MHC–Peptide Binding is Assisted by Bound Water Molecules

doi:10.1016/j.jmb.2004.02.039

J. Mol. Biol. (2004) 338, 419–435

MHC –Peptide Binding is Assisted by Bound Water Molecules Paula M. Petrone and Angel E. Garcia* Theoretical Biology and Biophysics Group, T-10 MS K710, Los Alamos National Laboratory, Los Alamos NM 87545, USA

Water plays an important role in determining the high affinity of epitopes to the class I MHC complex. To study the energy and dynamics of water interactions in the complex we performed molecular dynamics simulation of the class I MHC – HLA2 complex bound to the HIV reverse transcriptase epitope, ILKEPVHGV, and in the absence of the epitope. Each simulation was extended for 5 ns. We studied the processes of water penetration in the interface between MHC and peptide, and identified 14 water molecules that stay bound for periods longer than 1 ns in regions previously identified by crystallography. These water molecules in the interface perform definite “tasks” contributing to the binding energy: hydrogen bond bridges between MHC and peptide and filling empty spaces in the groove which enhance affinity without contributing to epitope specificity. We calculate the binding energy for interfacial water molecules and find that there is an overall gain in free energy resulting from the formation of water clusters at the epitope –MHC interface. Water molecules serving the task of filling empty spaces bind at the interface with a net gain in entropy, relative to their entropy in bulk. We conclude that water molecules at the interface play the role of active mediators in the MHC–peptide interaction, and might be responsible for the large binding affinity of the MHC complex to a large number of epitope sequences. q 2004 Elsevier Ltd. All rights reserved.

*Corresponding author

Keywords: hydration free energy; entropy driven binding; Gaussian model; water coordination; principal component analysis

Introduction Cytolytic immune response of an infected host cell is launched by specific proteins, which chop the viral material into pieces of 6 –15 amino acid residues long.1,2 These peptides or epitopes are transported to the cell surface and presented to the T-cell receptor by a protein known as the MHC complex. Different epitopes are found to bind in the groove of a single class I MHC molecule, through a combination of conserved and polymorphic contacts. Most humans can only express up to six different class I MHC molecules. Given the broad variety of different peptides coming from all possible pathogens, the MHC complex cannot afford specificity for a particular sequence. The MHC complex must be able to bind Abbreviations used: ER, endoplasmic reticulum; HC, heavy chain; PC, principal components; MSD, meansquare displacement. E-mail address of the corresponding author: [email protected]

“promiscuously” many different epitopes and present them to the T-cell repertoire. In contrast, the specificity of the T-cell receptor is such that it will react only against those epitopes recognized as foreign material (antigens). Thus, the MHC – peptide interaction is responsible for a complex which satisfies the dual requirements of strong peptide binding (high affinity) and, at the same time, allowing generous selectivity, in a way that many different peptides can fit in the binding groove of a single MHC molecule. Peptide – MHC complexes are extremely stable under physiological conditions and, given this high affinity, only a small fraction of the class I molecules are normally found on the cell surface without bound peptide.3 – 10 The assembly of the class I MHC inside the endoplasmic reticulum (ER) requires the presence of a series of chaperone proteins,11 that keep the MHC heavy chain (HC) and b2-microglobulin (b2m) together as a folding intermediate. Upon the binding of a peptide, all associated proteins are released and the MHC –peptide complex is transported to the cell surface. Spectroscopy

0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.

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and proteolysis studies of MHC-HCB7 provide clear evidence that the absence of a peptide in the binding groove makes the HC/b2m heterodimer adopt a different structural state, with properties similar to those of a molten-globule state: an increased tendency towards aggregation and a high content of native secondary structure.11 Moreover, increased susceptibility to proteolyses, especially in the epitope binding-site region, reflects a fluctuating structure. In addition, studies on the b2m dissociation rate6 suggest that the peptide-binding site and the b2m-binding site are mutually dependent, even though they are located on opposite sites of an eight-stranded b-sheet and that there is a linkage between antigenic peptide binding and HLA folding and/or assembly.8,10,12 X-ray structures of class I MHC complexes12 – 16 show that bound peptides are firmly attached by their ends, and arch away from the floor of the pocket. Comparison between different MHC alleles indicates that conserved side-chains of the MHC molecule hydrogen bond to main-chain polar atoms at both ends of the peptide, while the polymorphic residues bury the “anchor” peptide sidechains.3 – 5 Experiments with terminally modified peptides emphasize the importance of terminal contacts. Variations of these contacts can result in a conformational change at the center of the peptide.9 Fremont et al.17 compare the binding of peptides to MHC molecules with the packing of the core region of a typical globular protein. Peptides bind with high affinity (typically , 1028 M)18,19 by optimally complementing pockets in the core of the groove.20 A broad set of epitope sequences results in the same folded structure, although certain residues (“motifs”) are preferred in certain positions. The fundamental question arises: how can the MHC host such a broad collection of peptides and yet provide them all with a stable union? Alternate atomic packing can be achieved by rearrangements of secondary structure elements. At the same time, there is evidence that shows that water molecules in the interface play a crucial role in preserving this union.13,17,21,22 There is increasing awareness in the importance of water in the interface between protein and different ligands where solvent is an active participant and not an innocent bystander.23 – 29 Fixed or transient water may have structural or functional roles.23 Studies in DNA structures24,25 suggest that water molecules may increase the specificity towards a given sequence acting as an “extension” of the nucleic acids. Conserved water molecules in homologous enzymes seem to play an important role in the architecture of the active site.30 Reports of the kinetics and thermodynamics of the binding of a peptide to MHC show that for I MHC-HLAA2 binding to the Tax peptide (LLFGPVYV) the process is entropy-driven.31 This is surprising, since binding involves fixing of the peptide fluctuations and a reduction of the protein flexibility. However, hydrophobic hydration is characterized

MHC –Peptide Binding

by an increase in entropy. This increase in entropy has been interpreted in terms of the release of water around non-polar groups into the bulk solvent. We will show that there is also the possibility of increasing the entropy of water molecules at the peptide– MHC interface. The role of water, in the context of the MHC – peptide binding, is relevant to fill empty cavities and serve as hydrogen bonding bridges between the peptide and buried hydrophilic MHC residues. There is experimental evidence12 – 14,16,17 of tightly bound water molecules underneath the peptide that would help to stabilize the peptide mainchain by a complex network of hydrogen bonds to polymorphic class I MHC residues on the floor of the groove. The flexibility of a water network adds stability to the central region of the peptide where the greatest sequence and conformational variation is found. It has been suggested that this network of water will be one of the mechanisms through which the MHC might be able to adjust its binding surface to host different peptides.12 – 14,17,21,22 Here, we study the dynamics of the class I MHC – HLA2 complex bound to the HIV reverse transcriptase peptide ILKEPVHGV, and without the peptide by molecular dynamics simulations. These simulations are used to study the fluctuations in the system in the bound and unbound state, and the energy of interaction of water molecules at the MHC – peptide interface. Analysis of the fluctuations of the peptide-free MHC complex reports large fluctuations which deform the binding groove of the peptide-free MHC –HLA2, consistent with the experimental observations.11,31 – 33 We investigate the processes of water penetration in the interface, and we study the energetics of water molecules to learn about the role of solvent in this peculiar way of binding. For this purpose, we perform a 5 ns molecular dynamics simulation of the peptide-bound MHC (MHCP) and compare the results with those obtained in the system without peptide (MHCNP). Molecular dynamics simulations are used to study the spontaneous binding of water to pockets at the MHC – peptide interface, the density and residence time of water around these pockets. The localization of water at these pockets is in close agreement with crystallographic data.12 By tracking the instantaneous position of atoms, our simulations add further knowledge of the intimate structure and energetics of water clusters that is otherwise overseen by the resolution of experimental devices. We provide theoretical evidence of the role of bound interfacial water molecules in enhancing the association energy, while broadening the scope of peptide selectivity. Computer simulations for the class I MHC –HLA have been reported.21,22,34,35 Rognan et al. studied the binding of six different peptides to MHC I– HLA – Bp2705 and found that molecular dynamics simulations can discriminate between good binders and bad binders.35 Interestingly, they observed that inclusion of six crystallographic water

421

MHC –Peptide Binding

molecules in their simulations was not needed, since water molecules from the solvation shell could reproduce their binding role. However, simulations performed in vacuo never led to conformations and protein– peptide interactions present in the crystals. The MHC – peptide “dockingproblem” has been tackled by “growing” the ligand in the binding pocket in combination with simulated annealing.21 Water sneaking into the interface was observed. Molecular dynamics simulations have also been implemented for the MHC peptide system in which the binding groove has been filled with a predetermined number of water molecules.22,36 In the studies we present here, we do not bias the structure or number of water molecules at the protein –peptide interface by positioning crystallographically observed water. In our initial configuration the solvent fills cavities provided there is enough space available. The extensive sampling performed in our calculations enables us to study how water exchanges between bulk solvent and the protein interior takes place spontaneously, as the system evolves towards its equilibrium state. There is an evident free energy gain in the process of water binding to the interface, since it occurs spontaneously. In addition, we include in our simulations all chains that form the MHC heterotrimer such that we can observe the correlation that exists between the fluctuations of the MHC molecule and the presence of the peptide.31 – 33 Water molecules at the MHC – peptide interface observed by X-ray crystallography can be classified

Figure 1. MHC – HLA-A2 heterodimer is formed by b2microglobulin (b2m, blue), and HC. HC comprises domains a1 (red), a2 (yellow) and a3 (cyan). The epitope-binding cleft is located in the a1 and a2 domains. Chains H1 and H2 constitute its walls and the floor is a b-sheet.

into three categories: fixed, semi-fixed or variable, as suggested by Smith et al.13 Fixed water molecules are invariant between MHC alleles, like those found in the edge of the epitope-binding pocket which facilitates the conserved hydrogen bonding network to the peptide N terminus. Semifixed water molecules are those interchangeable in position with MHC or peptide side-chain atoms and form hydrogen bonds; these water molecules provide flexibility to peptide variability. The third category comprises the water molecules that fill the space between the peptide and the floor of the binding groove, acting as indirect anchors between the peptide main chain and MHC. Studying the trajectory of water molecules we support the existence of specific “tasks” that water plays in the interface. Water molecules that perform these tasks have very definite sites in the MHC binding groove, which they occupy for extended periods of time (. 1 ns). These “designated tasks” of water are such that if a water molecule abandons the site, it is quickly replaced by another one that performs the same role. Bound water molecules in the interface have two main tasks: (1) fill empty spaces; or (2) make bridging hydrogen bonds between the MHC and the peptide.

Results and Analysis Principal components Principal component (PC) analysis provides information about the collective motions of atoms in a protein.37 Here, we compare the motions in the structures MHCNP and MHCP to establish which are the main differences that a bound peptide can make in the dynamics of the MHC structure. The MHC –HLA2 complex is a heterodimer (Figure 1). It consists of a HC, and a b2-microglubulin chain (b2M). The HC is divided into three domains: a1, a2, and a3. Domains a1 and a2 fold almost identically into a b-sheet limited by two opposite helical regions, which correspond to the floor and wall of a peptide-binding groove. Each helical region is formed by two helixes, H1 and H2, short and long, respectively. If we examine the mean-square displacement (MSD) (Figure 2(a)) of the Ca atoms in both structures it is clear that the larger fluctuations belong to the structure without peptide. Moreover, most of these fluctuations involve the helices H1 and H2 that enclose the binding-groove region, in both a1 and a2 domains. The principal components of the MHCP comprise collective motions of atoms that do not deform the bindinggroove; namely, relative displacements between the a1, a2, a3 and b-microglobulin domains. However, the structure MHCP bears large fluctuations in the residues 173 – 186, which link the a2 domain with the a3 domain. The mode describing the largest fluctuations (the first principal component) of the peptide-free structure MHCNP also corresponds to inter-domain motion, and does not have

422

MHC –Peptide Binding

Figure 2. PC analysis. (a) MSD for each Ca in MHC without peptide (purple) and with peptide (blue). Fluctuations in the four helixes H1 – H2 that contain the binding-groove (domains a1 and a2) are dominant in the MHCNP case. (b) PC2 and PC3 of the MHCNP produce deformation of the binding-groove (amplitude exaggerated for the sake of clarity). Colors highlight the most fluctuating residues. In (b.i) mode 2 corresponds to the displacement of the central hinge of a2. (b.ii) Mode 3 involves the opening and closing of the binding cleft.

a major effect in the size of the binding cleft. However, the subsequent principal components (in order of decreasing eigenvalue and contribution to the MSD) denote fluctuations of main-chain atoms that contort the shape and size of the bindinggroove. Figure 2(b) shows the displacement, from the average structure, corresponding to the fluctuations accounted by the second and third principal components restricted to the binding-cleft region. PC 2 (Figure 2(bi)) describes the motion of the kinked region between H1 and H2, in the a2 domain. PC 3 (Figure 2(bii)) describes the displacement of the two walls of the cleft, opening and closing in scissoring fashion. These results show that the presence of the peptide helps to maintain the rigidity of the cleft, consistent with experimental observations. In the absence of the peptide, the a1 and a2 domains are highly susceptible to proteolysis.11 It has been suggested that a confor-

mational change takes place in these domains, which leads from a more disordered state to a more rigid structure upon peptide binding.11 Our results also correlate with studies suggesting that allosteric interactions take place among the HC, b2m and peptides.10 The principal component analysis we report here and the large MSD fluctuations of the binding site of the epitope-free system add further evidence to support this hypothesis. The packing of bound water molecules at the MHC – epitope interface might also contribute to reduce the relative mobility of the MHCP residues. Water molecules at the MHC – epitope interface fill empty spaces and contribute with hydrogen bonds that may restrain fluctuations, particularly, those described by modes that open and close the binding site. The fact that the empty MHCNP binding pocket is flexible might suggest that a plastic MHC promotes the adaptation of the

423

MHC –Peptide Binding

Table 1. Hydrogen bonds between MHC and the peptide, except for the hydrogen bond labeled † between P3 and P4 Position

Contact peptide

Contact MHC

˚) Mean distance (A

Mean angle (8)

P1 P2 P2 P2 P3 P3 P4 P4 P4 P4 P6 P6 P6 P7 P7 P8 P8 P8 P8 P8 P8 P8 P8

Ile376 O Leu377 N Leu377 N Leu377 N Lys378 N Lys378 NZ Glu379 OE1 Glu379 OE2 Glu379 OE1 Glu379 OE2 His382 N His382 O His382 O Gly383 N Gly383 O Val384 N Val384 N Val384 N Val384 O Val384 O Val384 OXT Val384 OXT Val384 OXT

Tyr159 OH c Tyr159 OH c Glu63 OE1 v Glu63 OE2 v Tyr99 OH v Glu379 N † Arg65 NE v Arg65 NE v Arg65 NH2 v Arg65 NH2 v Arg97 NH1 v Arg97 NH1 v Arg97 NH2 v Trp147 NE1 c Trp147 NE1 c Trp147 NE1 c Asp77 OD1 v Asp77 OD2 v Tyr84 OH c Thr143 OG1 c Tyr84 OH c Thr143 OG1 c Tyr123 OH c

1.65 2.10 1.87 1.71 1.76 2.11 1.90 2.11 1.92 1.91 2.13 1.71 1.81 2.08 1.69 2.16 1.71 1.93 1.97 2.18 1.65 1.65 2.15

164.5 144.6 122.8 157.3 162.0 122.1 151.7 134.3 144.5 150.3 138.4 149.3 138.4 138.3 155.2 139.4 152.3 142.4 130.9 159.3 161.0 165.8 138.1

The hydrogen bond mean distance and mean angle were averaged over the last 4 ns of simulation, when water molecules have already penetrated the interface. Criteria for hydrogen bonds include strong and moderate interactions and are restricted to a mean ˚ between H and the acceptor and a mean angle of 120 –1808.54 We restrain the search to the species N, distance in the range 1.1– 2.5 A O and OH. (We use two labels to differentiate MHC residues: c, completely conserved; and v, variable12).

complex to different peptides.31 – 33 On the other hand, its rigidity, when the peptide is bound, might contribute to keep the union stable. Direct MHC – epitope hydrogen bonding From our MD simulation, we can see that most hydrogen bonds in the binding groove are formed with the peptide backbone, and are thus nonsequence-specific (Table 1). These data agree with the conclusions drawn from X-ray data in HLA-A2 and other species.14,16,17,38 The peptide is strongly bound by its edges to conserved residues in the MHC, and its middle residues arch away from the groove. The occurrence of hydrogen bonds between the central residues and the MHC is very low or non-existent, specially for P5:PRO. As we will show later, central residues are bonded to the MHC floor by water-mediated hydrogen bonds contributing to keep the peptide main chain in place. In what follows, we describe the shape and hydration of various pockets.

MHCP we found seven molecules that penetrate spontaneously from bulk and stay inside the MHC –peptide interface for periods longer than 1 ns (Figure 3). In addition, we identified seven water molecules that penetrate the interface and stay bound during the rest of the simulation; the position of some of these match with the three water molecules found in the crystal structure.12 ˚ structure of HLA-B2714 and Analysis of the 2.1 A ˚ 2.3 A structure of HLA-B5313 reports that the peptide is bound to water molecules underneath and alongside its center, which in turn are hydrogen bonded to the MHC polar floor. Twelve molecules have been identified for HLA-B27 and six for HLA-B53 crystals.

Water penetration events We calculate the instantaneous coordination of water ðNc Þ to identify water penetration and water escape events (see Methods: Instantaneous water coordination number).12 Nc is the number of ˚ ) to a solvent molecules coordinated (within 3.5 A given water molecule. A drop in this number indicates that the molecule becomes isolated from solvent, and hence binds to the surface or a protein cavity as “interior water”. In the simulations of the

Figure 3. MD trajectory of water molecule (blue) entering spontaneously into pocket A. Top view of class I MHC complex with peptide (green). Pockets: A (white), B (pink), C (cyan), D (yellow), E (orange), F (grey).

424

Water molecules seldom stay isolated (Nc ¼ 0) in the protein interior, but instead form clusters that perform different tasks inside the interface. These tasks are either to fill spaces between peptide and MHC or to contribute to the binding energy forming hydrogen bond bridges between MHC and peptide. As we will show later, molecules that share the same task have similar interaction energy distributions. In the system without peptide (MHCNP) we could identify only four water molecules that once bound to the peptide-binding site, stay for the rest of the simulation. However, none of these positions seems to be near the sites occupied by water in the peptide-bound complex. In MHCNP we observe a great amount of water that fills the peptide pockets, but have a short residence time. In this case, the hydrogen bond tasks are not as definite as in the case where there is a peptide in the groove. A simple Gaussian model to estimate free energies The free energy of bound water molecules can be estimated from our simulations by calculating histograms of the energy of interaction of these water molecules with the rest of the system. We calculate the interaction energy for selected water molecules (see Methods) and construct histograms with all the occurrences during the 4 ns production stage of our simulations. The energy distributions obtained in this way reveal that bound water molecules or clusters of water molecules in the

MHC –Peptide Binding

interior of the protein, in most instances, interact very differently to bulk solvent. The mean-value of these distributions is not necessarily lower than bulk, but they are clearly narrower. This behavior has been observed earlier in solvated nanotubes. Hummer et al.39 performed MD simulations of solvated nanotubes and reported spontaneous penetration of water molecules. The energy distributions of these water molecules present features analogous to those we present here; i.e. narrower than bulk water distributions but with a higher mean value. The free energy is dominated, not by the strength of water binding, but by the lower population of weakest states.39 Following this principle, a simple Gaussian model applied to these energy distributions allows us to approximately describe the free energy gain associated with the presence of water molecules in the interface. The Gaussian distribution is characterized by two parameters, the mean energy E; and the width of the distribution, s. While the mean value of the Gaussian fit estimates the mean energy of binding, a greater width of the distribution determines the population of a wider range of states. Figure 4 shows different examples of Gaussian distributions and their estimated chemical potential value. Figure 4(a) shows three water energy distributions for bulk water (blue), a distribution with the same width as bulk (s ¼ sB), but lower average energy ðE , EB Þ (green), and a distribution with a smaller width (s , sB), and higher mean energy ðE . EB Þ (red). The corresponding chemical potentials for these three

Figure 4. Relation between width (s), mean energy (E) and chemical potential (m) corresponding to Gaussian distributions. (a) Examples of Gaussian distributions. (b) Contour plot for chemical potential of Gaussian distributions in (a) relative to bulk parameters (EB ; sB, mB). Chemical potential depends on both width and mean value of the distributions.

MHC –Peptide Binding

distributions are lower than the value for bulk. The lower average energy (blue) distribution shows an instance in which the lower chemical potential is a consequence of lower binding energy (enthalpy driven), while the higher average energy (red) distribution shows an instance in which the lower chemical potential is a consequence of a narrower distribution (entropy driven). Figure 4(b) shows a contour plot of the chemical potential values (relative to bulk water) for water molecules having energy distributions characterized by mean energy E and width s, as a function of ðE 2 EB Þ and s/sB. Blue contour lines show values for which the chemical potential is lower than for bulk water.

425

The black line shows values for which the chemical potential is the same as bulk. Notice that narrower energy distributions with higher mean energy can have the same chemical potential as bulk. The narrowest distribution, with zero width and characterized by a delta function, should have mean energy equal to the bulk chemical potential to have the same chemical potential as bulk water. Deviations from the Gaussian distribution can change the average chemical potential of the water molecules. However, the accurate determination of higher moments and convergence of the cummulant expansion in moments of the distribution is tedious.39

Figure 5. Hydration of pocket A. (a) Trajectories of water inside the pocket. (b) Coordination number of bound water molecules. (c) Energetics of a water molecule upon hydration of the pocket.

426

The most common description of bound water includes instances in which the entrance of a water molecule inside a cavity may reduce the degrees of freedom of the molecule, leading to a possible entropic cost in free energy. However, the degree of immobilization of water molecules can be lower when clusters are formed. The decrease in entropy may be as large as 2 27 cal/mol-K for a fixed molecule.40 In some instances, however, water molecules at the MHC – peptide interface can have higher entropy than in bulk. Molecules in bulk have limited freedom due to their participation in a water network, while water molecules inside a slightly non-polar cavity may have more freedom than in bulk, resulting in higher entropy. If this entropy is balanced by the energy of interactions with protein/peptide atoms or by other water molecules in a small cluster, the chemical potential of the molecule can be lower than the bulk value. Water penetration events are thermally induced, and in some instances the penetration occurs as a high-energy fluctuation (with a positive relative chemical potential difference from bulk), and in other instances it is driven by a gain in free energy. In what follows we present a description of the energy distributions for water performing different tasks in the MHC pockets. The different tasks of water in the MHC pockets Six pockets have been defined in the class I MHC HLA-A2 binding groove (Figure 3).12 Some pockets form a well-defined cavity that can accommodate only one side-chain of the epitope, in others the boundary between the pockets is ambiguous.16 These regions vary between deep and shallow depressions in the protein –MHC interface. Water molecules can eventually penetrate to fill the spaces left by protein atoms. The shape and polarity (polar, charged or hydrophobic) of the pockets are the two factors that can make some peptide side-chains more suitable to fit in a pocket than others. In what follows, we describe the hydration structure and energetics of water molecules in the different pockets.

MHC –Peptide Binding

times. The site S1 is buried and occupied by the first water molecule entering the pocket (W14753), while site S2 is occupied by two water molecules. In both cases, water molecules occupy definite positions, and once bound, these water molecules do not exchange sites, and remain performing their tasks during the rest of the simulation. The site S3 is alternatively occupied by two water molecules. We see in the coordination-plot (Figure 5(b) (S3)) that these two water molecules take turns such that this task is always satisfied by either water molecule. Water molecules in sites S1 and S3 fulfil the same hydrogen bond requirements as water molecules identified in crystallography.12 These are the so-called conserved water molecules mentioned by Smith et al.13 to be present in pocket A, since they are bonded to conserved residues and have also been found in other MHC species.13 As we can see in Table 2, these water molecules form hydrogen bonds between the MHC conserved residues Tyr59, Tyr7 and Tyr171, and the peptide main-chain atom Ile N. Given that these water molecules are not bound to the peptide side-chain, but mediate between peptide backbone and MHC, there is no evidence they will make any contribution with regards to specificity for that particular amino acid, but will contribute to a stronger affinity in that region. In Figure 5(c) we plot the distribution of energy of water W14753, before (t1) and after the entrance of the other water molecules in the pocket, namely t2 and t3. We can see that the energy distributions are narrower than bulk and they drift to a lower mean energy, as the pocket becomes more populated by water molecules, indicating that the most favorable state of

Table 2. Pocket A Water molecule W14753

W7673

Pocket A Figure 5 shows the position of pocket A in the MHCP complex. Pocket A is highly polar and the residues forming the pocket are mostly conserved through many class I MHC alleles. Surrounding the rim we find residues Tyr59, Glu63, Lys66, Tyr99, Tyr159, Thr163 and Trp167. Residues Met5, Tyr171 and Tyr7 form a second layer of atoms around the pocket. This pocket is responsible for anchoring the first peptide amino acid residue ILE (P1), by means of strong hydrogen bonds between the MHC conserved residues and the peptide backbone atoms O and N (Table 1). There are three main sites for water in this pocket: S1, S2, S3 (Figure 5(a)). In our simulations five water molecules occupy this pocket with long residence

W11858 W16529 W16448

Hydrogen bond Tyr7 OH c Tyr59 OH c Glu63 OE1 v Ile376 N p Ile376 N p Tyr171 OH Tyr59 OH c Ile376 N p Wat7673 O Lys66 NZ v Glu63 OE2 v Lys66 NZ v

Probabilities

Mean distance ˚) (A

0.828 0.795 0.796 0.442 0.886 0.498 0.482 0.701 0.599 0.932 0.880 0.786

2.84 3.01 2.64 3.07 2.89 3.03 3.02 2.90 2.89 2.87 2.65 2.89

Hydrogen bonds between water–protein and water–water, mean-distance and probability of occurrence for each selected water molecule, averaged during their residence time inside the interface. Water molecules are highly dynamic in the interface, and fluctuations make them break and form several hydrogen bonds at the same time. Criteria for hydrogen bonds are to include only those whose mean distance between water oxygen and the ˚ and occur with a probability P . 0:4 heavy atom is less than 3.5 A during the water’s residence time inside the interface. (We use three labels to differentiate residues: c, completely conserved;12 v, variable;12 p, peptide).

427

MHC –Peptide Binding

Figure 6. Hydration of pocket B: (a) Trajectories and H-bonds of a bound water molecule inside the pocket. (b) Coordination number of bound water molecules. (c) Energetics of water molecules performing similar “tasks”.

W14753 involves the presence of additional water molecules in the interface. Pocket B Figure 6 shows the position of pocket B in the MHCP complex, the trajectory of a bound water molecule in this pocket, and its associated energy distribution. Pocket B is mainly hydrophobic (Val67, Phe9, Met45) surrounded by a polar rim that consists of residues: Glu63, Lys66, His70, Tyr99, Tyr77. During the simulation, three water molecules visit this site and stay bound for periods longer than 1.5 ns, suggesting that the hydrogen bond requirements of the site are quite strong (Figure 6(b)). This water site (containing water molecules W6941 – W34280 –W12101) is stabilized by three hydrogen bonds to His70, Lys66 and the peptide atom P2 Lys O (Table 2). Showing, once again, an example of water

mediation in the binding between the MHC and the peptide main chain. Given that Lys66 and His70 are variable residues, these water molecules belong to the category of semi-fixed.13 Because of

Table 3. Pocket B Water molecule

Hydrogen bond

Probabilities

Mean distance ˚) (A

W12101

Lys378 O p His70 ND1 v Lys66 O v Glu379 OE1 p Lys378 O p His70 ND1 v

0.784 0.627 0.544 0.406 0.701 0.612

2.84 3.12 3.23 2.70 2.87 3.12

W34280 W6941

Hydrogen bonds between water–protein, water–water, their mean-distance and probability of occurrence ðP . 0:4Þ; for each selected water molecule, during residence time inside the interface. Details in Table 2. We use two labels to differentiate residues: v, variable;12 p, peptide.

428

their stable hydrogen bonds to the polar atoms in the pocket and the peptide backbone, these water molecules directly contribute to the binding affinity of the peptide, and not merely occupy space in the interface. If we look into the energetics of these water molecules (Figure 6(c)), we find that the energy distributions of the first and last water molecules to visit (in blue, W6941 and W12101) are extremely similar, and higher in mean energy compared to bulk. W34280 visits for a short time and lingers in a neighboring position, and its

MHC –Peptide Binding

energy distribution (red) is different from the other two. The energy distribution histograms help to understand and differentiate these designated tasks of water, since molecules sharing a same task at different periods of time also have the same energy distribution (Table 3). Pockets C –E Figure 7 shows the position of pockets C – E in the MHCP complex. These pockets comprise the

Figure 7. Hydration of pocket C. (a) Trajectories of water inside the pocket. (b) Coordination number of bound water molecules. (c) Energetics of the cluster of water molecules inside the pocket.

429

MHC –Peptide Binding

Table 4. Pockets C – E Water molecule

Hydrogen bond

Probabilities

Mean distance ˚) (A

W20018

Arg97: NH1 v W33974 O W21722 O W35693 O W33974 O PRO380 O p W33974 O W33974 O

0.840 0.492 0.785 0.568 0.568 0.487 0.864 0.57

2.93 2.92 2.82 2.86 2.86 2.83 2.81 2.83

W33974 W35693 W21722 W27947

Hydrogen bonds between water–protein, water–water, their mean-distance and probability of occurrence ðP . 0:4Þ; for each selected water molecule during residence time inside the interface. (Details in Table 2). We use two labels to differentiate residues: v: variable,12 p: peptide.

center of the binding groove and they lie facing each other at both flanks of the peptide mainchain. Pocket C is located on the inner wall of the a1 domain opposite to pocket D that is located on the wall of the a2 domain. It is in this central region where the network of water molecules becomes more evident. As noticed by Smith et al. in HLA-B53,13 the residues in the floor of the MHC pocket provide a relatively polar surface. The peptide main chain does not have direct contact to these residues, but binding is enhanced by means of a network of hydrogen bonded water molecules that fills the space between the MHC and the peptide, and also helps keeping the peptide main chain in place. In Figure 7(a) we observe four water molecules that penetrate the cavity, one at a time, and adopt positions in a four-story

Figure 8. Hydration of pocket F. (a) Trajectories of water inside the pocket as an example of water mediating H-bonds and water filling empty spaces. (b) Coordination number of bound water molecules. (c) Energetics of three bound water molecules inside the pocket.

430

water wall in which they remain for the rest of the simulation. The first water molecule in this network is attached to the polar residues in the MHC floor, and the top water molecule is hydrogen bonded to the peptide central residue by its mainchain atom P5 Pro O (Table 4). As in pocket A, the fact that none of the water molecules in this cluster is bound to the peptide side-chain might show that this network does not contribute to the selectivity of central residues in the peptide, but accommodates to their given sequence enhancing its affinity. As shown by the decrease in their coordination numbers (Figure 7(b)), all these water molecules enter the cavity spontaneously and remain for the rest of the simulation, with the exception of W27947, which stays for only 1.5 ns and returns to bulk (black in Figure 7(b)). In our calculations we observe other transient water molecules, but they might not stay longer, given that the pocket capacity is full. To understand this phenomenon, we plot the mean energy distribution of the cluster of water (Figure 7(b)). When the water network has five members the mean energy distribution is still narrower than bulk, but most accessible states are higher in energy than those belonging to a cluster of four water molecules, which seems to suit better the volume of the pocket. As suggested by Ernst et al.,41 interior water molecules tend to form clusters because this reduces their free energy relative to isolated solvent molecules. The addition of a fifth water molecule to the pocket does not improve the energy of the system and prevents W27947 from staying in the cavity. Pocket F Figure 8 shows the position of pocket F in the MHCP complex, the trajectory of bound water molecules in pocket F, and their coordination numbers and energy distributions. Pocket F defines the other end of the cleft where the peptide C terminus is bound. It comprises residues Thr80, Tyr84, Thr143, Trp147, Asp77, Tyr116 on the rim. Inside the cavity we find residues Leu81, Tyr123 and Tyr116. In the rim of this pocket we observe two sites for water (Figure 8(a)) that are exposed to the solvent. The first site (orange) is hydrogen bonded to Asp77 OD1. W15410 lives in this site from the beginning of the simulation. Because of its hydrogen bonds, this water molecule can be identified with W944 found in the crystal structure.12 This water site is an example of a semi-fixed water performing the task of filing space. This water molecule does not provide hydrogen bonds to the peptide atoms but it is precisely located where the peptide bears a short side-chain (P8 Gly). Other peptides bound to HLA-A2 exhibit different sidechains (Thr, Ser and Tyr) in this position.12 In the first two cases these residues have two relatively short side-chains with polar OH groups that would take the place of W15410. This water site is an example of water filling the space that, otherwise, could have been occupied by a longer side-

MHC –Peptide Binding

chain with polar atoms. In the peptide that has a Tyr in this position the crystal structure shows that this longer side-chain requires a different arrangement of atoms. The histogram corresponding to W15410 (Figure 8(c)) shows an energy distribution which is much narrower than bulk, and drifted to lower energy. This shows that the most populated states in this site have an enthalpic gain. The second water site is occupied, alternatively, by two different water molecules (W15470, and W35669), which do not share the site simultaneously but satisfy the same hydrogen bond requirements at different times of the simulation, for periods of about 2.5 ns each. These water molecules clearly contribute to the binding energy of the peptide because they are closely bound simultaneously to both Asp77 and the backbone O of the peptide terminal residue Val, without any interaction with the peptide side-chain. The energy distributions associated with these molecules (Figure 8(c)) are very similar in width and mean value, which is reasonable given that they satisfy the same hydrogen bond contacts. At the same time, the energy distributions show that all populated states are energetically more favorable in energy than bulk; which explains the fact that there is always a water molecule occupying that specific site, and staying bound for an extended residence time (Table 5).

Table 5. Pocket F Water molecule

Hydrogen bond

Probabilities

Mean distance ˚) (A

W15410

Thr73 O v Asp77 OD1 v Asp77 OD1 v Val384 O p Thr80 OG1 v W15410 O Asp77 OD1 v Thr80 OG1 v Val384 O p Val384 N p

0.933 0.992 0.948 0.928 0.804 0.448 0.926 0.915 0.976 0.792

2.88 2.70 2.76 2.78 2.88 3.24 2.79 2.88 2.78 3.84

W15470

W35669

Hydrogen bonds between water–protein, water–water, their mean distance and probability of occurrence ðP . 0:4Þ; for each selected water molecule, during residence time inside the interface. See details in Table 2. We use two labels to differentiate residues: v, variable;12 p, peptide.

Bound water molecules and free energy gain Figure 9 shows contour plots of the chemical potential for water molecules in the different MHC pockets. The chemical potential has been estimated according to equation (1) (Methods: Gaussian model of free energy), for each of the energy distributions (shown in Figures 5– 8) of bound water molecules and clusters. Highlighted in black is the chemical potential of bulk water mex B . Two orange curves show changes lying in the

MHC –Peptide Binding

431

Figure 9. Relation between width (s), mean energy ðEÞ and chemical potential (m) corresponding to Gaussian distributions of water molecules in MHC pockets A, B, C, F. Water molecules in the interface have lower chemical potential than water in bulk. (a) Pocket A: time history of water W14753, periods of time t1, t2, t3 correspond to the entrance of two other water molecules in the pocket. The chemical potential of W14753 is lowered as the pocket is hydrated. (b) Pocket B: comparison between water molecules that occupy the same position for a short time (W34280) or for longer times (W6941 –W12101). W34280 has a free energy similar to bulk and its penetration in the interface could be attributed only to thermal fluctuations. W W6941 –W12101 are stabilized in the interface. (c) Pocket C: chemical potential of the cluster of water-molecules in the interface is lower than bulk when the pocket is populated by four molecules rather than five. (d) Pocket F: chemical potential of water molecules in positions S1 and S2 is similar.

range of energies accessible through thermal fluctuations: mex B ^ kT; and serve as reference curves that will indicate whether water molecules would gain or lose significant free energy by penetrating in the MHC – peptide interface. Each MHC pocket shows a different feature of the role of water in the MHC –peptide interface. In pocket A, we observe the decrease of free energy of a water molecule as the pocket is hydrated by other water molecules through consequent times t1, t2, t3. This shows how a water molecule that entered by fluctuations

of the system can be later stabilized in its position by the formation of a water cluster inside the cavity of the pocket. As the system evolves to its equilibrium state, W14753 becomes deeply buried, with a favorable free energy gain. This water occupies the position of conserved water molecule. In pocket B, we graph the chemical potentials of different water molecules that alternate in the same task at different times. W34280 stays for a short time in the interface and its penetration and exit are due to fluctuations of the system; its

432

chemical potential is within the range of thermal fluctuations around bulk free energy. However, W6941 and W12101 stay for a longer time period and their chemical potentials are similar and clearly lower than bulk. In the center of the groove, (pockets C – F), we observe how the system rejects the addition of a certain water molecule, when the capacity of the pocket is full. By incrementing the number of water molecules to five, the chemical potential of the cluster increases. Therefore, an increase of free energy in the system explains why W27947 does not stay bound in the pocket, and returns to bulk solvent. In pocket F, there are two sites for bound water molecules: S1 and S2. S1 is occupied by one water molecule during the whole simulation. S2 is occupied, alternatively, by two water molecules performing the same task, and their chemical potentials are similar. Both S1 and S2 have very low chemical potential, which can account for the water molecules staying strongly bound for very long times.

Conclusions We have shown that water molecules take definite positions in the peptide –MHC interface, with permanence of at least 1 ns or longer. Water molecules that occupy these positions perform specific tasks in the MHC – peptide interaction, such as filling spaces or making hydrogen bonds between the MHC floor and the epitope. These tasks are achieved alone or forming a hydrogen bond network of solvent molecules, according to the demand of space inside the cavity. Some water molecules are bound to conserved MHC residues, such as water molecules in pocket A and F, that bind to the terminal main-chain atoms of the peptide, enhancing the binding affinity without restricting the interaction to a particular side-chain. Other water molecules, such as W15410, fill spaces where there are shorter side-chains in the peptide. By filling empty spaces, water molecules add variability to the MHC surface, enabling the binding of a wider range of sequences. The role of water in the task of filling spaces was experimentally observed19 in cases where a peptide residue was mutated for a shorter side-chain, and a bound water molecule was observed in the expanded cavity, affecting the affinity of T-cell recognition. A third group of water molecules, such as those found in pockets C – D, take part in a network that connects the polar floor of the central region of the cleft with the main-chain atoms of the peptide. Their role is to hold in place the most exposed, variable region of the peptide. The mechanism which enables the promiscuity and high affinity of the MHC, can be summarized in the following: plasticity of the empty MHC pocket when the peptide is not bound, alternate packing of MHC residues and MHC hydrogen bonding to the peptide main-chain atoms. Our work presents evidence that water is a relevant agent in all these

MHC –Peptide Binding

mechanisms. Interfacial water molecules reduce its free energy and take an active role in the MHC – peptide binding. In some instances the reduction in free energy is entropy driven and the entropy of the bound water increases. This role of water is such that it enhances the affinity, without contributing to peptide selectivity.

Methods Description of the system and simulations Two separate simulations were performed in this work. One simulation (MHCP) involves the MHC – HLA2 complex (residues 1 – 375) bound to an HIVreverse transcriptase peptide (residues 376– 384). A second simulation (MHCNP) was carried out without the bound peptide. The initial conformation was a crys˚ resolution, retrieved from the tal structure with 2.6 A Brookhaven Protein Data Bank (pdb 1HHJ).15 Corresponding residues were removed for the peptide-free simulation (MHCNP). We used the all-atom force field of Cornell et al.42 Electrostatic interactions were modelled with the particle mesh Ewald (PME)43 algorithm ˚ was used implemented in Amber6.44 A cut-off of 10 A for non-bonded and real space electrostatic interactions. The Fourier space part of the Ewald potential was calculated on a grid of 72, 96 and 72 points on each side and interpolated over all space with a cubic spline. We simulated the system at constant N, P ¼ 1 atm, T ¼ 300 K, using a weak coupling to an external bath.45 Both simulations were subject to 500 minimization cycles, followed by 5.0 ns of molecular dynamics simulation. The integration step was 0.002 ps. The last 4 ns are included as the production run. Configurations are saved for subsequent analysis at a rate of one per picosecond of simulation. Water molecules present in the crystal structure were not included in the initial configuration. The total charge of the protein in the oxidized state is 28E: We included 54 Naþ and 46 Cl2 in the system, to neutralize the total charge in the protein and model a 0.1 M excess salt concentration. Ions were located at random sites ˚ £ 94 A ˚ £ 73 A ˚ 3. The box within a rectangular box of 73 A was solvated using TIP3P46 explicit water solvent. Water molecules filled up cavities in the protein, provided that the distance between atoms and the closest solute atom is less than the sum of the atoms’ van der Waals distances. There are 10,019 water molecules for the system with the bound peptide (MHCP) and 10,044 for the control system (without peptide, MHCNP). Protein atoms add up to 6132 (peptide-bound system), 5984 (peptidefree system). The resulting systems contain a total of 36,289 atoms (with peptide), 36,216 atoms (without peptide). Histidine residues modeled as neutral residues with hydrogen atoms at the delta position are: A3, A70, A74, A93, A114, A145, A188, A192, A197, B13, B51, C7, where A, B, C correspond to the HC, b-microglobulin and peptide, respectively. Histidine A-263 was modeled as charged with hydrogen atoms at the delta and eta positions, because of the closeness to acceptors in Asp183, Tyr209 and Glu212 in chain A. There are six S– S bonded cysteine residues which are kept during the simulation: A101 –A164, A203– A259, B25– B80. The RMS distance between the crystal structure and the final con˚ for MHCP and 2.82 A ˚ for MHCNP. figuration is 2.84 A During the simulation the peptide remained tightly bound to the MHC complex. The rmsd fluctuations of

433

MHC –Peptide Binding

˚ , with rmsd the peptide Ca atoms are smaller than 0.5 A ˚ , 0.31 A ˚ , 0.31 A ˚ , 0.37 A ˚ , 0.44 A ˚, fluctuations of 0.46 A ˚ , 0.43 A ˚ , 0.46 A ˚ , and 0.45 A ˚ , for positions P1– P9, 0.39 A respectively. The overall MSD displacements of the peptide relative to other regions of the protein are very small, as seen in Figure 2 (the peptide consists of amino acid residues 376– 384). Principal component analysis Principal component analysis provides information about the collective-motions of atoms in the protein, by means of a set of coordinates and directions that best represent (in a least-square sense) the fluctuations in the set of configurations recorded during the simulation. The construction of these coordinates has been described37,47 and is presented as an eigenvalue P equation, solved for the average configuration y0 ¼ ð1=NÞ Si¼1 ri ; (S is the total number of configurations ri ) and a set of ~ The l values are eigenvalues l and eigenvectors m: ranked by decreasing order of magnitude and the corre~ account for a different direction sponding 3N-vectors {m} of displacement from y0 : The mean-square fluctuations P of each atom are computed as rmsd ¼ ð1=NÞ i¼1 li where the sum is performed over all eigenvalues. In our work, we restrict our analysis to the motion of the Ca atoms, recorded in the last 4 ns of our simulation. Instantaneous water coordination number To identify water molecules that are bound to the protein surface or interior we analyze the instantaneous coordination of water. This number is obtained for every saved configuration by counting all water mole˚ from the water oxygen cules within a range of 3.5 A atom, which defines the first hydration shell of water.48 Water molecules in bulk have four to six water molecules in their first hydration shell. However, the instantaneous coordination number covers a range of 0 – 12 coordinated neighbors per water molecule, having its peak in Nc ¼ 5 for bulk water. In bulk solvent it is rare to find isolated water molecules for which their coordination number remains small for an extended period of time (2– 5 ps). In the presence of the protein the occurrence of water molecules isolated from other water molecules increases and the distribution of coordination number for water changes for Nc # 3. This way, water molecules presenting persistent Nc # 3 are regarded as protein-bound or interior water. Nevertheless, this description bears some limitations, and has to be reformulated for large cavities that accommodate major solvent clusters.48,49 In this work, we trace solvent molecules involved in the interface between MHC and bound peptide by following the time-history of their coordination number. In this way, we can identify water penetration and escape events. Interaction energy distribution for water molecules in the interface We characterize the energy of interaction of identified bound water molecules in the interface between MHC complex and peptide. We construct histograms of the energy of interaction between each of these selected water molecules and the rest of the system, for saved configurations of the simulation. The interaction energy of water was calculated using the Lennard – Jones potential and the Ewald method to solve the long-range Coulomb potential for all atoms in a box that satisfies

periodic boundary conditions. We set the convergence factor kappa to a value of 6.5 units of the box length, and use 750 k-vectors.50,51 The Lennard – Jones potential was computed using the Lennard – Jones coefficients provided by the Cornell et al. force field.42 The energy of these molecules fluctuates largely between 2 30 kcal and 0 kcal, due to their rotations and the different bonds that molecules establish with the surrounding atoms in the cavity. This calculation was also carried out in a cubic box of TIP3P water yielding an energy distribution similar to the one obtained by Hummer et al.39 Randomly chosen water molecules in the bulk phase of the system present similar bulk distributions. In the same way, we calculated energy distributions rbin ðuÞ for water molecules with relevant residence times inside the interface (t . 1 ns) and compared these distributions with those of bulk water in our system. Gaussian model of free energy The excess chemical potential of a water molecule is defined as mexc ¼ m0exc þ Dmexc, where m0exc is the free energy of inserting a hard sphere into a cavity and Dmexc is directly related39 to the distribution rbin ðuÞ of binding energies of that individual water molecules by: ð ð1Þ ebDmexc ¼ rbin ðuÞebu du The binding energy rbin ðuÞ is the potential energy difference of the system in a given configuration with and without that molecule, b ¼ 1=kB T; T is the temperature, and kB is the Boltzmann constant. The chemical potential defined in equation (1) can be expanded into a cummulant expansion in the moments of the energy distribution, rbin ðuÞ:52 In this expansion the chemical potential is given by: X mexc ¼ bn21 cn ð2Þ n

where cn is the nth cummulant. The first four cummulants are c1 ¼ kul; c2 ¼ kðu 2 kulÞ2 l ¼ s2 ; c3 ¼ kðu 2 kulÞ3 l ¼ s3 skewðuÞ; and c4 ¼ kðu 2 kulÞ4 l 2 3s4 ¼ 4 s kurtðuÞ: The water interaction energy distributions can be well approximated by Gaussian distributions, meaning that only the first two moments (the average, c1 ¼ kul and the width, c2 ¼ kðu 2 kulÞ2 l ¼ s2 ; of the distribution are needed. For a Gaussian distribution all cummulants of order higher than 2 are exactly zero. Higher-order corrections contribute to the free energy, but these are not easily obtained in nanosecond time simulations for complex systems. Likely modifications to the free energy can come from the skewness of the distribution (third moment) and from the kurtosis (fourth moment). Within the Gaussian approximation the excess chemical potential Dmexc depends on the width and mean-value of this distribution, and is given by: Dmexc ¼ m0exc þ kul þ

kðu 2 kulÞ2 l 2kB T

ð3Þ

To calculate the free energy of inserting a hard sphere in bulk water, m0exc ; it is reasonable to take a value for ˚ 53 which yields a the hard sphere diameter of s , 2.7 A value for m0exc of approximately 4 kcal/mol. Using formula (3) and this estimated value for m0exc ; we obtain mexc , 2 5.3 kcal/mol, which compares well with the chemical potential for TIP3P water obtained from combined histograms and particle insertion calculations:

434 mexc ¼ 2 6.07(^0.02) kcal/mol.39 Our bulk energy distributions is almost identical to that reported in Hummer et al.39 and the difference in chemical potential may arise from non-Gaussian contributions, omitted in this approximation, and from the asymmetry of inserting and extracting water molecules from bulk. Due to the ,1 kcal/mol energy difference obtained with the Gaussian approximation we take this as a typical error in our calculated free energy values. In the same way, we approximate the energy distributions of bound water molecules or clusters with a Gaussian function, and compare the Dmexc obtained for these selected water molecules to that estimated for bulk. This simple model helps to interpret the water energy distributions and explain water penetration in the interface as a process driven by free energy gain.

MHC –Peptide Binding

11.

12.

13.

14.

Acknowledgements This work was supported by the US Department of Energy under contract W-740-ENG-36 and the Los Alamos Directed Research and Development Program. The authors thank Dr Brian M. Baker, Dr Lawrence R. Pratt and Dr Catherine Macken for insightful comments.

15.

16.

17.

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Edited by I. Wilson (Received 17 July 2003; received in revised form 2 February 2004; accepted 2 February 2004)