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J. Chem. Inf. Comput. Sci. 2003, 43, 1584-1590
Investigation on an Orientation and Interaction Energy of the Water Molecule in the HIV-1 Reverse Transcriptase Active Site by Quantum Chemical Calculations Mayuso Kuno, Rungtiva Palangsuntikul, and Supa Hannongbua* Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand Received October 2, 2002
To obtain basic information such as interaction between the water molecule and amino acids in the active site of HIV-1 Reverse Transcriptase (HIV-1 RT), ab initio molecular orbital calculations and the two-layer ONIOM method were performed. The energetic results from different methods show that the ONIOM2 (MP2/6-311G**:HF/6-31G*//HF/6-31G**:HF/3-21G) can provide reliable results on the orientation of the water molecule in the HIV-1 RT active site. The interaction between the water molecule and Asp186 was found to be the most preferable. The obtained results from ONIOM2 calculations indicated that the active site model system included six amino acid residues (Asp186, Asp185, Met184, Tyr183, Leu187, and Tyr188) leading a preferable representation of the environment surrounding the water molecule in the more realistic model. The water molecule presented in the active site tends to form H-bonding with Asp186, Tyr183, and Tyr188 as indicated by the distances of O4-H2 ) 1.91 Å, O3-H7 ) 2.36 Å, and O3-H17 ) 1.73 Å, respectively. The stability of this complex system brings to the foundation of the estimated binding energy approximately -15.8 kcal/mol or -8.1 kcal/mol which is more stabilized relative to the smallest model complex. These observations revealed that the water molecule forms both a hydrogen bond donor and a hydrogen bond acceptor in the cavity and plays an important role in the specific conformation of the active site of HIV-1 RT. The H-bonding is a rather strong interaction; thus, the water might induce the conformation of the active site to fit the catalysis process and helpfully attract dNTP to elongate the viral DNA in the replication process of this enzyme. 1. INTRODUCTION
HIV-1 reverse transcriptase (HIV-1 RT) is known as the causative agent of acquired immunodeficiency syndrome (AIDS) and is an essential enzyme involved in the life cycle of the HIV virus responsible for copying the single-strand RNA viral genome into a double-stranded proviral DNA form for integration into the host genome. Therefore, it has been an important target of several antiviral therapeutic agents used in the treatment of AIDS.1-4 HIV-1 RT inhibitors have been developed and classified into two main categories: nucleoside and non-nucleoside analogues.5-11 The nucleoside RT inhibitors (NRTIs) such as AZT, ddC, and ddI have been widely used to treat AIDS patients. Unfortunately, these derivatives have suffered from loss of antiviral activity, high cellular toxicity, several side effects, and the emergence of drug-resistant viral variants.5,7,13,14 The other class of HIV-1 RT inhibitors is the non-nucleosides (NNRTIs) such as HEPT,15 TIBO,16 nevirapine,17 and efavirenz,18 which are highly specific for HIV-1 RT. The NNRTIs are much less toxic than the NRTIs; nevertheless, the emergence of drug-resistant viral strains has limited the therapeutic efficacy of NNRTIs.6,7,9,10,12,19 To date, the development of X-ray crystallography has provided more than 50 valuable crystal structures of HIV-1 RT, which are available in the Protein Data Bank (RCSB PDB, http://www.rcsb.org).20 The catalytic site for RT lies in a cleft in the palm of the p66 subunit and contains the * Corresponding author phone: +66-(2)-9428900; fax: +66-(2)5793955; e-mail:
[email protected].
sequence Tyr183, Met184, Asp185, and Asp186, which are highly conserved in retroviral RTs as well as in other DNA polymerase. Asp110 is also required for catalytic activity and is brought into the active site in the folded protein. The 3′-OH of the primer terminus is held close to the catalytically essential Asp110, Asp185, Asp186 residues; thus, this terminus is in a position for nucleophilic attack on the R-phosphate of an incoming nucleoside triphosphate.5,11 Observation of a hydrogen bond between the 3′-OH of the primer terminus and the side chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization.21 It was reported that the polymerase of HIV-1 RT requires divalent metal ions (preferably Mg2+) as cofactors and proposed that there are two Mg2+ bound to the active site aspartate residues in the active form of HIV-1 RT.21 Interaction between proximal oxygen atoms of Asp186 and the 3′-terminal phosphate may be mediated via either a metal ion or a water molecule. Such a water molecule might also be the ligand for one metal ion at the polymerase active site.22 Evidently, there are 27 crystal structures of HIV-1 RT complex with the existence of the water molecules in the polymerase active site cavity. It is questioned how important these water molecules are in the polymerase active site. Based on ab initio Molecular Dynamics Simulations23 study on HIV-1 RT, it was shown that water molecules present in the active site may be crucial for substrate recognition. Therefore, theoretical investigation can be used to obtain the basic information of the role of the water molecule and the interaction with the polymerase active site of HIV-1 RT.
10.1021/ci0203850 CCC: $25.00 © 2003 American Chemical Society Published on Web 07/04/2003
HIV-1 REVERSE TRANSCRIPTASE ACTIVE SITE
J. Chem. Inf. Comput. Sci., Vol. 43, No. 5, 2003 1585
Figure 1. The starting geometry of HIV-1 RT/water complex used in this study (the arrow indicates the direction of translation for the single point calculations scanning at the HF/3-21G level).
Recently, the multilayered integration method or ONIOM (Our own N-layered Integrated molecular Orbital + molecular Mechanics) has been introduced24 and has been proven to be a powerful tool for the theoretical treatment of large molecular systems where different levels of theory are applied to different parts of the molecule.25-28 The most important part of the molecule forms the innermost layer described at the highest level of theory. Subsequent layers are treated by using progressively computationally cheaper lower-level approaches. In this study, ab initio quantum chemical calculations29-33 and the two-layer ONIOM (ONIOM2) method were used to study the nature of the intermolecular interactions between the water molecule and amino acid residues in the HIV-1 RT active site. Our aim is to gain an insight into the key role of the water molecule, which is essential for the HIV-1 RT active site. This work determined the water-amino acid residues interactions in order to understand the water orientation and interaction energy of the water molecule in the active site of HIV-1 RT. 2. COMPUTATIONAL METHODS
2.1. Structural Active Site Model. The geometry of HIV-1 RT and a water complex was obtained from the Protein Data Bank (PDB) entry code: 1VRT.34 For the model system of the HIV-1 RT active site, four amino acid residues from p66 subunit were considered,35 as shown in Figure 1. These amino acid residues consist of Tyrosine183 (Tyr183), Methionine184 (Met184), Aspartic acid185 (Asp185), and Aspartic acid186 (Asp186). They are included in the Ω-loop of the active area of HIV-1 RT.35 In this HIV-1 RT structure, there is only one water molecule which lies in the active site and the distance from an oxygen atom of Asp186 is 2.94 Å. Hydrogen atoms were used as the terminated atoms to saturate the model system of amino acid residues in neutral form. 2.2. Configurations of the Water Molecule. Based on X-ray crystallographic data, there is no hydrogen position information; therefore, numerous configurations of the water molecule in the active site were generated. The interaction between the water molecule and the Asp186 was first investigated (Figure 1), based on single point calculations at the HF/3-21G level. All calculations were carried out using the Gaussian 98 program36 implemented in IBM/SP2 cluster. Starting configuration of water is kept constant and defined
Figure 2. The model system for the ONIOM2 method; the inner layer is displayed in bond and stick and the outer layer is displayed in stick.
as water gas-phase geometry in which O-H ) 0.96 Å and ∠H-O-H ) 104.5°. The angles ∠O3-O4-C5 (A1) and ∠H2-O3-O4 (A2) and the dihedral angles O3-O4-C5C6 (D1), H2-O3-O4-O5 (D2), and H1-O3-H2-O4 (D3) were optimized in the ranges 0° e A e 360° and 0° e D e 360° by the rotational step ∆D ) 15°. The translation of water molecule from the Asp186 was moved by a vector pointing from O4 to O3 within the range of O3-O4 distance (L); 1 Å e L e 5 Å. To get closer to the accurate configuration, translation steps with 0.5 Å were done, and additional steps with 0.1 Å between the optimum range were also reoptimized in order to search for a more precise configuration. Moreover, numerous configurations of the water molecule and the oxygen atoms of Tyr183 (atom O15) and the peptide bond between Tyr183 and Met184 (atom O16) were also considered and calculated in the same manner. Consequently, the optimal configuration of the water molecule in the active site was obtained. The binding energy (∆E) of the active site and the water complex structure were calculated at HF/3-21G level with basis set superposition error (BSSE) corrections. 2.3. ONIOM2 Calculations. To investigate the interaction between the water molecule and the active site of HIV-1 RT, the effect of amino acid residues surrounding the water molecule in the active site were taken into account. Therefore, Asp186, Asp185, Met184, Tyr183, Leu187, Tyr188, Trp229, and Met230 were added to the model system consecutively. These amino acid residues were located within a 7 Å diameter from oxygen atom of the water molecule. In the present ONIOM2 calculations, the inner layer consists of part of Asp186 and the water molecule (Figure 2), which were treated with the higher level calculations. The remaining amino acid residues surrounding the water molecule (outer layer) were considered as the more realistic system and calculated at the lower level. For the ONIOM2 calculations, the MP2/6-311G**, HF/6-31G**, HF/6-31G*, and HF/3-21G level of theory were applied. Consequently, the abbreviation of the combined methods are defined as the following: ONIOM2M ) (MP2/6-311G**:HF/6-31G*)//(HF/6-31G**:HF/3-21G) and ONIOM2H ) (HF/6-31G**:HF/3-21G). The orientation of water molecule in the inner layer was fully optimized, while those of other heteroatoms in the model system were kept
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Figure 3. The relation between the most stable configurations, varying O3-O4 distances and the interaction energies (D2 ) 175.4° and D3 ) -0.6°).
frozen. The extrapolated energy for the ONIOM2 approach is defined as shown in eq 1
ET AL.
Figure 4. The optimal orientation of the water molecule, interacting with Asp186. The optimal orientation: L (O3-O4) ) 2.80 Å, D2 (
