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Received Date : 28-Jun-2014 Revised Date : 13-Nov-2014 Accepted Date : 21-Nov-2014 Article type
: Research Article
Structural basis of why nelfinavir resistant D30N mutant of HIV-1 protease remains susceptible to saquinavir
Running title: Why D30N mutant of HIV-1 PR is susceptible to saquinavir
Key words: X-ray Crystallography, HIV-1 protease, Saquinavir, Nelfinavir, Drug-resistance, Susceptibility.
Vishal Prashar1, Subhash C. Bihani1, Jean-Luc Ferrer2 and M.V. Hosur 3
1
Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400085, India, 2 Institut de Biologie Structurale Jean-Pierre Ebel, Groupe Synchrotron, Commissariat a l’Energie Atomique et aux Energies Alternatives, Centre National de la Recherche Scientifique, Universite de Grenoble Alpes, 38027 Grenoble, France, 3 Present address: Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai-410210, India.
∗
Corresponding author, Tel: 91-22-25594688, Fax: 91-22-25505151.
E-mail address: [email protected]
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Abstract Although anti-HIV-1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant tethered HIV-1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main-chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N-NFV complex. Apart from maintaining these two main-chain hydrogen bonds, the P2-Asparagine of SQV forms an additional hydrogen bond to the mutated side-chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug-resistant HIV-1 protease.
Introduction Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS) (1, 2). According to a recent report by The Joint United Nations Programme on HIV/AIDS
(UNAIDS); about 35 million people worldwide are infected with HIV (3). Efforts that have been undergoing for the past many years to develop an effective vaccine against HIV have not yielded satisfactory results due to extreme variability of the virus and its escape mechanisms from the immune system (4). In this scenario, therapeutic approaches aimed at counteracting the molecules essential for the virus cycle have proved to be very useful (5). HIV-1 protease is one such major target enzyme because of the crucial role it plays in the processing of the viral poly-proteins (6). The introduction of HIV-1 protease inhibitors (PIs) in 1995 and the application of Highly Active Anti-Retroviral Therapy
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(HAART), which includes simultaneous use of the inhibitors of viral enzymes HIV-1 protease and HIV-1 reverse transcriptase, resulted in a vast decrease in mortality and a prolonged life expectancy of HIVpositive patients (7, 8). From 1996 to 2012, antiretroviral therapy prevented 6.6 million AIDS-related deaths worldwide (3). Besides, HAART also has a potential to limit the risk of HIV transmission (9). Until now, 9 PIs have been approved by the U.S. Food and Drug Administration (FDA) for use as drugs in the treatment of infected patients. But long term clinical efficacy of these drugs has been plagued by various issues (10, 11). Development of resistant strains of virus carrying mutated forms of HIV-1 protease enzyme is the major issue amongst them. Other problems include large dosages, undesirable side effects and patient non-adherence. With respect to these problems, the development of a new generation of PIs which possess improved pharmacokinetic properties and drug-resistance profiles is urgently required. In this regard, comparing the resistance profiles of the present anti-HIV-1 protease drugs and correlating them with their chemical structures is an important guide.
All the HIV-1 PIs, except tipranavir, in the market today are peptidomimetics. They mimic the peptide substrates of the enzyme, but the cleavable peptide bond has been replaced with hydroxyethylene or hydroxyethylamine based transition-state isosteres. Saquinavir (SQV) was the first HIV-1 PI released as drug by Hoffman-La-Roche (12). It was developed by replacing the Phe-Pro cleavage site of the peptide substrate with a non-cleavable hydroxyethylamine-based transition-state isostere (Figure 1A). The P1’proline was replaced with the dodecahydroisoquinoline (DIQ) bicyclic ring. At the P3 site, a quinoline ring was introduced while the P2’ site has tertiary (tert)-butylcarboxamide moiety. SQV is highly potent HIV-1 PI with nanomolar inhibitory constant but suffers from poor oral bioavailability. Nelfinavir (NFV) was developed at Agouron pharmaceuticals labs by the modification of SQV (Figure 1B). The P3quinoline ring was removed while the P2-Asparagine (Asn) was replaced with a 2-methyl-3-hydroxy
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benzamide moiety. At the P1 position, instead of a phenyl group, slightly longer S-phenyl side-chain was used. The rest of the substituents were same. These changes reduced the molecular weight and improved the oral bioavailability (13).
D30N is a major drug resistance mutation in HIV-1 protease against NFV (14). Compared to the wild-type protease, the binding of NFV by D30N mutant is decreased by 20-fold (15). One reported study found D30N mutation in 30.7% of patients failing first-line NFV-based HAART regimen (16). But this mutation does not affect SQV even though SQV and NFV have common functional groups. In fact, the D30N mutation does not exhibit cross-resistance with most of the other PIs (17, 18). To explore the reason for this, we solved the structure of HIV-1 protease D30N mutant in complex with the drug SQV. On comparing this structure with the D30N-NFV complex, we found differences in the hydrogen-bonding interactions between the drugs and the mutant enzyme. We propose that the presence of additional hydrogen bonds in the case of D30N-SQV complex is responsible for SQV's ability to maintain efficacy against the D30N mutant.
Materials and Methods Site directed mutagenesis Quick change site directed mutagenesis kit (Stratagene, La Jolla, CA) was used for making the D30N
mutation with oligonucleotide primer 5’-CTCCTCCAGTACAGTATTATCAGCACCGGTATCCAG-3’. Presence of the mutation was confirmed through DNA sequencing carried out by Eurofins genomics India Pvt. Ltd.
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Protein expression and purification HIV-1 protease tethered dimer used in the present study contains a five-residue linker, GGSSG,
covalently linking the two monomers. Expression and purification of HIV-1 protease tethered dimer followed the procedures reported earlier (19).
Differential scanning fluorimetry Eppendorf mastercycler ep realplex RT-PCR machine was used to carry out differential scanning fluorimetry to detect change in the protein melting temperature after the drug binding (20). The protein solutions were used to reach a concentration of 0.1-0.2 mg/ml in 50 mM sodium acetate buffer, pH 5.5 in 20 µL reaction mixtures. The final reaction mix also contained five fold molar excess of the respective drugs and 5 X SYPRO Orange dye (Sigma-Aldrich, Inc., St. Louis, MO). The RT-PCR machine was programmed to equilibrate the samples at 25°C for 10 minutes and then increase the temperature to 95°C at a rate of 1°C/minute, taking a fluorescence reading every 1°C using excitation and emission wavelengths of 470 nm and 605 nm respectively. The melting temperatures were obtained as maxima of the first derivatives of the melting curves.
Crystallization The single crystals were obtained by the hanging drop vapour diffusion method. The protein (5 mg/ml
in 50 mM sodium acetate, pH 4.5, containing 1 mM dithiothreitol) was reacted at the room temperature for 30 minutes with ten-fold molar excess of SQV dissolved in dimethyl sulfoxide. For crystallization, equal volumes of the reaction mixture and the reservoir solution (1% saturated ammonium sulfate, 200
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mM sodium phosphate, and 100 mM sodium citrate at pH 6.2) were mixed on a cover slip and sealed over the reservoir well at the room temperature.
X-ray data collection and refinement The crystals were equilibrated in the cryo-protectant (25% glycerol and 75% reservoir solution) before
flash-freezing, for exposure to X-rays on the FIP-BM30A beam-line (21). The diffraction data were indexed, integrated, and scaled by using the computer program XDS (22). The data processing statistics are given in Table 1.
Crystal structure was solved by difference Fourier method using the native coordinates (PDB ID 1LV1). The structure was refined in the computer program PHENIX by using the amplitude-based maximum likelihood target function (23). A test set containing 5.0% of randomly chosen reflections were reserved for the determination of Rfree (24). The simulated-annealed mFO-DFC omit electron density map for SQV
was calculated by omitting SQV atoms during simulated annealing as implemented in the program PHENIX, where the model was heated to a temperature of 2500K followed by cooling to 300K in 500 cooling steps. All interactive model building and molecular superpositions were carried out using the molecular modeling software O (25). The structural comparisons are based on superpositions of protein Cα atoms. All the figures were drawn using the computer program PyMOL (26). The atomic coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under the PDB ID 4Q5M.
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Results Differential scanning fluorimetry The melting temperatures (Tm) of the native and the D30N mutant of tethered HIV-1 protease, both with
and without the drugs NFV and SQV, were determined using differential scanning fluorimetry (Figure 2). With the native protease, both NFV and SQV showed a similar change in the Tm (22 ± 0.2 °C). But with
the D30N-NFV complex, shift in the Tm (∆ Tm) was 15.7 ± 0.9 °C while with the D30N-SQV complex it was 23 ± 0.8 °C suggesting that D30N mutation decreases the affinity of binding with NFV but remains susceptible to SQV.
Overall structure The crystal structure of D30N mutant of HIV-1 protease complexed with SQV was determined to a resolution of 1.79 Å in the space group P61. The results of crystallographic refinement are summarized in Table 1. The crystallographic asymmetric unit contains a tethered dimer of HIV-1 protease and one molecule of SQV. The residues in the first monomer are numbered as 1–99 and those in the second monomer are numbered 1001–1099. When the 2mFO–DFC electron density map was contoured at 1σ
level, there was no density for the linker region of the tethered dimer under study, suggesting that the linker region is not ordered. Alternate conformations were modeled for the side-chains of the residues 84 and 1084. The stereochemistry of the molecular model was quite good, with more than 90% of nonglycine residues occupying the most favored regions of the Ramachandran plot as defined in the program PROCHECK (27). Figure 3 shows the simulated-annealed mFO-DFC omit electron density map for
SQV. This density could be rationalized by SQV modeled in two orientations consistent with the approximate two-fold symmetry of the functional HIV-1 protease dimer.
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Drug-mutant interactions SQV binds to the mutant enzyme in an extended conformation through hydrogen bonds to the backbone atoms of the protein. The side-chain groups P3-P2’ of the drug interact with the residues in the corresponding sub-sites S3-S2’, mainly through hydrophobic interactions. The tert-butylcarboxamide moiety fits into the S2’ sub-site, while the DIQ bicyclic ring occupies the hydrophobic S1’ pocket. The central hydroxyl group binds to the catalytic aspartates of the enzyme through hydrogen bonds. The P1phenyl group occupies the S1 sub-site and the polar Asn side-chain of the inhibitor resides in the S2 pocket. The quinoline ring at P3 occupies the S3 sub-site. A water molecule bridges the P2 and P1’ carbonyls of the inhibitor to the Ile50 and Ile1050 main-chain amides in the flap region of the enzyme through hydrogen bonds in a manner analogous to that observed in the other HIV-1 protease inhibitor complexes. The Ile84 side-chain was located opposite to the inhibitor, and 84 CD1 forms van der Waals contacts with the P1-Phe and the P2’-tert-butylcarboxamide residue of SQV. In the second orientation of the inhibitor, the P1’-DIQ ring is located opposite to Ile84, and to optimize interactions with this moiety, Ile84 adopts an alternate conformation. The occupancy distribution of each conformation was 0.51 and 0.49 respectively, consistent with the occupancies of two-fold related conformations of the inhibitor.
Comparison with D30N-NFV complex The present complex was compared with the complex of NFV with D30N mutant of HIV-1 protease (PDB ID 2Q64) (15). The two structures were very similar with an rmsd, for 198 Cα atom pairs, of 0.2 Å. Relative positions of SQV and NFV in the active site are shown in Figure 4. Chemically, SQV and NFV are different on the non-primed side-chain groups (Figure 1). At the P2 site, there is an Asn residue in SQV while NFV has a 2-methyl-3-hydroxybenzamide moiety at this position. At the P1 site, the slightly longer S-phenyl side-chain of NFV partially extends into the S3 region and forms van der Waals contacts with
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Arg8. But in the case of SQV, the bulky quinoline ring at the P3 position would be in steric contact with Arg8/1008 depending upon the orientation, the inter-atomic separation between the C3 atom of quinoline ring and the NH2 atom of Arg8/1008 being only 2.1/2.0Å (Figure S1, supporting information). This has been avoided by a change in the conformation of the side-chain of Arg8/1008 in the SQV complex. Comparison of the torsion angles of Arg8/1008 in the two complexes is given in Table S1 (supporting information). The corresponding separation is now 3.4/3.3 Å. Similarly, there is a change in the main-chain torsion angles of the residue Gly48/1048 of the protein to avoid steric contacts with the P3-quinoline ring of SQV (Figure 4C), corresponding separation between the N1 atom of quinoline and the carbonyl oxygen of Gly48/1048 being increased from 2.8/2.7 Å to 3.3 Å. The P2-Asn of SQV forms a hydrogen bond with the Asn30 N of the protease backbone. In case of NFV, 2-methyl-3hydroxybenzamide moiety of the inhibitor occupies the S2 sub-site, with the m-phenol group hydrogenbonding to the Asn30 side-chain (Figure 4D). Carbonyl oxygen of the P3-quinaldic amide from SQV forms a hydrogen bond with the backbone nitrogen of Asp29 (Figure 4C). Thus, SQV makes two hydrogen bonds with the main-chain atoms of the protein. This was similar to what was previously observed in the crystal structure of SQV with multi-drug-resistant HIV-1 protease containing 20 mutations including D30N (PDB ID 3UFN) (28). Drug darunavir (DRV) also maintains backbone hydrogen bonds to the residues 29 and 30 in the D30N containing mutants (PDB IDs 2F80 and 3LZV) (29, 30). Although the aniline group on the other end of DRV has water-mediated interaction with the side-chain of Asn30 in these structures as compared to direct interaction with the side-chain of Asp30 in the native-DRV complex (PDB ID 2IEN) (31).
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Comparison with native HIV-1 protease-SQV complex Figure 5 shows the structural superposition of the present D30N-SQV complex on to the native HIV-1 protease-SQV complex (PDB ID 1HXB) (32). The two structures superpose to an rmsd of 0.2 Å over 198 Cα atom pairs. Interesting localized changes are observed in the conformation of the drug as well as of
the protein. There is a slight rotation around χ2 from -49° to -63° in Asn30 (from -51° to -68° in case of Asn1030) which enables formation of a hydrogen bond with the side-chain of Asn residue at the P2 position of SQV. The side-chain of P2-Asn of SQV also adjusts to form hydrogen bonds with both the main-chain and the side-chain of Asn30/1030 (Figure 5B). It may be pointed out that the hydrogen bonds from the inhibitor to the protein main-chain atoms are not disturbed by the mutation in the protein.
Discussion All the available anti-HIV-1 protease drugs are designed as competitive inhibitors. The substrate clearly forms a natural template for the design of such inhibitors. However, the peptidic inhibitors lack suitable physico-chemical properties and metabolic stability resulting in poor bioavailability. At the same time, to maintain the potency, the inhibitor molecules should maintain the interactions natural substrates make with the enzyme molecule. The design of first generation HIV-1 PIs included efforts to minimize the inhibitor molecular weight, and reduce its peptidic character while still imitating the interactions natural substrate made with the enzyme. NFV resulted from the optimization of SQV which suffers from poor oral bioavailability, because of its retained peptide character. However, resistance to NFV arises with the selection of D30N mutation in HIV-1 protease. The substitution of asparagine for aspartic acid at the position 30 is very specific to the NFV-resistant protease. The crystal structures of HIV-1 protease
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complexed with the peptide substrates showed that backbone atoms of the residues Asp25, Gly27, Ala28, Gly48, Asp29, and Asp30 are involved in making hydrogen bonds with the substrates (33, 34). Among these residues, Asp25, Gly27, Ala28, and Asp29 are well conserved. Very often, the Asp30 residue main-chain is involved in hydrogen-bonding to the P2/P2’ side-chains of various substrates. This interaction appears to be an important hydrogen bond whenever a polar side-chain is present at the P2/P2’ position. However, there is only one direct hydrogen-bonding interaction seen between the Asp30 side-chain of the enzyme and the P2 side-chain of NFV. This interaction with the D30N mutant is expected to be comparatively weaker or absent leading to reduced NFV affinity (35-37). On the contrary, SQV forms two direct hydrogen bonds with the main-chain atoms of the residues Asp29/1029 and Asp30/1030 (Table 2). When Asp30 is mutated to Asn30, the side-chain of Asn residue at the P2 position makes an additional hydrogen bond (Asn30/1030 ND2….P2-Asn OD1 = 3.1/3.1 Å) with the mutated sidechain of the enzyme (Figure 5B). These extra interactions are consistent with higher ∆Tm of the D30N-
SQV complex as compared to the D30N-NFV complex. The hydrogen bonds from SQV to the protein main-chain atoms are also observed in the structure of native HIV-1 protease-SQV complex. According to a recent hypothesis, the substrate-envelope defined as consensus volume occupied by the natural substrates, not only explains the substrate specificity but also the development of drug resistance (34, 38). The substrate-envelope hypothesis defined Asp30 amongst the vulnerable residues for drug resistance mutations in HIV-1 protease (38). Mutations into smaller or bulkier side chains at the vulnerable sites may impact drug binding leading to drug resistance (39). The D30N mutation does not affect the side-chain length but the nature of side-chain. This change results in the decrease of electrostatic binding energy between the m-phenol group of NFV and the 30th residue while the gain of a hydrogen bond between the P2-Asn of SQV and Asn30 of the protein. Although D30N mutation does not provide cross-resistance to most of the other PI drugs, but the accumulation of additional secondary mutations may affect the conformational distribution of HIV-1 protease leading to cross-resistance to
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other HIV-1 PIs (35, 40). Interestingly, in a multi-drug-resistant HIV-1 protease variant which carries 20 mutations including D30N, backbone hydrogen bonds are still maintained with SQV as in the present D30N-SQV complex (28). These observations support the previously-published conclusions that hydrogen-bonding interactions from the drug molecules to the main-chain protein atoms are less affected by mutations (31, 41-43).
Conclusions Comparing the resistance profiles of anti-HIV-1 protease drugs and correlating them to their chemical structures can provide important clues to the future drug design efforts. We report here the crystal structure of the complex between the drug SQV and D30N mutant tethered HIV-1 protease refined to 1.79 Å resolution. Structural comparison with the D30N-NFV complex showed following differences in the interactions: 1) hydrogen bonds from SQV are to the protein main-chain atoms, 2) The P2-Asn of SQV forms an additional hydrogen bond to the mutated side-chain of the residue 30. These differences could be the reason why D30N is not a drug resistance mutation against SQV. Further, the structure supports the previous studies which indicated that interactions with the main-chain atoms could be an important design principle to guide the development of new drugs that are effective against drugresistant mutants.
Acknowledgements We thank the National Facility for Macromolecular Crystallography, SSPD, BARC, for the X-ray diffraction and biochemistry equipment and Dr. D.R. Rao (CIPLA), for providing the drugs saquinavir and nelfinavir. We also thank Dr. Ajay Saini and Mr. S. R. Jadhav for technical help.
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Conflict of Interest The authors declare no conflict of interest.
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35. Clemente J.C., Hemrajani R., Blum L.E., Goodenow M.M., Dunn B.M. (2003) Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N. Biochemistry; 42:15029-15035. 36. Ode H., Ota M., Neya S., Hata M., Sugiura W., Hoshino H. (2005) Resistant mechanism against nelfinavir of human immunodeficiency virus type 1 proteases. J Phys Chem B; 109: 565574. 37. Kolli M., Lastere S., Schiffer C.A. (2006) Co-evolution of nelfinavir-resistant HIV-1 protease and the p1-p6 substrate. Virology; 347: 405-409. 38. King N.M., Prabu-Jeyabalan M., Nalivaika E.A., Schiffer C.A. (2004) Combating susceptibility to drug resistance: lessons from HIV-1 protease. Chem Biol; 11: 1333-1338. 39. Nalam M.N., Ali A., Reddy G.S., Cao H., Anjum S.G., Altman M.D., Yilmaz N.K., Tidor B., Rana T.M., Schiffer C.A. (2013) Substrate envelope-designed potent HIV-1 protease inhibitors to avoid drug resistance. Chem Biol; 20: 1116-1124. 40. de Vera I.M., Smith A.N., Dancel M.C., Huang X., Dunn B.M., Fanucci G.E. (2013) Elucidating a relationship between conformational sampling and drug resistance in HIV-1 protease. Biochemistry; 52: 3278-3288. 41. Koh Y., Nakata H., Maeda K., Ogata H., Bilcer G., Devasamudram T., Kincaid J.F., Boross P., Wang Y.F., Tie Y., Volarath P., Gaddis L., Harrison R.W., Weber I.T., Ghosh A.K., Mitsuya H. (2003) Novel bis-Tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI) UIC-94017 (TMC114) with potent activity against multi-PI-resistant human immunodeficiency virus in vitro. Antimocrob Agents Chemother; 47: 3123-3129.
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42. Ohtaka H., Freire E. (2005) Adaptive inhibitors of the HIV-1 protease. Prog Biophys Mol Biol; 88: 193–208. 43. Lin Y-C., Perryman A.L., Olson A.J., Torbett B.E., Eldera J.H., Stout C.D. (2011) Structural basis for drug and substrate specificity exhibited by FIV encoding a chimeric FIV/HIV protease. Acta Crystallogr D Biol Crystallogr; 67: 540–548.
Figure Legends Figure1. The chemical structures of (A) Saquinavir (SQV) (B) Nelfinavir (NFV) Figure2. The melting curves of the native and D30N mutant of HIV-1 protease with and without the drugs nelfinavir (NFV) and saquinavir (SQV) as recorded by using differential scanning fluorimetry. Figure3. The simulated annealed mFO-DFC omit electron density map for SQV contoured at 2.5 σ level
showing two orientations of SQV bound in the active site. Two conformations of SQV are shown in yellow and cyan carbon atoms, respectively. Figure4. (A) SQV (yellow carbon atoms) bound in the D30N mutant HIV-1 protease (cyan cartoon representation). Mutant residue Asn30 side-chain is shown in red color in both the sub-units of the enzyme. (B) NFV (magenta carbon atoms) bound in the D30N mutant HIV-1 protease (cyan cartoon representation). Mutant residue Asn30 side-chain is shown in red color in both the sub-units of the enzyme. (C) Structural comparison of the D30N-SQV complex (yellow carbons) with the D30N-NFV complex (magenta carbons) in the active site (D) Comparison of hydrogen-bonding interactions of the P2 residues in SQV and NFV in the D30N mutants. The D30N-SQV complex is shown in yellow carbons while the D30N-NFV complex is shown in magenta carbons. The distances from NFV to the protein atoms are
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shown in black solid lines and the distances from SQV to the protein atoms are shown in black dashed lines. All the distances are indicated in Å units. Figure5. (A) Superposition of the D30N-SQV complex (yellow carbons) and the native HIV-1 proteaseSQV complex (green carbons) showing conformation of SQV bound in the respective structures (B) Structural effects around the D30N mutation site: The native HIV-1 protease-SQV complex is shown in green carbons while the D30N-SQV complex is shown in yellow carbons. The distances from SQV to Asp30 in the native complex are shown in black solid lines while the distances from SQV to Asn30 in the mutant complex are shown in black dashed lines. All the distances are indicated in Å units.
Supporting Information Figure S1. The fit of Arg8 in the 2mFO-DFC map contoured at 1.0 σ. D30N-SQV complex is shown in
yellow carbons while D30N-NFV complex is shown in magenta carbons. Distances from NFV to the protein atoms are shown in black solid lines and distances from SQV to the protein atoms are shown in black dashed lines. All the distances are indicated in Å units. Table S1. Comparison of the conformation of Arg8/Arg1008 in the D30N-NFV and the D30N-SQV complex
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Table 1: Data processing and refinement statistics Space group
P61
Unit cell parameters (Å)
a = b = 62.84, c= 82.49
Resolution (Å)
45.4-1.79 (1.9-1.79)*
Number of unique reflections
17150 (2740)*
I/σ(I)
16.6 (2.4)*
Rmerge (%)
5.5 (56.2)*
Completeness (%)
99.1 (98.2)*
a
Refinement statistics b
Rwork / Rfree (%)
20.6 / 23.5 (26.8 / 33.7)*
RMS deviations from ideal values Bond lengths (Å)
0.007
Bond angles (o)
1.09
Ramachandran plot non-glycine residues in Most favoured region (%)
96.2
Additionally allowed region (%)
3.8
Generously allowed region (%)
0.0
Disallowed region (%)
0.0
*Data for highest resolution shell are given in the parenthesis. a
Where, Ihkl,j is the intensity of an observation and is the mean value for its unique reflection.
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b
Where, Fobs and Fcalc are the observed and calculated structure factor amplitudes. Rfree is calculated exactly as Rwork using a random 5% of the reflections omitted from refinement.
Table 2: Hydrogen bonds between the protein main-chain atoms and SQV
Protein atom
SQV atom
Length (Å)
First orientation Asp29 N
P3 O
3.0
Asn30 N
P2-Asn OD1
3.0
Second orientation Asp1029 N
P3 O
3.0
Asn1030 N
P2-Asn OD1
3.1
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Received Date : 28-Jun-2014 Revised Date : 13-Nov-2014 Accepted Date : 21-Nov-2014 Article type
: Research Article
Structural basis of why nelfinavir resistant D30N mutant of HIV-1 protease remains susceptible to saquinavir
Running title: Why D30N mutant of HIV-1 PR is susceptible to saquinavir
Key words: X-ray Crystallography, HIV-1 protease, Saquinavir, Nelfinavir, Drug-resistance, Susceptibility.
Vishal Prashar1, Subhash C. Bihani1, Jean-Luc Ferrer2 and M.V. Hosur 3
1
Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai – 400085, India, 2 Institut de Biologie Structurale Jean-Pierre Ebel, Groupe Synchrotron, Commissariat a l’Energie Atomique et aux Energies Alternatives, Centre National de la Recherche Scientifique, Universite de Grenoble Alpes, 38027 Grenoble, France, 3 Present address: Advanced Centre for Treatment, Research and Education in Cancer, Kharghar, Navi Mumbai-410210, India.
∗
Corresponding author, Tel: 91-22-25594688, Fax: 91-22-25505151.
E-mail address: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12494 This article is protected by copyright. All rights reserved.
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Abstract Although anti-HIV-1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant tethered HIV-1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main-chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N-NFV complex. Apart from maintaining these two main-chain hydrogen bonds, the P2-Asparagine of SQV forms an additional hydrogen bond to the mutated side-chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug-resistant HIV-1 protease.
Introduction Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome (AIDS) (1, 2). According to a recent report by The Joint United Nations Programme on HIV/AIDS
(UNAIDS); about 35 million people worldwide are infected with HIV (3). Efforts that have been undergoing for the past many years to develop an effective vaccine against HIV have not yielded satisfactory results due to extreme variability of the virus and its escape mechanisms from the immune system (4). In this scenario, therapeutic approaches aimed at counteracting the molecules essential for the virus cycle have proved to be very useful (5). HIV-1 protease is one such major target enzyme because of the crucial role it plays in the processing of the viral poly-proteins (6). The introduction of HIV-1 protease inhibitors (PIs) in 1995 and the application of Highly Active Anti-Retroviral Therapy
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(HAART), which includes simultaneous use of the inhibitors of viral enzymes HIV-1 protease and HIV-1 reverse transcriptase, resulted in a vast decrease in mortality and a prolonged life expectancy of HIVpositive patients (7, 8). From 1996 to 2012, antiretroviral therapy prevented 6.6 million AIDS-related deaths worldwide (3). Besides, HAART also has a potential to limit the risk of HIV transmission (9). Until now, 9 PIs have been approved by the U.S. Food and Drug Administration (FDA) for use as drugs in the treatment of infected patients. But long term clinical efficacy of these drugs has been plagued by various issues (10, 11). Development of resistant strains of virus carrying mutated forms of HIV-1 protease enzyme is the major issue amongst them. Other problems include large dosages, undesirable side effects and patient non-adherence. With respect to these problems, the development of a new generation of PIs which possess improved pharmacokinetic properties and drug-resistance profiles is urgently required. In this regard, comparing the resistance profiles of the present anti-HIV-1 protease drugs and correlating them with their chemical structures is an important guide.
All the HIV-1 PIs, except tipranavir, in the market today are peptidomimetics. They mimic the peptide substrates of the enzyme, but the cleavable peptide bond has been replaced with hydroxyethylene or hydroxyethylamine based transition-state isosteres. Saquinavir (SQV) was the first HIV-1 PI released as drug by Hoffman-La-Roche (12). It was developed by replacing the Phe-Pro cleavage site of the peptide substrate with a non-cleavable hydroxyethylamine-based transition-state isostere (Figure 1A). The P1’proline was replaced with the dodecahydroisoquinoline (DIQ) bicyclic ring. At the P3 site, a quinoline ring was introduced while the P2’ site has tertiary (tert)-butylcarboxamide moiety. SQV is highly potent HIV-1 PI with nanomolar inhibitory constant but suffers from poor oral bioavailability. Nelfinavir (NFV) was developed at Agouron pharmaceuticals labs by the modification of SQV (Figure 1B). The P3quinoline ring was removed while the P2-Asparagine (Asn) was replaced with a 2-methyl-3-hydroxy
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benzamide moiety. At the P1 position, instead of a phenyl group, slightly longer S-phenyl side-chain was used. The rest of the substituents were same. These changes reduced the molecular weight and improved the oral bioavailability (13).
D30N is a major drug resistance mutation in HIV-1 protease against NFV (14). Compared to the wild-type protease, the binding of NFV by D30N mutant is decreased by 20-fold (15). One reported study found D30N mutation in 30.7% of patients failing first-line NFV-based HAART regimen (16). But this mutation does not affect SQV even though SQV and NFV have common functional groups. In fact, the D30N mutation does not exhibit cross-resistance with most of the other PIs (17, 18). To explore the reason for this, we solved the structure of HIV-1 protease D30N mutant in complex with the drug SQV. On comparing this structure with the D30N-NFV complex, we found differences in the hydrogen-bonding interactions between the drugs and the mutant enzyme. We propose that the presence of additional hydrogen bonds in the case of D30N-SQV complex is responsible for SQV's ability to maintain efficacy against the D30N mutant.
Materials and Methods Site directed mutagenesis Quick change site directed mutagenesis kit (Stratagene, La Jolla, CA) was used for making the D30N
mutation with oligonucleotide primer 5’-CTCCTCCAGTACAGTATTATCAGCACCGGTATCCAG-3’. Presence of the mutation was confirmed through DNA sequencing carried out by Eurofins genomics India Pvt. Ltd.
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Protein expression and purification HIV-1 protease tethered dimer used in the present study contains a five-residue linker, GGSSG,
covalently linking the two monomers. Expression and purification of HIV-1 protease tethered dimer followed the procedures reported earlier (19).
Differential scanning fluorimetry Eppendorf mastercycler ep realplex RT-PCR machine was used to carry out differential scanning fluorimetry to detect change in the protein melting temperature after the drug binding (20). The protein solutions were used to reach a concentration of 0.1-0.2 mg/ml in 50 mM sodium acetate buffer, pH 5.5 in 20 µL reaction mixtures. The final reaction mix also contained five fold molar excess of the respective drugs and 5 X SYPRO Orange dye (Sigma-Aldrich, Inc., St. Louis, MO). The RT-PCR machine was programmed to equilibrate the samples at 25°C for 10 minutes and then increase the temperature to 95°C at a rate of 1°C/minute, taking a fluorescence reading every 1°C using excitation and emission wavelengths of 470 nm and 605 nm respectively. The melting temperatures were obtained as maxima of the first derivatives of the melting curves.
Crystallization The single crystals were obtained by the hanging drop vapour diffusion method. The protein (5 mg/ml
in 50 mM sodium acetate, pH 4.5, containing 1 mM dithiothreitol) was reacted at the room temperature for 30 minutes with ten-fold molar excess of SQV dissolved in dimethyl sulfoxide. For crystallization, equal volumes of the reaction mixture and the reservoir solution (1% saturated ammonium sulfate, 200
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mM sodium phosphate, and 100 mM sodium citrate at pH 6.2) were mixed on a cover slip and sealed over the reservoir well at the room temperature.
X-ray data collection and refinement The crystals were equilibrated in the cryo-protectant (25% glycerol and 75% reservoir solution) before
flash-freezing, for exposure to X-rays on the FIP-BM30A beam-line (21). The diffraction data were indexed, integrated, and scaled by using the computer program XDS (22). The data processing statistics are given in Table 1.
Crystal structure was solved by difference Fourier method using the native coordinates (PDB ID 1LV1). The structure was refined in the computer program PHENIX by using the amplitude-based maximum likelihood target function (23). A test set containing 5.0% of randomly chosen reflections were reserved for the determination of Rfree (24). The simulated-annealed mFO-DFC omit electron density map for SQV
was calculated by omitting SQV atoms during simulated annealing as implemented in the program PHENIX, where the model was heated to a temperature of 2500K followed by cooling to 300K in 500 cooling steps. All interactive model building and molecular superpositions were carried out using the molecular modeling software O (25). The structural comparisons are based on superpositions of protein Cα atoms. All the figures were drawn using the computer program PyMOL (26). The atomic coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under the PDB ID 4Q5M.
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Results Differential scanning fluorimetry The melting temperatures (Tm) of the native and the D30N mutant of tethered HIV-1 protease, both with
and without the drugs NFV and SQV, were determined using differential scanning fluorimetry (Figure 2). With the native protease, both NFV and SQV showed a similar change in the Tm (22 ± 0.2 °C). But with
the D30N-NFV complex, shift in the Tm (∆ Tm) was 15.7 ± 0.9 °C while with the D30N-SQV complex it was 23 ± 0.8 °C suggesting that D30N mutation decreases the affinity of binding with NFV but remains susceptible to SQV.
Overall structure The crystal structure of D30N mutant of HIV-1 protease complexed with SQV was determined to a resolution of 1.79 Å in the space group P61. The results of crystallographic refinement are summarized in Table 1. The crystallographic asymmetric unit contains a tethered dimer of HIV-1 protease and one molecule of SQV. The residues in the first monomer are numbered as 1–99 and those in the second monomer are numbered 1001–1099. When the 2mFO–DFC electron density map was contoured at 1σ
level, there was no density for the linker region of the tethered dimer under study, suggesting that the linker region is not ordered. Alternate conformations were modeled for the side-chains of the residues 84 and 1084. The stereochemistry of the molecular model was quite good, with more than 90% of nonglycine residues occupying the most favored regions of the Ramachandran plot as defined in the program PROCHECK (27). Figure 3 shows the simulated-annealed mFO-DFC omit electron density map for
SQV. This density could be rationalized by SQV modeled in two orientations consistent with the approximate two-fold symmetry of the functional HIV-1 protease dimer.
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Drug-mutant interactions SQV binds to the mutant enzyme in an extended conformation through hydrogen bonds to the backbone atoms of the protein. The side-chain groups P3-P2’ of the drug interact with the residues in the corresponding sub-sites S3-S2’, mainly through hydrophobic interactions. The tert-butylcarboxamide moiety fits into the S2’ sub-site, while the DIQ bicyclic ring occupies the hydrophobic S1’ pocket. The central hydroxyl group binds to the catalytic aspartates of the enzyme through hydrogen bonds. The P1phenyl group occupies the S1 sub-site and the polar Asn side-chain of the inhibitor resides in the S2 pocket. The quinoline ring at P3 occupies the S3 sub-site. A water molecule bridges the P2 and P1’ carbonyls of the inhibitor to the Ile50 and Ile1050 main-chain amides in the flap region of the enzyme through hydrogen bonds in a manner analogous to that observed in the other HIV-1 protease inhibitor complexes. The Ile84 side-chain was located opposite to the inhibitor, and 84 CD1 forms van der Waals contacts with the P1-Phe and the P2’-tert-butylcarboxamide residue of SQV. In the second orientation of the inhibitor, the P1’-DIQ ring is located opposite to Ile84, and to optimize interactions with this moiety, Ile84 adopts an alternate conformation. The occupancy distribution of each conformation was 0.51 and 0.49 respectively, consistent with the occupancies of two-fold related conformations of the inhibitor.
Comparison with D30N-NFV complex The present complex was compared with the complex of NFV with D30N mutant of HIV-1 protease (PDB ID 2Q64) (15). The two structures were very similar with an rmsd, for 198 Cα atom pairs, of 0.2 Å. Relative positions of SQV and NFV in the active site are shown in Figure 4. Chemically, SQV and NFV are different on the non-primed side-chain groups (Figure 1). At the P2 site, there is an Asn residue in SQV while NFV has a 2-methyl-3-hydroxybenzamide moiety at this position. At the P1 site, the slightly longer S-phenyl side-chain of NFV partially extends into the S3 region and forms van der Waals contacts with
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Arg8. But in the case of SQV, the bulky quinoline ring at the P3 position would be in steric contact with Arg8/1008 depending upon the orientation, the inter-atomic separation between the C3 atom of quinoline ring and the NH2 atom of Arg8/1008 being only 2.1/2.0Å (Figure S1, supporting information). This has been avoided by a change in the conformation of the side-chain of Arg8/1008 in the SQV complex. Comparison of the torsion angles of Arg8/1008 in the two complexes is given in Table S1 (supporting information). The corresponding separation is now 3.4/3.3 Å. Similarly, there is a change in the main-chain torsion angles of the residue Gly48/1048 of the protein to avoid steric contacts with the P3-quinoline ring of SQV (Figure 4C), corresponding separation between the N1 atom of quinoline and the carbonyl oxygen of Gly48/1048 being increased from 2.8/2.7 Å to 3.3 Å. The P2-Asn of SQV forms a hydrogen bond with the Asn30 N of the protease backbone. In case of NFV, 2-methyl-3hydroxybenzamide moiety of the inhibitor occupies the S2 sub-site, with the m-phenol group hydrogenbonding to the Asn30 side-chain (Figure 4D). Carbonyl oxygen of the P3-quinaldic amide from SQV forms a hydrogen bond with the backbone nitrogen of Asp29 (Figure 4C). Thus, SQV makes two hydrogen bonds with the main-chain atoms of the protein. This was similar to what was previously observed in the crystal structure of SQV with multi-drug-resistant HIV-1 protease containing 20 mutations including D30N (PDB ID 3UFN) (28). Drug darunavir (DRV) also maintains backbone hydrogen bonds to the residues 29 and 30 in the D30N containing mutants (PDB IDs 2F80 and 3LZV) (29, 30). Although the aniline group on the other end of DRV has water-mediated interaction with the side-chain of Asn30 in these structures as compared to direct interaction with the side-chain of Asp30 in the native-DRV complex (PDB ID 2IEN) (31).
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Comparison with native HIV-1 protease-SQV complex Figure 5 shows the structural superposition of the present D30N-SQV complex on to the native HIV-1 protease-SQV complex (PDB ID 1HXB) (32). The two structures superpose to an rmsd of 0.2 Å over 198 Cα atom pairs. Interesting localized changes are observed in the conformation of the drug as well as of
the protein. There is a slight rotation around χ2 from -49° to -63° in Asn30 (from -51° to -68° in case of Asn1030) which enables formation of a hydrogen bond with the side-chain of Asn residue at the P2 position of SQV. The side-chain of P2-Asn of SQV also adjusts to form hydrogen bonds with both the main-chain and the side-chain of Asn30/1030 (Figure 5B). It may be pointed out that the hydrogen bonds from the inhibitor to the protein main-chain atoms are not disturbed by the mutation in the protein.
Discussion All the available anti-HIV-1 protease drugs are designed as competitive inhibitors. The substrate clearly forms a natural template for the design of such inhibitors. However, the peptidic inhibitors lack suitable physico-chemical properties and metabolic stability resulting in poor bioavailability. At the same time, to maintain the potency, the inhibitor molecules should maintain the interactions natural substrates make with the enzyme molecule. The design of first generation HIV-1 PIs included efforts to minimize the inhibitor molecular weight, and reduce its peptidic character while still imitating the interactions natural substrate made with the enzyme. NFV resulted from the optimization of SQV which suffers from poor oral bioavailability, because of its retained peptide character. However, resistance to NFV arises with the selection of D30N mutation in HIV-1 protease. The substitution of asparagine for aspartic acid at the position 30 is very specific to the NFV-resistant protease. The crystal structures of HIV-1 protease
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complexed with the peptide substrates showed that backbone atoms of the residues Asp25, Gly27, Ala28, Gly48, Asp29, and Asp30 are involved in making hydrogen bonds with the substrates (33, 34). Among these residues, Asp25, Gly27, Ala28, and Asp29 are well conserved. Very often, the Asp30 residue main-chain is involved in hydrogen-bonding to the P2/P2’ side-chains of various substrates. This interaction appears to be an important hydrogen bond whenever a polar side-chain is present at the P2/P2’ position. However, there is only one direct hydrogen-bonding interaction seen between the Asp30 side-chain of the enzyme and the P2 side-chain of NFV. This interaction with the D30N mutant is expected to be comparatively weaker or absent leading to reduced NFV affinity (35-37). On the contrary, SQV forms two direct hydrogen bonds with the main-chain atoms of the residues Asp29/1029 and Asp30/1030 (Table 2). When Asp30 is mutated to Asn30, the side-chain of Asn residue at the P2 position makes an additional hydrogen bond (Asn30/1030 ND2….P2-Asn OD1 = 3.1/3.1 Å) with the mutated sidechain of the enzyme (Figure 5B). These extra interactions are consistent with higher ∆Tm of the D30N-
SQV complex as compared to the D30N-NFV complex. The hydrogen bonds from SQV to the protein main-chain atoms are also observed in the structure of native HIV-1 protease-SQV complex. According to a recent hypothesis, the substrate-envelope defined as consensus volume occupied by the natural substrates, not only explains the substrate specificity but also the development of drug resistance (34, 38). The substrate-envelope hypothesis defined Asp30 amongst the vulnerable residues for drug resistance mutations in HIV-1 protease (38). Mutations into smaller or bulkier side chains at the vulnerable sites may impact drug binding leading to drug resistance (39). The D30N mutation does not affect the side-chain length but the nature of side-chain. This change results in the decrease of electrostatic binding energy between the m-phenol group of NFV and the 30th residue while the gain of a hydrogen bond between the P2-Asn of SQV and Asn30 of the protein. Although D30N mutation does not provide cross-resistance to most of the other PI drugs, but the accumulation of additional secondary mutations may affect the conformational distribution of HIV-1 protease leading to cross-resistance to
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other HIV-1 PIs (35, 40). Interestingly, in a multi-drug-resistant HIV-1 protease variant which carries 20 mutations including D30N, backbone hydrogen bonds are still maintained with SQV as in the present D30N-SQV complex (28). These observations support the previously-published conclusions that hydrogen-bonding interactions from the drug molecules to the main-chain protein atoms are less affected by mutations (31, 41-43).
Conclusions Comparing the resistance profiles of anti-HIV-1 protease drugs and correlating them to their chemical structures can provide important clues to the future drug design efforts. We report here the crystal structure of the complex between the drug SQV and D30N mutant tethered HIV-1 protease refined to 1.79 Å resolution. Structural comparison with the D30N-NFV complex showed following differences in the interactions: 1) hydrogen bonds from SQV are to the protein main-chain atoms, 2) The P2-Asn of SQV forms an additional hydrogen bond to the mutated side-chain of the residue 30. These differences could be the reason why D30N is not a drug resistance mutation against SQV. Further, the structure supports the previous studies which indicated that interactions with the main-chain atoms could be an important design principle to guide the development of new drugs that are effective against drugresistant mutants.
Acknowledgements We thank the National Facility for Macromolecular Crystallography, SSPD, BARC, for the X-ray diffraction and biochemistry equipment and Dr. D.R. Rao (CIPLA), for providing the drugs saquinavir and nelfinavir. We also thank Dr. Ajay Saini and Mr. S. R. Jadhav for technical help.
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Conflict of Interest The authors declare no conflict of interest.
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Figure Legends Figure1. The chemical structures of (A) Saquinavir (SQV) (B) Nelfinavir (NFV) Figure2. The melting curves of the native and D30N mutant of HIV-1 protease with and without the drugs nelfinavir (NFV) and saquinavir (SQV) as recorded by using differential scanning fluorimetry. Figure3. The simulated annealed mFO-DFC omit electron density map for SQV contoured at 2.5 σ level
showing two orientations of SQV bound in the active site. Two conformations of SQV are shown in yellow and cyan carbon atoms, respectively. Figure4. (A) SQV (yellow carbon atoms) bound in the D30N mutant HIV-1 protease (cyan cartoon representation). Mutant residue Asn30 side-chain is shown in red color in both the sub-units of the enzyme. (B) NFV (magenta carbon atoms) bound in the D30N mutant HIV-1 protease (cyan cartoon representation). Mutant residue Asn30 side-chain is shown in red color in both the sub-units of the enzyme. (C) Structural comparison of the D30N-SQV complex (yellow carbons) with the D30N-NFV complex (magenta carbons) in the active site (D) Comparison of hydrogen-bonding interactions of the P2 residues in SQV and NFV in the D30N mutants. The D30N-SQV complex is shown in yellow carbons while the D30N-NFV complex is shown in magenta carbons. The distances from NFV to the protein atoms are
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shown in black solid lines and the distances from SQV to the protein atoms are shown in black dashed lines. All the distances are indicated in Å units. Figure5. (A) Superposition of the D30N-SQV complex (yellow carbons) and the native HIV-1 proteaseSQV complex (green carbons) showing conformation of SQV bound in the respective structures (B) Structural effects around the D30N mutation site: The native HIV-1 protease-SQV complex is shown in green carbons while the D30N-SQV complex is shown in yellow carbons. The distances from SQV to Asp30 in the native complex are shown in black solid lines while the distances from SQV to Asn30 in the mutant complex are shown in black dashed lines. All the distances are indicated in Å units.
Supporting Information Figure S1. The fit of Arg8 in the 2mFO-DFC map contoured at 1.0 σ. D30N-SQV complex is shown in
yellow carbons while D30N-NFV complex is shown in magenta carbons. Distances from NFV to the protein atoms are shown in black solid lines and distances from SQV to the protein atoms are shown in black dashed lines. All the distances are indicated in Å units. Table S1. Comparison of the conformation of Arg8/Arg1008 in the D30N-NFV and the D30N-SQV complex
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Table 1: Data processing and refinement statistics Space group
P61
Unit cell parameters (Å)
a = b = 62.84, c= 82.49
Resolution (Å)
45.4-1.79 (1.9-1.79)*
Number of unique reflections
17150 (2740)*
I/σ(I)
16.6 (2.4)*
Rmerge (%)
5.5 (56.2)*
Completeness (%)
99.1 (98.2)*
a
Refinement statistics b
Rwork / Rfree (%)
20.6 / 23.5 (26.8 / 33.7)*
RMS deviations from ideal values Bond lengths (Å)
0.007
Bond angles (o)
1.09
Ramachandran plot non-glycine residues in Most favoured region (%)
96.2
Additionally allowed region (%)
3.8
Generously allowed region (%)
0.0
Disallowed region (%)
0.0
*Data for highest resolution shell are given in the parenthesis. a
Where, Ihkl,j is the intensity of an observation and is the mean value for its unique reflection.
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b
Where, Fobs and Fcalc are the observed and calculated structure factor amplitudes. Rfree is calculated exactly as Rwork using a random 5% of the reflections omitted from refinement.
Table 2: Hydrogen bonds between the protein main-chain atoms and SQV
Protein atom
SQV atom
Length (Å)
First orientation Asp29 N
P3 O
3.0
Asn30 N
P2-Asn OD1
3.0
Second orientation Asp1029 N
P3 O
3.0
Asn1030 N
P2-Asn OD1
3.1
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