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. 2015 Jan 20;54(2):422-33.
doi: 10.1021/bi501088e. Epub 2015 Jan 7.

Defective hydrophobic sliding mechanism and active site expansion in HIV-1 protease drug resistant variant Gly48Thr/Leu89Met: mechanisms for the loss of saquinavir binding potency

Nathan E Goldfarb  1 , Meray Ohanessian, Shyamasri Biswas, T Dwight McGee Jr, Brian P Mahon, David A Ostrov, Jose Garcia, Yan Tang, Robert McKenna, Adrian Roitberg, Ben M Dunn
Affiliations

Defective hydrophobic sliding mechanism and active site expansion in HIV-1 protease drug resistant variant Gly48Thr/Leu89Met: mechanisms for the loss of saquinavir binding potency

Nathan E Goldfarb et al. Biochemistry. .

Abstract

HIV drug resistance continues to emerge; consequently, there is an urgent need to develop next generation antiretroviral therapeutics.1 Here we report on the structural and kinetic effects of an HIV protease drug resistant variant with the double mutations Gly48Thr and Leu89Met (PRG48T/L89M), without the stabilizing mutations Gln7Lys, Leu33Ile, and Leu63Ile. Kinetic analyses reveal that PRG48T/L89M and PRWT share nearly identical Michaelis-Menten parameters; however, PRG48T/L89M exhibits weaker binding for IDV (41-fold), SQV (18-fold), APV (15-fold), and NFV (9-fold) relative to PRWT. A 1.9 Å resolution crystal structure was solved for PRG48T/L89M bound with saquinavir (PRG48T/L89M-SQV) and compared to the crystal structure of PRWT bound with saquinavir (PRWT-SQV). PRG48T/L89M-SQV has an enlarged active site resulting in the loss of a hydrogen bond in the S3 subsite from Gly48 to P3 of SQV, as well as less favorable hydrophobic packing interactions between P1 Phe of SQV and the S1 subsite. PRG48T/L89M-SQV assumes a more open conformation relative to PRWT-SQV, as illustrated by the downward displacement of the fulcrum and elbows and weaker interatomic flap interactions. We also show that the Leu89Met mutation disrupts the hydrophobic sliding mechanism by causing a redistribution of van der Waals interactions in the hydrophobic core in PRG48T/L89M-SQV. Our mechanism for PRG48T/L89M-SQV drug resistance proposes that a defective hydrophobic sliding mechanism results in modified conformational dynamics of the protease. As a consequence, the protease is unable to achieve a fully closed conformation that results in an expanded active site and weaker inhibitor binding.

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Figures

Figure 1
Figure 1
(A) Superposition of the crystal structures for the closed (1HVR) and open (2PC0) forms of HIV PR. The ligand for the closed form has been omitted for clarity. The different mobile regions of PR are color coded with the open form in lighter shades: elbow flaps (39–57; blue), cantilever (58–75; orange), and fulcrum (11–22; red). Black arrows indicate proposed directions of movements of these regions during flap opening. (B) 90° rotation of PR shown in (A). Only monomer B is shown for simplicity. Green arrows represent regions of hydrophobic sliding during flap opening and closing. (C) Crystal structure of PRG48T/L89M-SQV (cyan) superposed with PRWT-SQV (1HXB) (silver). Red indicates regions where the r.m.s.d. of Cα carbons is >0.75 Å. Yellow spheres indicate the locations of the mutations Gly48Thre and Leu89Met. Mutations are show in only one monomer for clarity.
Figure 2
Figure 2
Molecular structure of the FDA approved protease inhibitor, saquinavir.
Figure 3
Figure 3
(A) Superposition of PRG48T/L89M-SQV (cyan) with PRWT-SQV (1HXB) (silver). In monomer A of PRG48T/L89M-SQV, hydrogen bonds between Gln18 and Gly16, as well as between Gln18 and Ser37 are lost resulting in approximately 1.5 and 2 Å displacement of the ‘teens and 30’s strand, respectively. The displaced regions are stabilized by new vdw interactions between Leu38, Ile15, and Tyr59. Hydrogen bonds are indicated as dashed lines (black). Vdw interactions are shown as dashed lines for PRG48T/L89M-SQV (red) and PRWT-SQV (gray). (B) Superposition of PRG48T/L89M-SQV (cyan) with PRWT-SQV (silver). Monomer B of PRG48T/L89M-SQV exhibits a greater displacement in the ‘teens region (2.5 Å) and smaller displacement in the 30’s strand (1.25 Å) compared to monomer A. Black dashes indicate hydrogen bonds. In PRG48T/L89M-SQV, there is a new hydrogen bonding interaction between the carbonyl oxygen of Ser37′ and the Nε of Gln18′. The displaced 30’s strand is further stabilized by a new hydrogen bonding interaction between Oε of Gln18′ and the Nζ of Lys20′.
Figure 4
Figure 4
(A) Superposition of the “flap” region of PRG48T/L89M-SQV (cyan/orange) and PRWT-SQV (silver). Monomer A is indicated by cyan/silver and Monomer B colored in orange/silver. PRG48T/L89M-SQV residues are rendered as dark red sticks and PRWT-SQV as silver sticks. Vdw interactions and distances are shown in red for PRG48T/L89M-SQV and gray for PRWT-SQV. Repositioning of the flap of monomer B of PRG48T/L89M-SQV is stabilized by new vdw interactions between Phe53′ and Thre48′. There is an increase in distance between the side chains of Ile54 and Gly51′ in PRG48T/L89M-SQV compared to PRWT-SQV resulting in a loss of a vdw interaction between the flaps of monomer A and B in PRG48T/L89M-SQV. Vdw contacts are also lost between Gly51 and Gly52′ and between between Ile50 and Thre48′ in PRG48T/L89M-SQV. (B) The root mean squared fluctuations of the Cα atoms of PRWT-SQV and PRG48T/L89M-SQV. The average structure of the ensemble was used as the reference. The differences between the RMSF values of the PRWT-SQV and PRG48T/L89M-SQV were calculated. A positive number indicates that a particular residue of PRWT-SQV fluctuates more than PRG48T/L89M-SQV and vice versa. (C) ΔRMSF values in (B) mapped onto PR illustrating where the largest differences between PRWT-SQV and PRG48T/L89M-SQV occur. (Red signifies residues that are less flexible in PRG48T/L89M-SQV compared to PRWT-SQV. Yellow signifies residues that are more flexible in the PRG48T/L89M-SQV relative to PRWT-SQV.
Figure 5
Figure 5
Stereo images of hydrophobic core residues in monomer B of (A) PRWT-SQV and (B) PRG48T/L89M-SQV. PR is color coded to represent the fulcrum (magenta: 11–22), cantilever (orange: 58–78), elbow flap (blue: 39–57), and 30’s strand (red). Vdw interactions are shown as black dashed lines. In PRWT-SQV, vdw interactions are amenable for hydrophobic sliding; however, in PRG48T/L89M-SQV a redistribution of vdw forces results in altered strand interactions and modified hydrophobic sliding.
Figure 6
Figure 6
(A) PRWT-SQV is shown in silver and PRG48T/L89M-SQV is shown in cyan and magenta. In PRG48T/L89M-SQV, the side chain of Val82′ is relocated 156° away from the P1 Phe of saquinavir and the guinidinium group of Arg8′ is rotated 117° away from the active site. The repositioning of Val82′ and Arg8′ results in the opening of the base of the S1/S3 subsite in the PRG48T/L89M-SQV structure and results in the loss of three vdw contacts between the P1 Phe of SQV and S1 of PRG48T/L89M-SQV. In the S3 subsite, introduction of the threonine side chain at position 48 creates new 2.9 Å noncononical O–H---π interactions between the Thr48 OH and the C3 and C4 of Phe53 resulting in the loss of a hydrogen bond between N1 of SQV and the main chain carbonyl of Gly48. Only one O–H---π interaction is shown for clarity. (B) Surface representation of PRWT-SQV (gray). Arg8′ is colored blue andVal82′ is colored orange. (C) Surface representation of PRG48T/L89M-SQV.
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