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. Author manuscript; available in PMC: 2007 Oct 29.
Published in final edited form as: Structure. 2007 Feb;15(2):225–233. doi: 10.1016/j.str.2007.01.006

Hydrophobic Sliding: A Possible Mechanism for Drug Resistance in Human Immunodeficiency Virus Type 1 Protease

Jennifer E Foulkes-Murzycki 1,†, Walter Robert Peter Scott 2,†, Celia A Schiffer 1,*
PMCID: PMC2044563  NIHMSID: NIHMS18427  PMID: 17292840

Summary

Hydrophobic residues outside the active site of HIV-1 protease frequently mutate in patients undergoing protease inhibitor therapy, however, the mechanism by which these mutations confer drug resistance is not understood. From analysis of molecular dynamics simulations, 19 core hydrophobic residues appear to facilitate the conformational changes that occur in HIV-1 protease. The hydrophobic core residues slide by each other, exchanging one hydrophobic van der Waal contact for another, with little energy penalty, while maintaining many structurally important hydrogen bonds. Such hydrophobic sliding may represent a general mechanism by which proteins undergo conformational changes. Mutation of these residues in HIV-1 protease would alter the packing of the hydrophobic core, affecting the conformational flexibility of the protease, thereby impacting the dynamic balance between processing substrates and binding inhibitors thus contributing to drug resistance.

Keywords: HIV protease, molecular dynamics, conformational changes, drug resistance

Introduction

Human Immunodeficiency Virus type 1 (HIV-1) protease is a symmetric homodimeric aspartyl protease that cleaves a series of ten non-homologous and asymmetric substrate sites in the Gag and Gag-Pro-Pol polyproteins allowing the virus to mature and become infectious. In the liganded conformation of HIV-1 protease, two flaps close over the active site, thereby encompassing the ligand (Figure 1b). A crystal structure of the unliganded protease (Spinelli et al., 1991) shows the flaps of the enzyme extended, yet still touching each other at Ile50 (Figure 1a). However, this conformation is possibly stabilized by a crystal contact from a symmetry related molecule (Scott and Schiffer, 2000; Spinelli et al., 1991). While it is clear that these flaps must separate for a substrate to access the active site, the method by which the substrate gains access to the active site remains unclear. Numerous studies have explored the movement of the flap region (Bandyopadhyay and Meher, 2006; Collins et al., 1995; Freedberg et al., 2002; Harte et al., 1992; Hornak et al., 2006; Ishima et al., 1999; Katoh et al., 2003; Meagher and Carlson, 2005; Perryman et al., 2004; Perryman et al., 2006; Scott and Schiffer, 2000; Toth and Borics, 2006a; Toth and Borics, 2006b) but few have explored the role of domains outside the active site and flap region in substrate binding (Ishima et al., 2001b; Muzammil et al., 2003; Ohtaka et al., 2003; Perryman et al., 2004; Perryman et al., 2006; Rose et al., 1995; Zoete et al., 2002). Many residues outside the active site and flap region are often associated with drug resistance and, somehow have a role in ligand binding. This role may be to facilitate the conformational changes that occur in HIV-1 protease to allow ligand binding.

Figure 1.

Figure 1

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Conservation of hydrophobic residues. Only the hydrophobic residues in HIV-1 protease are displayed. Residues colored red are completely conserved, those colored blue only mutate to other hydrophobic residues, and cyan colored residues mutate to hydrophilic residues. Yellow circles are used to highlight Leu33. a. Unliganded crystal structure of HIV-1 protease (PDB: 1HHP). b. Liganded crystal structure of protease (PDB: 1F7A)

Each monomer of HIV-1 protease is made up of 99 residues, 40 of which are hydrophobic (Figure 1). Some of these hydrophobic residues line the active site of the protease making close van der Waal contacts with the ligand, while others are in the flap region. A third category of residues encompasses the hydrophobic core within each monomer of the protease. Within the set of core residues are seven isoleucines, whose side chains can adopt a large number of conformations (Ishima et al., 2001a; Ishima et al., 2001b). As these isoleucines change conformations, they could initiate conformational changes in the protease through interdependent exchanges of van der Waal contacts. Many residues within the hydrophobic core are associated with drug resistance (Johnson et al., 2004; Rhee et al., 2005). For example, Leu33Phe is a secondary mutation for all other inhibitors except saquinavir and nelfinavir (Johnson et al., 2005). Protein crystal structures show that this residue is not in contact with either substrates or inhibitors, but is packed deeply into the hydrophobic core (Figure 1). Therefore its role in drug resistance is not obvious. The role of other such core residues in the development of drug resistance is similarly unclear.

We hypothesize that the hydrophobic residues within the core, such as Leu33, facilitate the large conformational change between liganded and unliganded conformations of the protease as one hydrophobic surface slides by another with a minimal energetic expense. Through an analysis of molecular dynamics (MD) simulations, we found 19 residues in the core region of HIV-1 protease that had limited solvent accessibility throughout the simulation. Further analysis of these MD simulations revealed that these hydrophobic core residues facilitate the opening of the active site cavity to allow for substrate binding. Mutations in the core likely affect the dynamic properties of HIV-1 protease and potentially affect the ability of protease to bind inhibitors and substrates. Therefore, mutations in this region that preferentially affect inhibitor binding over substrate binding may explain the role of these residues in the development of drug resistance.

Results

Obtaining an atomic view of the concerted movements of a protein is not easily achievable by any experimental technique. Therefore, solvated molecular dynamics simulations were performed to observe the conformational flexibility of HIV-1 protease under three different starting conditions. Two simulations were started from the crystal structure of a substrate complex of HIV-1 protease (PDB entry code 1F7A) (Prabu-Jeyabalan et al., 2000; Prabu-Jeyabalan et al., 2002), one with the substrate (MDSUB) and the other without the substrate (MDNOSUB). These simulations were run for 5 nanoseconds and appeared to have reached a local minimum. The other simulation, MD1HHP, was started from the unliganded crystal structure (PDB entry code 1HHP). This simulation was performed for 9 nanoseconds. Though MDNOSUB and MD1HHP were both started from unliganded structures, the protease in MDNOSUB was in the closed conformation, while the protease in MD1HHP was in the semi-open conformation. While MDNOSUB had slightly larger fluctuations in peripheral regions of the protein relative to MDSUB, the flexibility of the protease in MD1HHP was much greater than either of the two simulations started from the substrate bound coordinates in the time period sampled (Figure 2). The closed conformation without substrate (MDNOSUB) appears to be an energetically favorable conformation and did not substantially rearrange in the timescale of this simulation. The flap region in MD1HHP and the amino terminus of protease in MDSUB had the largest fluctuations, consistent with what has been seen by NMR (Freedberg et al., 2002; Hong et al., 2000; Ishima et al., 1999; Ishima et al., 2001a; Todd et al., 2000). Based on the correlation between MD1HHP, MDSUB and NMR results, we concluded that the force field is reasonable and the conformations sampled in the MD1HHP are not likely an artifact of the simulation. To further examine the significant changes observed in MD1HHP, this simulation was continued for another 5 nanoseconds using the AMBER8 software package (Figure 3). By examining the protease in two force fields we determined that the changes observed were not an artifact of the GROMOS simulation. Additionally, because the two monomers that started in the same conformation underwent different conformational changes, we were able to examine two possible pathways for protease dynamics. In order to determine what was causing these changes, we began a detailed analysis of atomic interactions in these simulations.

Figure 2.

Figure 2

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Relative fluctuations (Å) of the alpha-carbon atoms during 1 – 5 ns. The fluctuations of each structure relative to the starting coordinates were mapped onto a ribbon diagram of each structure using coordinates from the simulations. The backbone is colored based on the degree of fluctuations. Residues colored blue had greater than 0.4 Å fluctuations, those colored purple greater than 0.55 Å fluctuations, magenta 0.7 Å, orange 0.85 Å, and yellow greater than 1 Å fluctuations. a. MDSUB. b. MDNOSUB. c. MD1HHP.

Figure 3.

Figure 3

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Snapshots of HIV-1 protease during the MD1HHP simulation. The catalytic aspartic acids are displayed and colored black. Residues 11–22 are colored magenta, 31–38 colored red, 39–57 colored blue, 58–78 colored cyan. 1–10, 22–24, 25–32, and 79–99 are colored grey. a. Starting coordinates. b. After 9 ns in the GROMOS96 force field. c. After an additional 5 ns in the AMBER force field.

Maintenance of hydrogen bonds provides structural stability

As a protein undergoes conformational changes it might be possible for one set of hydrogen bonds to be exchanged with another, or with solvent, with very little energy cost to the protein, as long as the total number of bonds remains constant. However this is not what is observed in the simulations where conserved hydrogen bonds appeared to stabilize the secondary structure. Throughout the MDSUB and MDNOSUB simulations, and the first 5 ns of the MD1HHP simulation, any hydrogen bonds that existed were tracked using geometrical considerations of distance and angle between the donor and acceptor atoms over time. In Figure 4 all the hydrogen bonds that were both formed greater than 50% of the time between the first and fifth nanoseconds of the trajectory and found in all three simulations are shown. Despite the increased fluctuations of the protease in MD1HHP, many hydrogen bonds were still maintained, although for a slightly smaller percentage of the time than in the other simulations. Most of these hydrogen bonds were among backbone atoms. In fact, the bonds appeared to maintain the secondary structure of the protease as the enzyme undergoes conformational changes.

Figure 4.

Figure 4

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Hydrogen bonds maintain the structure of HIV-1 protease. Hydrogen bonds are colored based on the percentage of time they existed. Bonds colored black existed for more than 80% of the time, those colored green for more than 60% of the time, and yellow for more than 50% of the time. Nitrogen atoms are colored blue, and oxygen atoms are colored red. Side chains involved in conserved hydrogen bonds are displayed and colored cyan. These include Thr26, Thr31, His69, Asn83, and Asn88. a. Liganded HIV-1 protease simulated with the substrate bound. The grey box represents the area displayed in part d. b. Liganded HIV-1 protease simulated with the substrate removed. c. Unliganded HIV-1 protease. d. Hydrogen bonds involving residues Thr31, Asn83, and Asn88. The carbon atoms in these residues are colored magenta. The residues of the hydrophobic core are also shown with van der Waal spheres.

In order to compare the simulations with experimentally determined structural information, six crystal structures of HIV-1 protease bound to substrates were examined for side chain hydrogen bonds that were conserved between the structures (Prabu-Jeyabalan et al., 2000; Prabu-Jeyabalan et al., 2002). Only five side chains in each monomer maintained conserved hydrogen bonds among these structures and these same side chains maintained hydrogen bonds in all three simulations more than 50% of the time in both monomers. These five residues, Asp/Asn25, Thr26, Thr31, Asn83 and Asn88, are highly conserved among viral isolates. Only Asn88 mutates 9% of the time, usually to aspartic acid or serine, under the pressure of inhibitor therapy (Shafer et al., 1999a). Two of the residues, the catalytic Asp25 and Thr26 (which is involved in a "fireman's grip" with itself on the dimer interface), have been extensively studied by other groups (States et al., 1982; Wallqvist et al., 1998). The other three residues, Thr31, Asn83, and Asn88, form a network of conserved side chain hydrogen bonds that effectively cap off the hydrophobic residues within the core of each monomer from the solvent environment (Figure 4d) and are likely essential to the structural integrity of the protease.

Residues within the hydrophobic core

While many significant hydrogen bonds were maintained throughout the simulations, the unliganded protease undergoes a considerable conformational change in MD1HHP. In order to determine the mechanism of this change, we thoroughly examined all the inter-residue contacts in the MD1HHP simulation. The examination of this simulation revealed that hydrophobic residues in the core of protease exchange contacts with each other and that these exchanges may lead to the conformational changes observed. To determine which residues comprised the hydrophobic core, a detailed structural analysis was performed at every 0.5 ns in the MD1HHP simulation to look for residues whose sidechains were buried for the majority of the simulation. This analysis revealed nineteen hydrophobic residues with especially limited solvent accessibility. These residues, along with their mutational frequencies in patient sequences, are shown in Table 1. There are seven isoleucines in the core of each monomer. Isoleucine, whose sidechain has more possible conformations than leucine or valine, appears to provide increased flexibility to the monomer. As the isoleucine sidechains rotated through different conformations, they facilitated dramatic changes in the hydrophobic core observed in these simulations. Five of these isoleucines mutate in the patient population, regardless of inhibitor therapy (Table 1). However, they only mutate to other hydrophobic residues, most frequently valine. This implies that maintaining hydrophobic interactions in this core is essential to HIV-1 protease function. In order to examine why these hydrophobic interactions are important we examined the changes in the hydrophobic core over time using MD simulations.

Table 1.

Frequency of mutation of the residues in the hydrophobic core among untreated and treated patients (Johnson et al., 2005; Rhee et al., 2005; Shafer et al., 1999b). Numbers are listed as the percentage of sequences that had another amino acid at this site. Those listed in red cause drug resistance, those in blue contribute to drug resistance, and those in bold italics are invariant.

Position 5 11 13 15 22 24 33 36 38 62 64 66 75 77 85 89 90 93 97
Wild Type Amino Acid L V I I A L L M L I I I V V I L L I L
Untreated Patients (%) 0 0 38 24 0 0 2 55 0 12 15 0 0 15 0 47 0 30 0
Treated Patients (%) 0 0 27 23 0 5 6 43 0 26 21 1 0 26 3 18 31 36 0
Most Frequent Mutation - - V V - I F I - V V F - I V M M L -
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Rearrangement occurs within the hydrophobic cores

The conformational changes of the protease in the MD1HHP simulation included a variety of changes throughout the protein. In order to aid in the analysis and description of these movements, four loops were highlighted in different colors in Figures 3 and 5. The flap region, which consists of residues 39–57, was colored blue. This region is not part of the hydrophobic core, though it is attached at either end to loops involved in the core. Just before the flap region is a short loop comprising residues 31–38 (red). A larger loop made up of residues 58–78 (cyan), stems from the other end of the flap region. The last loop consists of residues 11–21 (magenta). The two monomers, which were in the same conformation at the start of the simulation, rearranged differently indicating that more than one physically reasonable conformation of the protease was accessible. Within the first 9 ns, the flap region slid back and the tips of each flap curled into each active site. This coincided with a significant rearrangement of the hydrophobic core as the other three loops all dropped relative to their starting positions (Figure 3b). Additional changes were observed when the simulation was continued for an additional 5 ns in the AMBER simulation package. By examining the changes in atomic interactions within each monomer, we can determine how these conformational changes within the protease occur.

Figure 5.

Figure 5

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Stereo images of the hydrophobic core during the MD1HHP simulation. Residues 11–22 are colored magenta, 31–38 colored red, 39–57 colored blue, 58–78 colored cyan. 1–10, 22–24, 25–32, and 79–99 are colored grey. a. Unliganded HIV-1 protease at the start of the simulation when both monomers are in the same conformation. The same image is shown above both columns. b. Monomer A after 9 ns. c. Monomer B after 9 ns. d. Monomer A after 14 ns. e. Monomer B after 14 ns.

To compare the differences in conformation between monomers that occur during the simulation it is easiest to focus on changes relative to a single residue. Ile15 in the magenta loop is highlighted in Figure 5, which uses stereo images to illustrate the changes in each monomer at 9 and 14 ns relative to the starting positions. In the unliganded crystal structure Ile15 made van der Waal contacts with Leu33, Met36, and Leu38 in the red loop, and Ile62, Ile64, and Val75 in the cyan loop (Figure 5a). After 9 ns of molecular dynamics, however, this was no longer the case and the two monomers adopt very different conformations. In one monomer, residues Leu33 and Leu38 in the red loop slid over Ile15, increasing the contact between Leu38 and Ile15 (Figure 5b). Simultaneously, the cyan loop slid downward as Ile15 exchanged contacts with Ile64 and Val75 for contacts with Ile62 and Val77. The correlated movements between the residues in the red and cyan loops, which are attached to either end of the flap region, facilitate the movement of the flap region from the active site (Figure 3b). While correlated movements of the red and cyan loops occurred in the second monomer, changes in the red loop revolved around Ile13 instead of Ile15. In the second monomer residues Leu33 and Leu38 packed around Ile13, which left Ile15 partially solvated (Figure 5c). The contacts that were made by Ile15 with the cyan loop were split between Ile13 and Ile15 in this monomer. On one side of the cyan loop, Ile13 gained the contacts lost by Ile15 with Val75. On the other side of this loop, Ile15 maintained contact with Ile62 and Ile64. In this way, the red and cyan loops facilitated the movement of the flap region from the active site over the first 9 ns.

Additional changes in both monomers were observed after continuing the simulation for 5 ns in another force field. In the first monomer, the flap region appeared to be moving back toward the active site. This change was facilitated by the upward movement of the cyan loop as Val75 slid over Ile15 (Figure 5d). In the second monomer, Ile15 moved back into the hydrophobic core displacing Ile13 as Leu38 slid over Ile15 (Figure 5e). The red and cyan loops in this monomer moved together through the direct contact between Leu38 and Ile62, pulling the flap further from the active site. The movements of these residues as they alter side chain conformations led to correlated movements of the various loops in protease. These changes are likely to be physically reasonable as they are primarily rearrangements of the hydrophobic residues and there should be very little energetic penalty for exchanging one set of hydrophobic van der Waal contacts for another.

Discussion

HIV-1 protease undergoes a large conformational change in order for substrates (or inhibitors) to access the active site. While the mechanism by which the flaps come apart has been widely examined, in this study we have shown that an extensive rearrangement of the core region may facilitate conformational changes in protease. This rearrangement was not due to the exchange of inter-residue hydrogen bonds in the core. As shown in Figure 4, many hydrogen bonds were conserved between MD1HHP and the less flexible simulations MDSUB and MDNOSUB. The backbone hydrogen bonds appeared to stabilize the structure, while some of the side chain hydrogen bonds protected the hydrophobic core from the solvent (Figure 4d). Further examination of the MD1HHP simulation revealed that the mechanism of the observed conformational changes was the exchange of van der Waal contacts within the hydrophobic core.

Nineteen residues from each monomer were found to comprise the hydrophobic core. Seven of the nineteen residues in the core are isoleucines (Table 1), which have more possible conformations than other hydrophobic residues such as phenylalanine. The rotation of these isoleucines through various rotamers assists in the sliding of these residues over one another. As demonstrated in Figures 3 and 5, the exchange of van der Waal contacts between residues in the hydrophobic core is correlated with dynamic changes in the protease structure. Many sidechains within the hydrophobic core also have been shown by NMR to have significant internal motions on the sub-nanosecond timescale (Ishima et al., 2001b). When any of the residues within this core region mutate, they only mutate to other hydrophobic residues (Figure 1). This implies that maintaining these hydrophobic contacts is important to the protease structure or function. Based on these observations, we propose that the conservation of hydrophobic residues is required for the hydrophobic sliding that allows the conformational changes required for protease function.

Of the 19 residues in the hydrophobic core, 6 are invariant (< 1% mutation rate) among either treated or untreated patients (Table 1) (Shafer et al., 1999a). Two of these invariant residues, Leu5 and Leu97, are at the structurally conserved dimer interface, and the sensitivity of these residues to mutation has already been shown (Choudhury et al., 2003; Loeb et al., 1989). The other 4 invariant residues are Val11, Ala22, Leu38, and Val75. Val11 and Ala22 do not interact with each other or with any of the other invariant residues but are in constant contact with other highly variable residues in the hydrophobic core. The remaining two invariant residues, Leu38 and Val75, are in separate loops in the protease but they were in contact with each other throughout the simulation. However, their positions relative to each other changed as the flap region slides down the side of protease (Figures 3 and 5). The sidechains of these two residues also have been reported to be highly dynamic by NMR (Ishima et al., 2001b). The contact between Leu38 and Val75 may be a necessary component of the flap movement as Leu38 slides over Val75 facilitating the relative movement of the cyan and red loops. Facilitating this movement may explain why Leu38 and Val75 are invariant.

Many other residues in the hydrophobic core, however, are correlated with drug resistance (Table 1) (Johnson et al., 2004; Rhee et al., 2005; Shafer et al., 1999a). One such is Leu33.While Leu33 is still conserved in 98% of the untreated and 94% of the treated patient population (Table 1), the development of mutations at this site, particularly Leu33Phe, appears to be related to the most recent generation of protease inhibitors. Leu33 is packed into the core of each monomer throughout the simulation and interacts with many different residues (Figure 5). Mutation to phenylalanine would alter the packing of the core as the other residues shifted to accommodate the larger sidechain. Additionally, phenylalanine has different properties than leucine. The aromatic ring, while non-polar, is capable of forming hydrogen bonds. Therefore, this change would likely affect the conformational flexibility of the monomer both by altering the packing of the core and by potentially forming hydrogen bonds with backbone atoms. Interestingly, in sequences with Leu33Phe mutations, additional residues within the hydrophobic core have an increased frequency of mutation. These changes include increases relative to the general population of Ile13Val from 34% to 51%, Ile62Val from 16% to 48%, and Val77Ile from 18% to 35%. While each of these mutations is correlated with drug resistance, their role has not previously been uncovered. However, the interactions of these residues with each other in the hydrophobic core may explain the potential interdependence of these mutations. Based on the cooperativity of their movements and their mutational frequencies, we hypothesize that the role of these sites in drug resistance is to modify the conformational flexibility of HIV-1 protease. Mutation at sites within the core to other hydrophobic residues could affect the conformational range of the protease and, therefore, the ease by which the inhibitors or substrates can bind. This could explain the role of these residues in either causing or contributing to drug resistance.

Drug resistance in HIV-1 protease is one of the greatest challenges in developing new inhibitors. Using MD simulations, this study elucidated the motion of 19 hydrophobic residues outside the active site that form the hydrophobic core. The extensive rearrangement of this core to facilitate a conformational change has not been previously described and is not easily observed experimentally. However, such a change could occur with minimal energetic expense because the changes only involve the exchange of hydrophobic interactions. The association between protease and its substrate is a highly dynamic process. Protease undergoes a large conformational change to bind its substrate, cleave a peptide bond, and then release the protein product. Ideally, the binding of inhibitors to the protease should be less dynamic because the inhibitor should stay bound to the protease for a longer period of time than the substrate. Mutations that increase the flexibility of HIV-1 protease may detrimentally impact inhibitor binding by increasing the rate of dissociation between the protease and the inhibitor. Additionally, mutations that alter the conformational flexibility may affect the balance between unliganded and liganded states of the protease, thereby specifically detrimentally impacting the recognition of inhibitor over substrates. In order to fully understand their role of these residues in drug resistance, we must further examine their role in the dynamic conformational changes in HIV-1 protease.

More generally, the mechanism of hydrophobic sliding probably represents a common mechanism by which protein undergo conformational changes. Most proteins have either hydrophobic cores or hydrophobic surfaces, which are often explored to understand protein folding. These hydrophobic regions likely also facilitate dramatic conformational changes, at a minimal energetic expense, that are necessary for the protein function. Thus, this study proposes both a novel mechanism for protein dynamics and an explanation for the role of non-active site mutations in the development of drug resistance.

Experimental Procedures

GROMOS molecular dynamics simulation

Three separate MD simulations were run using the GROMOS96 (Scott et al., 1999; van Gunsteren et al., 1996) simulation package and the 43A1 force field with the SPC water model (Berendsen et al., 1987) were used in all simulations. Bond lengths were constrained using the SHAKE algorithm (Ryckaert et al., 1977) with a relative geometric tolerance of 10−4. A twin-range pairlist of 8 and 14 Å was employed in the non-bonded force calculations that was updated every 10 fs. A Poisson-Boltzmann reaction field (Tironi et al., 1995) correction term was employed in the Coulomb interactions. Temperature was maintained at 300K and pressure was maintained at 1 atm using the Berendsen weak coupling approach (Berendsen et al., 1984). A time step of 2 fs was employed in the leapfrog integration scheme (van Gunsteren and Berendsen, 1977). In the first simulation, the crystal structure of 1HHP was solvated with 5843 water molecules in a truncated octahedron periodic box (MD1HHP). The second and third MD simulations were started from the crystal structure of 1F7A. First, the structure was solvated with 7334 waters in a truncated octahedron periodic box (MDSUB). In a separate simulation, the substrate from this crystal structure was removed and the protein was solvated with 7381 waters in a truncated octahedron periodic box (MDNOSUB). A steepest descent minimization was performed with the protein atoms harmonically restrained to their positions in the starting crystal structure. Initial atom velocities corresponding to a temperature of 300 K were generated from a Maxwellian distribution for an equilibration molecular dynamics (MD) run of 10 ps, keeping the harmonic restraints in place. The system was equilibrated for a further 500 ps without harmonic restraints. A simulation of 5 ns was performed for data collection, with coordinates and energies saved every 1 ps. The MD1HHP was carried out for another 4 ns.

The first nanosecond of all three simulations was considered equilibration time and was not included in the analysis. The backbone alpha-carbon fluctuations relative to the starting structure were calculated for all three simulations between 1–5 ns. Using the PROAHB analysis tool within GROMOS96, the coordinates at each ps were also examined to look for the presence of hydrogen bonds between 1–5 ns in all the simulations. A hydrogen bond was defined by a distance between the hydrogen and the acceptor atom of less than 2.5 Å and a donor-hydrogen-acceptor angle of greater than 135º. The percentage of time these hydrogen bonds existed were calculated and mapped onto coordinates from the simulation. In order to determine which residues were part of the hydrophobic core, coordinates at every 0.5 nanoseconds throughout the entire MD1HHP simulation were examined for hydrophobic residues with limited solvent accessibility. Hydrophobic residues within the core region of the protease were inspected for limited solvent accessibility using the graphic modeling program MIDAS (Ferrin et al., 1988). Hydrophobic residues not solvent-accessible for the majority of the simulation were defined as part of the hydrophobic core.

AMBER molecular dynamics simulation

The 9 ns coordinates of the GROMOS simulation were used as the starting structure for this simulation performed with the AMBER8 software package (Case et al., 2004). These coordinates were stripped of the solvent and 200 cycles of restrained steepest descent energy minimization were performed restraining the protease with a harmonic force constant of 9.55 kcal mol−1 Å−2. The protease was then solvated using a TIP3P water box to allow for at least 8 Å of water on each face of protease. The initial periodic box dimensions were 82.2 by 82.6 by 80.4 Å3. Approximately 14,000 water molecules were added to each system and 1000 cycles of steepest descent energy minimization were performed keeping the protease coordinates fixed. Equilibration of the system continued with 5000 steps of restrained MD with a 9.55 kcal mol−1 Å−2 force constant, and constant temperature (300 K) and pressure (1 atm). This equilibration was followed by 25,000 steps of unrestrained MD. The data collecting portion of the simulation was performed at constant temperature and pressure for 5 ns using 1 fs time steps. Coordinates at every 0.5 ns were examined for changes in hydrophobic contacts.

Acknowledgments

The authors gratefully acknowledge the assistance of Dr. James Caldwell. This research was supported by NIH grant P01-GM66524.

We would like to dedicate this work to the memory of Dr. Peter A. Kollman.

Footnotes

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