structures, regarded as the best
models of the invisible state in slow exchange with the apo form were also included.
In general, the most favorable complexes were obtained when GCA was docked first,
followed by the docking of GCDA (box diagrams in Fig 5). This scenario did not result in a
successful ternary complex for only one of the proposed hidden structures, HD1, corresponding
to an intermediate position between the apo and holo ensembles along Type I opening. Ligand
binding provides the highest stabilization for the partially unfolded structures corresponding
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Fig 3. PCA scatter plot of the apo MUMO (red dots) and holo MUMO (blue dots) ensembles along with the conformer pool (purple hollow squares) used
to select the structures best corresponding to the NMR-derived invisible state. Structures with a mean correlation between |??| (15N) values and calculated
chemical shift differences above a threshold of 0.35 are shown with black dots (left panel). Structures with an RMSD between |??| (15N) values and calculated
chemical shift differences lower than 0.00603 are depicted with green dots (right panel). Selected structures are also depicted and linked to their corresponding
points in the PCA scatter plots. These hidden conformations are termed HD1-HD5.
Fig 4. Secondary structure of the conformations inferred from our simulations (rows). Each column represents one amino acid. Extended ?-strands are
colored yellow, ?-helices are brown, the rest of the residues are colored black. (A) All of the conformations. (B) The high correlation conformations (subset of
conformations of panel A). (C) Conformations with lowest RMSD (another subset of panel A). The analysis was performed with DSSPCont [
]. Note the
shortening of secondary structure elements in some structures, especially in B) and C).
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Fig 5. Relative energies of docked structures relative to the ligand-free conformations. Differences between the starting conformations of the
molecular dynamics-derived structures relative to 2MM3 are depicted with colored bars. White boxes: only GCDA docked, stripped white boxes:
GCDA docked first, GCA docked second, gray boxes: only GCDA docked, stripped gray boxes: GCA docked first, GCDA docked second. HM
denotes a representative conformer from the holo MUMO ensemble, HN (holo narrow) and HW (holo wide) are selected extreme structures from the
holo MUMO ensembles corresponding to Type II opening. In addition, three from the high correlation hidden conformers are selected (HD1, HD3
to a larger opening along a motion resembling Type II opening. Comparing the relative
estimated energies of the corresponding apo structures (colored bars in Fig 5) suggests a complex
energetic landscape where conformational states and ligand binding contribute to stability in
an interdependent manner. Our results are compatible with a scenario where ligand entry
occurs in an open, partially unfolded state followed by subsequent structural compaction,
completing a transition along Type I rearragement along a pathway including a different, Type
It should be noted that we do not regard the docking energies reported here as reliable ones
in their absolute values, but as ones that can offer some insight to the binding process when
HM (a representative conformer from the holo MUMO ensemble), HW (holo wide), HN (holo narrow), corresponding to selected extreme conformations along Type II
opening in the holo MUMO ensembles (Fig 2D). HD1, HD3 and HD5are selected hidden conformers (see Fig 3).
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used in a comparative evaluation. More elaborated methods, like free energy calculations [
on the different complexes or umbrella sampling [
] could yield more precise and reliable
estimates of the energetics of the binding process. It should be noted, however, that for the
partially unfolded structures it is not trivial to set up and run such simulations and thus these are
outside the scope of the present study, which focuses on ensembles generated on the basis of
Comparison of ligand positions in the different structures (Table 3) and the total energies
of the complexes (Fig 5) might lead to the conclusion that open gastrotropin structures can
bind ligands with a high structural versatility while maintaining high affinity, although
additional investigations are needed to fully prove this statement. These above results suggest that
ligands might undergo dynamic reposition even in the binary and ternary complexes.
We have generated structural ensembles that are in agreement with available NMR parameters
reporting on the structure and fast time-scale dynamics of human gastrotropin. The two types
of barrel opening identified are in agreement with previous observations of the iLBP family.
We propose a refined model of ligand entry that is compatible with the portal hypothesis,
namely, that the structural transition from the apo to the holo state, termed Type I opening,
proceeds along an indirect route involving partial unfolding of the helical cap structure. In our
model this unfolding is related to and facilitated by another mode of barrel opening, termed
Type II, that is present in both the apo and holo states.
S1 Fig. Experimental and back-calculated S2 order parameters of different ensembles. (A)
Apo MUMO ensembles by amino acids
(B) Apo MUMO ensembles as correlation plots
(C) Holo MUMO ensembles by amino acids
(D)Holo MUMO ensembles as correlation plots
(E) Unrestrained ensembles by amino acids
(F) Unrestrained ensembles as correlation plots
The PDB ensembles are depicted in all panels as references. All the plots were generated by the
S2 Fig. C? atom distance correlations. (A) C? atom distance correlation matrix with PC1
(above diagonal) and PC2 (below diagonal) for each amino acid pair. Helical regions are
labeled with beige rectangles, ? strand regions with gray rectangles
(B) The highest (red, brown lines) and lowest (blue, gray lines) distances depicted on the
structure correalted with PC1
(C) The lowest (blue, gray lines) distances depicted on the structure correalted with PC2.
S3 Fig. Investigation of the docked frames. (A) Tanimoto distances of the simulation frames
and the docked frames based on ligand contact data (see text)
(B) Comparison of the average of the number of ligand heavy atoms (vertical axis) being closer,
than 4 ? to each amino acid (horizontal axis) of CHO (blue all frames, black docked frames).
(C). Comparison of the average of the number of ligand heavy atoms (vertical axis) being
closer, than 4 ? to each amino acid (horizontal axis) of GCH: green simulated frames, red
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docked frames. Correlations are listed on the top.
S4 Fig. Histograms of PC2 (Type II motion) of the MUMO ensembles along PC1 and PC2.
S1 Table. The number of frames in each simulation, where the respective hydrogen bonds
are present. The first column contains the specification of the hydrogen bonds in the
S1 Movie. The movement along the first PCA mode PC1.
S2 Movie. The movement along the second PCA mode PC2.
S1 File. Topology files for the the 6 ns MUMO simulations.
S1 Appendix. Main features of protein-ligand interactions are similar in the simulated and
Conceptualization: Orsolya T?ke, Zolta?n Ga?spa?ri.
Formal analysis: Zita Harmat, Andra?s L. Szabo?, Zolta?n Ga?spa?ri.
Investigation: Zita Harmat, Andra?s L. Szabo?, Zolta?n Ga?spa?ri.
Methodology: Zita Harmat, Andra?s L. Szabo?, Orsolya T?ke, Zolta?n Ga?spa?ri.
Project administration: Zolta?n Ga?spa?ri.
Resources: Zolta?n Ga?spa?ri.
Software: Zolta?n Ga?spa?ri.
Supervision: Zolta?n Ga?spa?ri.
Validation: Andra?s L. Szabo?, Zolta?n Ga?spa?ri.
Visualization: Zita Harmat, Andra?s L. Szabo?.
Writing ? original draft: Zita Harmat, Andra?s L. Szabo?, Orsolya T?ke, Zolta?n Ga?spa?ri.
Writing ? review & editing: Zita Harmat, Andra?s L. Szabo?, Orsolya T?ke, Zolta?n Ga?spa?ri.
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