Abstract
The success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.
1 Introduction
The current treatment of choice to suppress in vivo HIV replication is highly active antiretroviral therapy (HAART) since the mid 199[1,2,3]. However, neither a cure nor vaccine is available for HIV infection [4,5]. Three or more drugs are combined in HAART regimen, for example two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTIs) or one protease inhibitor (PI). Despite the success of HAART, the drug resistance is emerging rapidly as a result of the absence of proofreading function of reverse transcriptase [6,7,8], which makes it urgent to develop novel drugs to combat against the drug resistance [9,10,11,12,13].
HIV-1 protease (HIV PR) is an aspartic protease, a symmetric homodimer with 99 amino acids in each monomer [14], that cleaves newly synthesized HIV-1 polyproteins (nine major cleavage sites) to generate the mature protein components of an infectious HIV-1 virion. The HIV-1 gag gene codes for structural proteins: matrix protein (MA), capsid protein (CA), and nucleocapsid protein (NC) while the gag-pol gene encodes both structural proteins (MA, CA and NC) and enzymes such as: protease (PR), reverse transcriptase (RT), RNAse H (RH), and integrase (IN). Without effective HIV-1 PR, HIV-1 virions remain uninfectious, hence making HIV-1 protease inhibitors the most potent anti-HIV drugs and essential therapeutic components of HAART [1,11].
Many crystal structures are available in literature with WT HIV-1 protease or limited drug resistant HIV-1 mutants complexed with various substrate peptides [15,16,17,18,19,20]. The analysis of these structures demonstrated a conserved asymmetric binding pattern of different substrate peptides with various sequences. Nonetheless, the crystallographic information is limited analyzing the binding pattern of the nine substrate peptides to MDR HIV-1 protease.
To understand the structural characteristics of MDR HIV-1 protease substrate peptide complexes, we chose a clinical isolate, MDR769 HIV-1 protease, as our study model. The high resolution crystal structure of MDR769 HIV-1 protease was solved by our group and it showed an expanded active site cavity with mutations at positions 10, 36, 46, 54, 62, 63, 71, 82, 84, 90 [21,22,23]. The nine hepta-peptides corresponding to the natural cleavage sites P3 to P4′ were co-crystallized with inactive MDR769 HIV-1 protease and the structures were reported recently[24].
Further detailed structural analysis revealed that the highly conserved hydrogen bonds between the protease and substrate hepta-peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and the protease stabilization. In addition, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease. All these findings may provide a novel strategy to design HIV-1 protease inhibitors with excellent resistance profiles.
2 Materials and methods
2.1 Substrate Peptides Preparation
The nine substrate hepta-peptides were purchased from SynBioSci Corporation, Livermore, CA. (Table 1). All the hepta-peptides were purified by HPLC to purity higher than 98%. Peptide powder was dissolved in DMSO to prepare stock solution of 20 mM concentration and the samples were stored at -20 °C.
Table1. Sequences of the nine sites within the HIV-1 Gag and Pol polyproteins that are cleaved by HIV-1 protease.
| Cleavage Site | P3 | P2 | P1 | P1′ | P2′ | P3′ | P4′ | PDB code |
|---|---|---|---|---|---|---|---|---|
| MA/CA | Gln | Asn | Thr | Pro | Ile | Val | Gln | 3OTS |
| CA/p2 | Arg | Val | Leu | Phe | Glu | Ala | Met | 3OUD |
| p2/NC | Thr | Ile | Met | Met | Gln | Arg | Gly | 3OUC |
| NC/p1 | Gln | Ala | Asn | Phe | Leu | Gly | Lys | 3OUB |
| p1/p6 | Gly | Asn | Phe | Leu | Gln | Ser | Arg | 3OUA |
| TF/PR | Phe | Asn | Phe | Pro | Gln | Ile | Thr | 3OU4 |
| PR/RT | Leu | Asn | Phe | Pro | Ile | Ser | Pro | 3OU3 |
| RT/RH | Glu | Thr | Phe | Tyr | Val | Asp | Gly | 3OTY |
| RH/IN | Lys | Val | Leu | Phe | Leu | Asp | Gly | 3OU1 |
The cleavage sites are named by the proteins released after the sites are cleaved: matrix (MA), capsid (CA), nucleocapsid (NC), trans frame peptide (TF), protease (PR), reverse transcriptase (RT), RNAse H (RH), and integrase (IN)
2.2 Protein Purification and Co-Crystallization
The MDR769 HIV-1 A82T protease was over expressed by using a T7 promoter expression vector in conjunction with the E. coli host, BL21 (DE3). Details of protein expression and purification were discussed in our previous research [22,24].
The substrate hepta-peptides were mixed with 2.5 mg/ml MDR769 HIV-1 A82T and diluted to 0.4 mM final concentration. The hanging drop vapor diffusion method was used to form the bi-pyramidal crystals of the MDR769 protease, with crystallization conditions published before [24].
2.3 Data Collection and Crystallographic Refinement
Protease crystals were dipped in 30% glucose for cryoprotection and flash frozen in liquid nitrogen. The diffraction data were collected at 1.00 Å wavelength at the Advanced Photon Source (APS) (LS-CAT 21), Argonne National Laboratory (Argonne, IL). Data were reduced to structure amplitudes with CrystalClear (CrystalClear: An Integrated Program for the Collection and Processing of Area Detector Data, Rigaku Corporation, 1997–2002). In all cases the crystals belonged to the same space group P41. Molecular replacement was performed with Molrep-autoMR in CCP4 [25] with model previously solved in our lab [26]. Initial refinements were performed without substrate hepta-peptides using Refmac5 [27,28]. The nine hepta-peptides were built into the difference electron density maps as the refinement processed in program COOT[29]. Crystallographic waters were added with program ARP/wARP[30]. The structures were refined to resolution 1.6 to 2.0 Å in Refmac5. The final stereochemical parameters were checked using PROCHECK[31]. Images were generated in PyMol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) DeLano Scientific, Palo Alto, CA, USA.)
2.4 Analysis
Hydrogen bonds docking hepta-peptides were analyzed by Pisa Server[32]. Conserved hydrogen bonds within protease were identified with program Ligplot [33]. Temperature factor analysis was done with the CCP4 program Temperature Factor Analysis. Protease substrate complexes were superimposed based on protease residues 1 to 99 Cα and RMSD of Cα was analyzed with the CCP4 program Superpose Molecules [34]. Solvent accessible area was calculated with the CCP4 program Accessible Surface Areas. Water molecules located within 2.0 Å among nine complex structures were considered conserved. The criterion was extended to 3 Å if the water molecules formed similar interaction pattern among nine complexes. All analysis was visualized using PyMol.
3 Results
3.1 Determination of crystal structures of MDR769 HIV-1 protease-substrate complexes
The inactive MDR769 HIV-1 protease was co-crystallized with nine heptapeptides representing the nine natural substrate cleavage site sequences in Gag and Gag-Pol polyproteins. These complexes crystallized into space group P41, in which each asymmetric unit contained one biologically relevant HIV-1 protease dimer complexed with one ligand. The structures were refined to 1.6 - 2.0Å resolution. The hepta-peptides were named after the proteins released upon cleavage: matrix (MA), capsid (CA), nucleocapsid (NC), trans frame peptide (TF), protease (PR), reverse transcriptase (RT), RNAse H (RH), and integrase (IN). The sequence of these peptides and the PDB accession codes are shown in Table 1.
3.2 Ligand backbone specific hydrogen bonds
The ligand backbone specific hydrogen bonds refer to the hydrogen bonds connecting the substrate backbone atoms and the protease atoms, which mimic the conformation of β strands. They are either conserved (present in at least six complexes out of nine) or non-conserved (present in less than six complexes out of nine) hydrogen bonds among the nine protease substrate complexes as demonstrated in Table 2 and Figure 1.
Table 2A. Conserved ligand backbone specific (substrate peptide) hydrogen bonds and length (Å).
| Substrate Atom | Protein Atom | MA/CA | CA/p2 | p2/NC | NC/p1 | p1/p6 | TF/PR | PR/RT | RT/RH | RH/IN |
|---|---|---|---|---|---|---|---|---|---|---|
| P3 O | Asp 29 N | 2.8 | 2.87 | 2.94 | 3.13 | 2.73 | 2.81 | 2.75 | ||
| P3 N | Asp 29 OD1 | 2.57 | 2.73 | 2.81 | 2.49 | 2.58 | 3.2 | |||
| P1 O | Asn 25 ND2 | 3.02 | A 3.42 B 3.13 |
3.11 | 2.49 | 3.00 | A 2.89 B 3.18 |
|||
| P1 N | Gly 27 O | 3.51 | 3.42 | 3.2 | 3.26 | 3.1 | 3.71 | 3.49 | 2.90 | 3.01 |
| P2′ O | Asp 29 N | 3.16 | 3.3 | 2.96 | 2.73 | 3.15 | 3.13 | 2.90 | ||
| P2′ N | Gly 27 O | 2.93 | 3.14 | 3.13 | 3.55 | 2.96 | 2.83 | 2.85 | 2.83 | 3.04 |
| Table 2B Non- conserved ligand backbone specific (substrate peptide) hydrogen bonds and length (Å) | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| P2 O | Asn 25 ND2 | 2.64 | ||||||||
| P2 N | ||||||||||
| P1′ O | Asn 25 ND2 | 3.68 | 3.79 | |||||||
| P1′ N | ||||||||||
| P3′ O | Gly 48′ N | 2.96 | 3.49 | 3.13 | ||||||
| P3′ N | Asp 29′ OD1 | 3.86 | ||||||||
| P4′ O | Gly 48′ N | 3.66 | 2.83 | 2.97, OXT 2.89b | ||||||
| P4′ N | Asp 29′ OD1 | 2.66 | 3.76 (Gly 48 O)a | 3.23 | 2.41 | |||||
P4′ N of NC/p1 forms hydrogen bond with Gly 48 O instead of Asp 29 OD1
P4′ O of p1/p6 forms hydrogen bond with both Gly 48 N and OXT
Figure 1. Ligand backbone specific hydrogen bonds and crystallographic waters involved in substrate peptide recognition.
The substrate backbone was shown in purple with stick model, and the MDR HIV-1 protease monomers are shown in green and cyanosis with stick model. The crystallographic water molecules are shown with grey sphere model. The conserved ligand backbone specific hydrogen bond and non-conserved ligand backbone specific hydrogen bonds between the substrate peptide and MDR HIV-1 protease are presented with black and cyanotic dash line respectively. The hydrogen bonds mediating substrate recognition via crystallographic water molecules are shown with orange dash line.
Compared to the WT HIV-1 protease substrate complexes, the amount of both conserved and non-conserved ligand backbone specific hydrogen bonds are reduced, which may be one of the major reasons of weaker substrate binding to the MDR HIV-1 protease.
Six conserved ligand backbone specific hydrogen bonds are observed; Table 2A shows the residues involved in these hydrogen bonds, which bridge the backbone atoms of hepta-peptides at sites P3, P1, P2′ and the atoms from protease residues Asn 25, Gly 27 and Asp 29.
The non-conserved ligand backbone specific hydrogen bonds are present between the substrate backbone and the protease residues Asn 25, Asp 29′, Gly 48′. The oxygen atom of p1/p6 C-terminal (OXT) forms a hydrogen bond with Gly 48′. Notably, in NC/p1, the nitrogen atom at P4′ forms a hydrogen bond with carbonyl oxygen of Gly 48 instead of residue Asp 29′ (Table2B). P2 and P1′ sites lack both conserved and non-conserved ligand backbone specific hydrogen bonds. P1′ backbone atoms form hydrogen bonds in RT/RH and RH/IN complexes, but the length of these hydrogen bonds are too long to contribute significantly to the interaction. Only CA/p2 forms a hydrogen bond between P2 carbonyl oxygen atom and the protease atom Asn 25 ND2 (Table 2B).
3.3 Ligand side chain specific hydrogen bonds
Besides fewer hydrogen bonds involving substrate peptide backbone atoms, fewer hydrogen bonds arising from side chains of the substrate peptides may also weaken the binding for ligands to MDR HIV-1 protease (Table 3). With the exception of NC/p1, all the hepta-peptides are docked to the protease by two to six ligand side chain specific hydrogen bonds. Most of the side chain specific hydrogen bonds (24 out of 29 hydrogen bonds in all nine complexes) involve side chains of P3, P2, and P2′, whereas P1, P1′, P3′ and P4′ form fewer hydrogen bonds (only 5 hydrogen bonds). On the protease, both the side chains of Arg 8, Asp 30, Thr 82, Asp 25 and the backbones of Asp 29, Asp 30, Pro 81, Leu 46, Gly 48 are involved in hydrogen bonds with hepta-peptide side chain atoms. In addition, in CA/p2 complex, Arg 1 NH1 (P3) forms hydrogen bond with Thr 82 OG1, due to the A82T mutation.
Table 3. Observed side chain (substrate peptide) to side chain (protease) and side chain (substrate peptide) to backbone (protease) hydrogen bonds.
| Side Chain to Side Chain Interaction | Side Chain to Backbone Interaction | |||||
|---|---|---|---|---|---|---|
| Cleavage Site | Substrate Atom | Protein Atom | Length (Å) |
Substrate Atom | Protein Atom |
Length (Å) |
| MA/CA | Gln 1 OE1 | Arg 8 NH2 | 3.07 | Asn 2 OD1 | Asp 29 N | 3.12 |
| Asn 2 ND2 | Asp 30 OD1 | 3.79 | Asn 2 OD1 | Asp 30 N | 2.94 | |
| CA/p2 | Arg 1 NH1 | Thr 82 OG1 | 3.35 | Arg 1 NH1 | Pro 81 O | 3.26 |
| Glu 5 OE1 | Asp 29 N | 3.29 | ||||
| Glu 5 OE1 | Asp 30 N | 2.96 | ||||
| p2/NC | Met 3 SD | Asn 25 ND2 | 3.5 | Gln 5 NE2 | Asp 30 O | 3.80 |
| NC/p1 | ||||||
| p1/p6 | Gln 5 NE2 | Asp 30 OD1 | 3.62 | Arg 7 NE | Leu 46 O | 2.68 |
| Asn 2 OD1 | Asn 25 ND2 | 3.5 | ||||
| TF/PR | Gln 5 NE2 | Asp 30 OD1 | 3.0 | Gln 5 OE1 | Asp 29 N | 2.93 |
| Thr 7 OG1 | Asp 30 OD1 | 2.92 | Gln 5 OE1 | Asp 30 N | 2.90 | |
| Asn 2 OD1 | Asp 29 N | 3.14 | ||||
| Asn 2 OD1 | Asp 30 N | 3.08 | ||||
| PR/RT | Asn 2 ND2 | Asp 30 OD1 | 3.85 | Asn 2 OD1 | Asp 29 N | 2.95 |
| Asn 2 OD1 | Asp 30 N | 2.92 | ||||
| RT/RH | Glu 1 OE2 | Arg 8 NH1 | 3.17 | Thr 2 OG1 | Asp 30 O | 3.87 |
| Asp 6 OD2 | Arg 8 NH2 | 2.54 | Thr 2 OG1 | Asp 29 N | 3.20 | |
| Thr 2 OG1 | Asp 30 N | 3.22 | ||||
| RH/IN | Asp 6 OD1 | Arg 8 NH2 | 2.76 | Lys 1 NZ | Gly 48 O | 3.00 |
3.4 The conserved flap-ligand bridging water reported for the wild type protease is missing in the MDR-ligand complex
The flap-ligand bridging water molecule is missing, while it is conserved in WT HIV-1 protease substrate peptide complexes tethering the protease flap (Ile 50N and Ile 50′ N) with ligands (P2 O and P1′O). This missing water molecule plays a crucial role in substrate cleavage by forming a transitional molecule in the WT HIV-1 protease substrate complexes; in addition, it contributes to the substrate recognition and stabilization in the WT HIV-1 protease substrate complexes. Without this key water molecule, the binding of substrate peptide to MDR769 HIV-1 protease is weakened and cleavage mechanism of the substrates may be different. As a result, HIV-1 protease inhibitors based on different mechanism may be required to overcome the drug resistance imposed by MDR769 HIV-1 protease.
3.5 The conserved water network contributes to the recognition and stability of the ligands in MDR protease substrate complexes
Eleven highly conserved water molecules are found in the MDR HIV-1 protease active site cavity with different functions in substrate recognition, five of which are also observed in WT HIV-1 protease substrate complexes. It is not surprising to have more conserved water molecules in the active site cavity of MDR HIV-1 protease substrate complexes, considering its expanded active site cavity and substrate envelope. Based on their functions, the water molecules fall into five categories: I. Directly bridge substrate and protease; II. Directly and indirectly bridge substrate and protease; III. Indirectly bridge substrate and protease; IV. Stabilize the water network in protease active site cavity; V. Fill the space without obvious functional indication. First, W1 /W1′ (symmetric) connect P3O/P2′O with Gly 27 O, Asp 29 OD2, Arg 8′ NE / Gly 27′ O, Asp 29′ OD2, and Arg 8 NE respectively. Second, W3 (asymmetric) connects W1 directly to protease at residue Arg 8′ NE and Arg 8′ NH1. But the finding that W3 also connects directly to hepta-peptides in MA/CA, CA/p2, NC/p1, TF/PR, and RH/IN complexes renders W3 the capacity to stabilize directly the hepta-peptides conformation. Third, W2/W2′ (symmetric) form hydrogen bonds with W1/W1′ and Thr 26 O, Arg 87 NE/Thr 26′ O, Arg 87′ NE respectively. Fourth, W4 (symmetric to W5) connects to W3, stabilizing W3. In contrast, in MA/CA, NC/p1, p1/p6, TF/PR, and RT/RH complexes, W4 connects to protease at Thr 82′ OG1, showing the significance of the mutation A82T in substrate recognition. Fifth, W5, W6, W7, W8, W9 connect only to protease and seem to fill the space only without significant functional role in substrate recognition. W5, W6, W7, W8, and W9 are far away from hepta-peptides, but reside within the active site cavity. Figure 1 presents the conserved water molecules from the first four categories based on their function.
3.6 The conserved water network contributes to the stability of the MDR HIV-1 proteases
Along with the highly conserved hydrogen bonds are the highly conserved crystallographic water molecules among the nine protease-substrate complexes. 193 to 299 crystallographic water molecules are identified in each of the nine complexes, among which 125 are completely conserved (Figure 2A).
Figure 2. Conserved crystallographic water molecules among the nine MDR HIV-1 protease substrate complexes.
Panel A. Overview of the highly conserved crystallographic water molecules. Grey spheres represent the crystallographic water molecules observed in all the nine complexes. The MDR HIV-1 protease monomers and substrate peptide are shown with cyanotic, green and purple ribbons respectively. The box 1 and box 2 are in the flap region and active site cavity which is investigated in more details in panel B and C.
Panel B. Detailed analysis of the crystallographic water molecules in the flap region.
Panel C. Detailed analysis of the crystallographic water molecules in the active site cavity.
Among the 125 conserved water molecules, 52 form at least two hydrogen bonds with protease residues, 43 are on the surface of the protease forming only one hydrogen bond with protease residues, 12 are on the surface of the flaps, 6 are involved in substrate recognition. In addition, 12 water molecules are on the surface of the protease without direct interaction with protease, instead they interact with other fixed water molecules to form a water network.
Among the 125 completely conserved water molecules, 114 are two-fold symmetrically distributed in the dimer. The distribution of these asymmetric water molecules are described as the following. Two are asymmetrically distributed at the dimerization interface, one is involved in the substrate binding, three are on the flap area, three are around residues 60-67, one is between residues 61 and 40, and one is around the α helix. Of these 11 asymmetric water molecules, six are on one monomer, four are on the other, and one is between two monomers.
Conserved water molecules are crucial in the tertiary structure stabilization. First, in both monomers, W10/W10′ bridge the carbonyl oxygen of Val 78/Val 78′ (preceding 80s loop) with NH1 and NE of Arg 57/Arg 57′ (downstream the flap area). These water molecules fix the N terminus of 80s loop and the C terminus of the flap, which are crucial in the substrate recognition and binding. Second, Ile 50 O, Ile 50 N, Gly 49 N and Gly 52 O in each monomer are connected through a hydrogen bond network contributed by the conserved water molecules W6/W7. The internal hydrogen bonds by W6/W7 stabilize the conformation of the flap tips (Figure 2B). Third, water network W11, W11′ and W12 is important in the interaction between the flap tips from different monomers, although the flap tips do not interact directly. W12 is conserved in eight complexes with one exception RH/IN; W11 is conserved in seven complexes, while W11′ is present in six complexes. The three water molecules form an isosceles triangle with W12 in the middle pointing down to the active site cavity. W12 forms indirect hydrogen bonds with the protease through W11/ W11′ water molecules, which connect directly to Ile 50/50′ O and Gly 51/51′ N respectively. Thus the water network actually links the tips of the flaps together (Figure 2B). Fourth, the active site loop is also stabilized by water molecules. W13/W13′ bridge Gly 27 O/ Gly 27′ O with Asp 29 OD2/ Asp 29′ OD2 and W14/W14′ bridge Thr 26 O/ Thr 26′ O with Arg 87 NE/Arg 87′ NE. Fifth, on the dimerization interface, six conserved water molecules connect the two monomers together. W15/ W15′ connect together Asn 98′ O, Gly 94 O, Thr 96 OG1/ Asn 98 O, Gly 94′ O, Thr 96′ OG1 respectively. W16/W16′ connect together Cys 95 O, Leu 5′ N / Cys 95′ O, Leu 5 N respectively. W17/W17′ connect together Ile 93 O, Phe 99′OXT / Ile 93′ O, Phe 99 OXT respectively (Figure 3). Sixth, there are four conserved water molecules that stabilize the N terminal loop around the dimerization area: W18/ W18′ connect together Pro 9 N, Gln 7 O, Thr 4 O / Pro 9′ N, Gln 7′ O, Thr 4′ O respectively, and W19 / W19′ connect together Gln 7 N, Trp 6 N, Thr 4 O / Gln 7′ N, Trp 6′ N, Thr 4′ O respectively (Figure 3).
Figure 3. The conserved crystallographic water molecules stabilizing the dimerization interface of MDR HIV-1 protease substrate complexes.
The MDR HIV-1 protease monomers are shown in stick model and the crystallographic water molecules are shown in grey spheres. The black dash line represents the interaction between the crystallographic water molecules and the MDR HIV-1 protease.
4 Discussion
4.1 Missing conserved flap-ligand bridging water in MDR complex that is critical in substrate cleavage
Wild type HIV-1 protease studies show a bridging water molecule, critical in substrate cleavage, connecting the protease flaps Ile 50 N / Ile 50′ N with P2 O / P1′ O. In the present study, this water molecule is missing in all the nine MDR complexes. Moreover, in eight out of nine MDR complexes, there is one water molecule located between the flaps, but can not reach either Ile 50 N / Ile 50′ N or P2 O / P1′ O, due to the wide opening of the flaps. Nevertheless, this water molecule can still interact indirectly with the MDR protease flaps, suggesting its stabilization role to the flap area.
It is possible that the cleavage mechanism of the MDR complexes is different from the one of WT complexes, with the absence of the flap-ligand bridging water. This may explain why MDR HIV-1 protease is resistant to current FDA approved HIV-1 protease inhibitors. As a result, new HIV-1 protease inhibitors are required, that do not need the flap-ligand bridging water to mimic the transitional state.
4.2 Reduced hydrogen bonds between the ligand and protease backbone imply drug resistance
An extensive hydrogen-bonding interaction between the inhibitors and the HIV-1 protease backbone is believed to be one of the keys to overcome drug resistance [35,36]. However, our crystal structure analysis demonstrates significantly reduced hydrogen bonds between the substrate peptide and the MDR HIV-1 protease backbone atom, compared with those found in WT protease substrate peptide complexes. Among the nine MDR HIV-1 protease substrate peptide complexes, there are four conserved and two non-conserved hydrogen bonds between the ligand and protease backbone atoms, while eight highly conserved hydrogen bonds are found in the WT counterparts. This finding may imply the importance to restore the extensive protease backbone hydrogen bonding in order to overcome the drug resistance brought by the mutations. In addition, the loss of protease backbone hydrogen bonds may explain the significant drug resistance of MDR HIV-1 protease against peptidomimetic-based HIV-1 protease. This conjecture is further supported by fact that Darunavir, a less peptidomimetic-based HIV-1 protease, shows relatively less drug resistance to MDR HIV-1 protease[13].
Conserved hydrogen bond and water among MDR HIV-1 protease substrate complexes
Hydrogen bonds, waters crucial in substrate recognition, protease stabilization
flap-ligand bridging water is missing in MDR HIV-1 protease substrate complexes
Acknowledgments
This research was supported by the National Institutes of Health grant AI65294 and a grant from the American Foundation for AIDS Research (106457-34-RGGN).
Abbreviations
- MDR
Multi-drug resistant
- HIV
human immunodeficiency virus
- WT
wild type
- HAART
highly active antiretroviral therapy
- PR
protease
- MA
matrix
- CA
capsid
- NC
nucleocapsid
- RT
reverse transcriptase
- IN
integrase
- RH
RNase H
- RMSD
root mean square deviation
Footnotes
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