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PDBsum entry 2pym
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References listed in PDB file
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Key reference
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Title
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Molecular analysis of the HIV-1 resistance development: enzymatic activities, Crystal structures, And thermodynamics of nelfinavir-Resistant HIV protease mutants.
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Authors
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M.Kozísek,
J.Bray,
P.Rezácová,
K.Sasková,
J.Brynda,
J.Pokorná,
F.Mammano,
L.Rulísek,
J.Konvalinka.
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Ref.
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J Mol Biol, 2007,
374,
1005-1016.
[DOI no: ]
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PubMed id
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Abstract
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Human immunodeficiency virus (HIV) encodes an aspartic protease (PR) that
cleaves viral polyproteins into mature proteins, thus leading to the formation
of infectious particles. Protease inhibitors (PIs) are successful virostatics.
However, their efficiency is compromised by antiviral resistance. In the PR
sequence of viral variants resistant to the PI nelfinavir, the mutations D30N
and L90M appear frequently. However, these two mutations are seldom found
together in vivo, suggesting that there are two alternative evolutionary
pathways leading to nelfinavir resistance. Here we analyze the proteolytic
activities, X-ray structures, and thermodynamics of inhibitor binding to HIV-1
PRs harboring the D30N and L90M mutations alone and in combination with other
compensatory mutations. Vitality values obtained for recombinant mutant
proteases and selected PR inhibitors confirm the crucial role of mutations in
positions 30 and 90 for nelfinavir resistance. The combination of the D30N and
L90M mutations significantly increases the enzyme vitality in the presence of
nelfinavir, without a dramatic decrease in the catalytic efficiency of the
recombinant enzyme. Crystal structures, molecular dynamics simulations, and
calorimetric data for four mutants (D30N, D30N/A71V, D30N/N88D, and D30N/L90M)
were used to augment our kinetic data. Calorimetric analysis revealed that the
entropic contribution to the mutant PR/nelfinavir interaction is less favorable
than the entropic contribution to the binding of nelfinavir by wild-type PR.
This finding is supported by the structural data and simulations; nelfinavir
binds most strongly to the wild-type protease, which has the lowest number of
protein-ligand hydrogen bonds and whose structure exhibits the greatest degree
of fluctuation upon inhibitor binding.
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Figure 1.
Figure 1. Superimposition of structures of HIV-1 PR mutants
in complex with nelfinavir. The positions of mutations are
indicated by spheres and numbered. The protease is shown in
ribbon representation. The D30N/N88D mutant is colored red, the
D30N/A71V mutant yellow, the D30/L90M mutant green and the D30N
mutant blue. Nelfinavir is represented as a stick model and its
solvent accessible surface is colored according to electrostatic
potential: blue, positive; red, negative (calculated with DS
ViewerPro 6.0 (Accelerys Software Inc.)).
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Figure 3.
Figure 3. Crystal structures of PR mutants in complex with
NFV. (a) Two orientations of NFV in the mutant PR active site.
The 2F[o]–F[c] electron density map is contoured at the 1.0σ
level. The carbon atoms for the two NFV conformations are
colored yellow and light red, respectively. Oxygen, nitrogen and
sulfur atoms are colored red, blue and gold, respectively.
Catalytic aspartate residues are shown with carbon atoms colored
gray. (b) Superposition of D30N mutant with wild-type complex
structure. Top view of the PR active site. Nelfinavir with
residues 30 are shown in stick representation with the carbon
atoms are colored gray and light red for the wild-type and D30N
mutant, respectively. Oxygen, nitrogen and sulfur atoms are
colored red, blue and gold, respectively. (c) Close van der
Waals contact of the M90 side-chain with the main-chain of the
catalytic residue. Van der Waals surfaces are shown as meshes
for catalytic aspartate and residue 90 in the D30N mutant and
the D30N/L90M mutant. (d) Hydrogen bond network formed by
residue 88 in wild-type and D30/N88D PR. The wild-type structure
is represented by gray carbon atoms, while the mutant structure
is represented by green carbon atoms. Water molecules (shown as
spheres) are colored correspondingly. Hydrogen bonds are
represented by dotted lines. The water mediated contact between
D88 and N30 in the mutant structure is represented by red lines.
(e) Plot showing the RMSDs for the positions of main-chain and
side-chain atoms of individual residues after superimposition of
the D30N PR and D30N/A71V PR structures.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2007,
374,
1005-1016)
copyright 2007.
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