International Immunology, Vol. 9, No. 9, pp. 1339–1346
© 1997 Oxford University Press
A model of water structure inside the
HLA-A2 peptide binding groove
Wilson S. Meng, Hermann von Grafenstein and Ian S. Haworth
Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue,
Los Angeles, CA 90033, USA
Keywords: HLA-A2, MHC, molecular dynamics, peptide, protein cavity, water
Abstract
Based on molecular dynamics simulations, it is proposed that water within the binding groove of
the human MHC class I molecule HLA-A2 plays a role in the formation of its complex with the
influenza matrix protein (residues 58–66; GILGFVFTL) peptide. In these simulations, a loosely
structured network of water molecules is present in the binding groove between the peptide and
the MHC molecule, and may be important in completing the peptide–MHC interface. In two
independent 400 ps simulations where groove-based water molecules were included, the peptide
remained essentially in the conformation observed in the crystal structure. In contrast, in a 400 ps
simulation in which no water molecules were placed between the peptide and the MHC molecule,
the crystal structure conformation was rapidly lost. The basis for this behavior appears to be that
the groove-based water molecules help to maintain the appropriate orientation of the Arg-97 side
chain of HLA-A2 and, in turn, the conformation of the central part of the peptide.
Introduction
Class I MHC molecules bind short peptide fragments of
intracellular proteins and present them at the surface of
infected cells to cytotoxic CD81 T cells (1,2). This type of
antigen presentation operates in most nucleated cells, and
provides a means for the immune system to recognize and
eliminate intracellular pathogens or cells with mutated
intracellular proteins (for a review, see 3). The peptides are
derived from intracellular antigens by proteolytic cleavage
(for a review, see 4).
The classical class I MHC molecules are encoded at three
loci in the human genome and comprise a large number of
different molecules. Despite the extensive polymorphism of
the MHC class I molecules, each individual can express only
up to six such variants and this small set is responsible for
presenting fragments of protein antigens from all potential
pathogens that can be cleared by CD81 T cells. Therefore
each of the class I molecules has to be able to bind a set of
peptides with diverse sequences.
The binding preference of HLA-A2 has been studied by a
variety of experimental (for a review, see 5) and theoretical
approaches (6–19). HLA-A2 shows a strong preference for
binding to nonameric peptides with Ile or Leu at position 2
and Leu or Val at position 9 (20). This motif is consistent with
crystallographic data that show regions of HLA-A2 (labeled
the B and F pockets) have hydrophobic characters well suited
to binding of hydrophobic side chains at positions 2 and 9
respectively (21). Although the presence of this motif is
usually necessary for high-affinity binding and stabilization of
individual MHC molecules, it does not define the specificity
of MHC–peptide interactions (22). The ability of each MHC
allelic variant to bind a number of diverse peptides with high
affinity suggests a requirement for flexibility in the chemical
environment of the MHC binding groove. Paradoxically,
despite being promiscuous in selecting their ligands, MHC
molecules are able to discriminate chemical changes as
minor as a glycine to alanine mutation in a given peptide
sequence (23,24).
Two structural features of HLA-A2 have been suggested to
facilitate sequence-degenerate binding. (i) Side chains of
the MHC molecule can adjust their conformations and/or
orientations in response to the different peptide side chains.
This conclusion has largely been reached by comparing
crystal structures of HLA-A2 complexed with five different
peptides (25). (ii) It has been proposed that water can be
situated inside the peptide binding groove and modify the
chemical surface presented to the bound peptide (26–29).
The role of water in contributing to the peptide–MHC
interface is becoming increasingly recognized and several
crystallographic reports have shown bound water molecules
in the peptide binding groove (26–29). Fremont et al. have
Correspondence to: I. S. Haworth or H. von Grafenstein
Transmitting editor: E. Sercarz
Received 11 June 1996, accepted 2 June 1997
1340 Water network in the peptide binding groove of a class I MHC molecule
solved structures of H-2Kb complexed with two different viral
peptides (26). In both structures, water molecules mediated
hydrogen bonds between the peptide and the MHC molecule.
Matsumura et al. (27) further discussed the potential role of
bound water molecules in facilitating the binding of peptides
to class I MHC molecules. More recently, two other studies
have shown that bound, groove-based water molecules allow
intrapeptide side chain–side chain interplay (28) and provide
the MHC molecule with the flexibility needed to accommodate
different peptides (29).
Based on these data, it is probable that groove-based
water molecules can be an important factor in the binding of
peptides to MHC molecules. As part of a continuing theoretical
study of the binding of peptides to HLA-A2, we have considered this possibility in the GILGFVFTL/HLA-A2 complex.
Using the X-ray structure (25) as a starting point, we have
analyzed the role of water in the central and widest portion
of the peptide binding groove. This region is partially filled
by the side chains of both conserved and polymorphic
residues of HLA-A2, but considerable space remains between
the peptide and the MHC molecule. Based on molecular
dynamics simulations, in this paper we propose that a flexible
network of water molecules may fill this region and contribute
to the formation of the GILGFVFTL/HLA-A2 complex.
Methods
All calculations were performed using the AMBER 4.0 allatom force field (30) on a Silicon Graphics Indigo2 workstation.
Graphic display and analysis of the trajectories were performed using the molecular modeling package QUANTA
version 4.0 (31). The starting structure for all simulations was
the X-ray crystal structure of the class I molecule HLA-A2
complex with a peptide derived from the influenza matrix
protein (32) [residues 58–66; one letter code amino acid
sequence GILGFVFTL; Brookhaven entry 1hhi (25)]. Coordinates of the peptide and the α1 and α2 domains of the complex
were used in the calculations.
Using AMBER, hydrogen atoms were added to the X-ray
coordinates of the GILGFVFTL/HLA-A2 complex (25). Following relaxation of the hydrogen atoms, the complex was placed
in a 25 Å radius TIP3P water sphere (33) centered upon the
center of mass of the peptide. Any water molecule within
2.0 Å of any solute atom was discarded from the calculation.
As a result, 1185 water molecules were added, forming a
solvent shell which fully solvated all the surface residues of
the binding groove to a depth of at least 20 Å. Other surface
residues of the MHC molecule were solvated to a lesser
extent. No water molecules were added to the inside of the
binding groove in this process.
The added solvent was first subjected to an equilibration
phase in which the solvent shell was minimized for 2000 steps
and then subjected to 50 ps of molecular dynamics. The final
structure of the molecular dynamics trajectory was then
minimized for 2000 steps. Using the solvent-equilibrated
system, two different molecular dynamics simulations were
performed. These will be referred to in the text as MDGW and
MDNW (‘molecular dynamics; groove water’ and ‘molecular
dynamics; no groove water’ respectively). For MDGW, 12
TIP3P water molecules were placed inside the binding groove
based on void space calculated by the program MS (34).
The specific locations of these buried water molecules at the
start of the simulation were chosen in an arbitrary fashion and
subjected to the same equilibration process described above.
In MDNW, no groove-based water was included and the
simulation was carried out only with the outer solvent shell.
A third simulation, referred to as MDGW2, and also including
groove-based water molecules, was performed using a
different protocol for groove solvation. In this case, the peptide
was removed from the binding groove and the empty HLAA2 molecule was then solvated in the manner described
above. In the absence of the peptide, the binding groove
region was filled with water in this process. The peptide was
then put back into the binding groove in its crystal structure
location. The solvent only was then subjected to 2000 steps
of minimization followed by a 5 ps dynamics simulation, which
resulted in 11 water molecules in the binding groove between
the peptide and the MHC molecule.
The remaining protocol for each of the three molecular
dynamics simulations was as follows. First, the solvent equilibrated structures were minimized for 2000 steps. The minimized
coordinates were then subjected to a 400 ps molecular
dynamics simulation, including an initial heating phase from
0 to 298 K in 10 ps, using a time step of 2 fs. Current
simulation approaches are largely limited to such timescales.
Coordinates were saved every 0.4 ps and a residue-based
non-bonded cut-off of 6 Å was used in all calculations.
In the molecular dynamics simulations, the backbone atoms
of the protein were loosely restrained to their minimized
positions by a force constant of 2 kcal/mol/Å. Computational
practicality demands that, since the β2-microglobulin (β2m)
and α3 domains are not directly interacting with the peptide,
these are omitted to reduce the overall size of the system.
However, the β2m domain is important, experimentally, in
stabilizing the α1 and α2 domains, and, in its absence, position
restraints on the α1 and α2 backbones are necessary to
maintain the overall structural integrity of these domains. We
emphasize that the restraints are very light and do not prevent
motion of the side chains of the protein.
In general, in performing simulations of class I MHC–
peptide complexes using the AMBER force field, we also find
it necessary to include light constraints to maintain conserved
hydrogen bonds between the peptide termini and the MHC
molecule (18). Specifically, we include constraints of 10 kcal/
mol/Å for the peptide N-terminal to Y171 (N–H · · · O–H, 2 Å),
for the peptide C terminal backbone to D77 (N · · · Cγ, 4.0 Å,
taken from ref. 25) and for the penultimate C-terminal residue
of the peptide to W147 (C5O · · · H–N, 2 Å). Each of these
interactions is conserved across all HLA-A2/peptide complexes for which X-ray structures have been solved (25).
We acknowledge that the inclusion of constraints for these
interactions indicates a deficiency in our methodology, but
we consider that maintenance of the conserved interactions
is important in drawing conclusions from the simulations.
Results
Conformational changes in the MHC–peptide complex
The essential difference between the simulations was that
without groove-based water (MDNW), the X-ray structure
Water network in the peptide binding groove of a class I MHC molecule
1341
Fig. 1. (Upper panel) The structure of the peptide GILGFVFTL and the location of key residues of HLA-A2 (Y99, H70, H74, H114, R97, Y116
and D77) after 200 ps of a molecular dynamics simulation in which no groove-based water molecules were included (MDNW). During the
simulation, the peptide F5 side chain moved away from its initial, crystallographic location and is now oriented into the solvent region. Also
shown is the HLA-A2 R97 side chain, which has adopted a C-terminal orientation and lost its indirect interaction with the F5 carbonyl group.
(Lower panel) A representative structure (after 200 ps) from a simulation in which groove-based water molecules were included (MDGW). The
water molecules are displayed as space-filled models and are colored in red. In contrast to the structure from MDNW, the crystal structure
conformation of the PF5 side chain and the orientation of the R97 side chain were preserved in this simulation. Note that in the lower plate the
R97 side chain is oriented towards the F5 carbonyl group, but that this interaction is indirect and mediated by a water–water bridge.
1342 Water network in the peptide binding groove of a class I MHC molecule
Fig. 2. (a–c) The motion of the PF5 χ torsion angle (around the Cα–Cβ bond). The conformation of PF5 changed from g1 to g– in MDNW
~60 ps into the simulation. In contrast, the PF5 side chain X-ray conformation was preserved in MDGW and MDGW2. (d–f) Distance between
the side chain of HLA-A2 residue R97 and peptide residue PF5 (N · · · O5C) showing the location of the R97 side chain with respect to PF5
of the peptide. Only the first 20 ps of the simulation are shown, because no significant changes occurred in these distances after this point.
The motion in MDNW represents the reorientation of the R97 side chain towards the direction of the C-terminus of the peptide. In MDGW and
MDGW2, the side chain of R97 remained in the initial orientation, towards the N-terminal of the peptide.
conformation of the peptide was lost rapidly, whereas in the
simulations with groove-based water (MDGW and MDGW2),
the X-ray conformation persisted for 400 ps. The difference
in the conformations generated with and without groove-
based water is shown in Fig. 1, contrasting the MDNW and
MDGW results. The most important difference is the orientation
of the F5 side chain (PF5; the peptide residues are henceforth
abbreviated as PG1, PI2, etc.).
Water network in the peptide binding groove of a class I MHC molecule
Table 1. Water occupied sites in the GILGFVFTL/HLA-A2
binding groove
Site
Site
Site
Site
Site
Site
1
2
3
4
5
6
Site residuesa
Occupying waterb
F9, H70, Y99
R97, PF5, PL3
T73, PV6
D77
PF7
Y116, PL9
Wat6, Wat9
Wat2 (Wat6), Wat8
Wat4, Wat12
Wat4, Wat5
Wat3, Wat10
Wat10 (Wat1)
aSites are defined by the backbone and/or side chain atoms of the
residues indicated (see text).
bWater molecules replacing the primary water during the simulation
are in parentheses.
1343
Molecular dynamics without groove-based water
Reorientation of the PF5 side chain around the χ torsion angle
(around the Cα–Cβ bond) occurs after ~60 ps of the MDNW
simulation [Figs 1 (upper panel) and 2a]. In the GILGFVFTL/
HLA-A2 X-ray structure, the backbone carbonyl group of PF5
is oriented towards the R97 side chain. In the MDNW simulation,
R97 moved away from its initial orientation [described as the
N-terminal orientation by Madden et al. (25)] towards the
C-terminal of the peptide [Figs 1 (upper panel) and 2d]. This
results in a loss of the PF5–R97 association and an increased
conformational mobility of the side chain of PF5.
Molecular dynamics with groove-based water
In comparing the complexes with and without groove-based
water molecules, we focus on the MDGW simulation, and on
Fig. 3. The X-ray conformation of the HLA-A2 complexed with GILGFVFTL showing the locations of the sites of water occupation in the binding
groove. Only the protein and peptide residues that are involved in the water network and the backbone of the α helices of the MHC molecule
are shown. In (a) the GILGFVFTL/HLA-A2 complex is viewed from above such that the N-terminus of the peptide is on the left. HLA-A2 residue
R97 is located centrally and is oriented towards the viewer. Site 2 is located between R97 and peptide residue PF5. Sites 1, 3 and 4 are also
visible in this orientation, close to H70, T73 and D77 respectively. In (b), the complex is rotated through 90° and shows a side view looking
into the binding groove. Site 2 can easily be located between R97 and PF5, and Site 5 is now visible below PF7.
1344 Water network in the peptide binding groove of a class I MHC molecule
the motion of the PF5 and R97 side chains. As is evident in
Fig. 2, the behavior of these side chains and the role of the
groove-based water molecules in maintaining their X-ray
conformations was very similar in the MDGW and MDGW2
simulations (Fig. 2b, c, e and f). Hence, all further description
of the groove-based water behavior will be based upon
observations made in the MDGW simulation.
Although the R97 side chain is oriented towards the carbonyl
group of PF5 and that of PF7, in the X-ray structure, a direct
interaction between R97 and either carbonyl group is not
possible because of the distances separating them from
R97. The presence of groove-based water molecules would,
however, allow for these interactions to occur indirectly via
water bridges [see Fig. 1 (lower panel)]. The MDGW simulation
showed that the inclusion of these water molecules appears
to be important in stabilizing the native orientation of the R97
side chain in the GILGFVFTL/HLA-A2 complex. During the
MDGW simulation, the change in orientation of R97 and PF5
side chains described for MDNW did not occur (Figs 1 and
2). All other inwardly oriented side chains in the binding
groove showed only slight motion and essentially remained
in their starting conformations/orientations over the entire
simulation (data not shown).
In MDGW, we found the groove-based water molecules
occupy well-defined sites with respect to the peptide and
protein. However, these water molecules are not fixed,
because they exhibit exchange among these sites, even in the
relatively short 400 ps simulation time. Many such examples of
this behavior were observed. A site is defined based on the
proximity of the site to protein and/or peptide residues (Table
1). For each site, one or more primary occupying water
molecule(s) is/are defined and the average location of the
site is calculated by the averaged van der Waals’ volume
occupied by the primary water molecule(s) during the simulation. The relationship of the sites to the MHC and peptide
amino acids is shown in Fig. 3 and summarized in Table I.
The water molecules themselves are arbitrarily labeled
Wat1–Wat12.
The water-mediated interaction between PF5 and R97
provides a good example of the dynamic nature of the water
network. We defined this particular part of the network as Site
2 (Fig. 3). This site is located directly above the side chain
of R97, underneath the backbone atoms of PF5, and surrounded by the hydrophobic side chains of PL3 and PF5.
Water molecules found in this region are associated with the
side chain of R97 and/or the groove-oriented carbonyl of PF5.
This site was occupied primarily by Wat2 and Wat8 with Wat2
hydrogen bonded to R97 (H–N) and Wat8 to PF5 (C5O) (Fig
4a and c). These two water molecules also formed a water–
water hydrogen bond (Fig. 4d). This is an important interaction
because it allows R97 to interact indirectly with PF5 of the
peptide, through the R97–Wat2–Wat8–PF5 network. This water
bridge allows the peptide to be anchored to the MHC molecule
in the center of the groove, reinforcing the anchoring role
played by the termini of the peptide at the ends of the binding
groove (35).
An example of water exchange in the groove is illustrated
by the replacement of Wat2 by Wat6 in the R97–Wat–Wat–
PF5 network during the 230–340 ps period of the simulation
(Fig. 4). Wat6 moved into this site from Site 1 and Wat9
Fig. 4. Hydrogen bond distances between (a) R97(HN1) and Wat2,
(b) R97(HN1) and Wat6, (c) PF5 (C5O) and Wat8, (d) Wat2 and
Wat8, and (e) Wat6 and Wat8. These hydrogen bonds show how a
water–water bridge facilitates the interaction between R97 and PF5.
This bridge primarily involves Wat2 and Wat8, but, due to the dynamic
nature of the water structure in the binding groove, Wat2 can be
replaced by Wat6 during 230–340 ps without disturbing the overall
R97–PF5 interaction.
Water network in the peptide binding groove of a class I MHC molecule
1345
Fig. 5. The observed variations in hydrogen bond pattern in MDGW are shown at 50, 150, 250 and 350 ps in the simulation. Hydrogen bonds
are defined by a distance between hydrogen donor (D) and acceptor (A) ,2.5 Å and a D–H · · · A angle between 140° and 180°. The defined
sites (see text) for each time point are shown in the first row and water molecules are listed in the first column (water molecules are abbreviated
as Wat1, Wat2, etc.; the numbers are chosen arbitrarily). Selected protein and peptide residues are shown for each site. The presence of a
hydrogen bond contact is indicated by a closed box. A black box indicates that the water molecule is the hydrogen bond acceptor and a
gray box indicates that the water is the hydrogen bond donor. If a water molecule is an acceptor and donor concurrently with a given amino
acid, the box is filled with a cross-section pattern.
substitutes for Wat6 in Site 1 (data not shown) during the
230–340 ps period. This is illustrative of the structured but
fluid nature of the water network.
Figure 5 provides an overview of the water to peptide–
protein hydrogen bond contacts and illustrates the exchange
processes occurring between water molecules occupying the
defined sites. It should be noted that the side chains in the
various sites remained largely in their starting conformations
throughout the simulation. The water molecules can be
grouped into two categories with respect to their relative
residence time (over the length of a 400 ps simulation) in a
particular site: fixed and mobile. The first category is defined
as those water molecules that maintained a contact with a
specific side chain for at least two-thirds of the trajectory.
Wat2, Wat3, Wat4, Wat5, Wat8 and Wat12 can be considered
to be in this category of water molecules. Each of these fixed
water molecules fulfilled the hydrogen bonding requirements
in their respective sites and participated in the backbone of
the overall water structure in the groove.
The second category includes water molecules that explicitly
moved between two or more of the defined sites in the simulation. Wat1, Wat6, Wat9 and Wat10 are considered to be in this
category. For example, Wat6 initially resided in Site 1 before
moving into Site 2 in the second half of the simulation, whereas
Wat9 moved from Site 2 to Site 1 (Fig. 5). The fluidity of the
water network is characterized by this group of water molecules
moving within and among the sites. The function of these water
molecules may be to satisfy hydrogen bond deficiencies created by other motions of the water structure, and motions of the
peptide and of the protein side chains. The two categories
accounted for 10 of the original groove-based water molecules
at the start of the MDGW simulation. Wat7 escaped from the
binding groove into the bulk solvent early in the simulation and
did not play any role in the water network. Wat11 remained near
the floor of the groove throughout the simulation and was not
involved in the water network.
Discussion
From the above results, we propose that a dynamic, groovebased water network contributes to the formation of the
GILGFVFTL/HLA-A2 complex. Although specific sites can be
identified within this network that are constantly occupied by
water molecules, the fluidity of the network is suggested by the
water–water exchanges between such sites that occur in the
simulation. The site located between PF5 and R97 is of interest
because it potentially mediates an indirect peptide to protein
interaction. However, the presence of water in this site alone is
insufficient to maintain the PF5 and R97 orientations, and other
sites also need to be occupied to complete the hydrogen bonding network. We have drawn this conclusion by conducting
simulations with only two or three water molecules in the binding
groove, between PF5 and R97. Although we have not discussed
these data in detail in this paper, the motions of PF5 and R97
1346 Water network in the peptide binding groove of a class I MHC molecule
side chains were very similar to those in the simulation where
no water molecules were placed in the binding groove.
The groove-based water adds a further level of complication
to the mechanism of the GILGFVFTL/HLA-A2 interaction. For
interactions of HLA-A2 with diverse peptide sequences, a flexible network of water molecules in the binding groove might
allow protein side chains to adjust their conformation in
response to different peptides. In addition, changes in the
chemical nature of the bound peptide might impose a different
pattern of water sites to that suggested here for GILGFVFTL/
HLA-A2. Hence, the presence of groove-based water may play
a role in determining the affinity of specific peptide–MHC interactions.
Acknowledgements
This work was supported by a grant from the Pharmaceutical Research
and Manufacturers of America Foundation to H. v. G. W. S. M is supported by the Krown Fellowship and a grant from the American Foundation for Pharmaceutical Education (AFPE). We also thank the USC Norris
Cancer Center Molecular Graphics Facility (supported by the National
Cancer Institute, IRG-21-33) for generous allocation of computer time.
Abbreviations
β2m
MDGW
MDNW
β2-microglobulin
molecular dynamics; groove water
molecular dynamics; no groove water
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