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{{STRUCTURE_3ggu| PDB=3ggu | SCENE= }}


== '''Description''' ==
<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene> is a drug resistant [[HIV-1 protease]]. Shown is a patient's variant in complex with [[darunavir]].

HIV proteases (PR) are essential for the functioning of the retrovirus that causes AIDS. HIV needs active proteases to process gag & gap - polymerase polyprotein precursors into mature structural proteins and replicative enzymes. 
[[HIV-1 protease]]s contain a highly conserved region Asp - Thr - Gly (<scene name='Sandbox_Reserved_712/Activesiteasp25/1'>Asp25</scene>, <scene name='Sandbox_Reserved_712/Activesitethr26/1'>Thr26</scene> and 
<scene name='Sandbox_Reserved_712/Activesitegly27/2'>Gly27</scene>), with the aspartic residue being the <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene>
in the aspartyl protease.<ref> PMID:3290901 </ref>

Because of its importance for the life-cycle of the retrovirus, [[HIV-1 protease]]s are the major target for anti-HIV treatment. 
HIV protease inhibitors are the most potent agents used in anti-HIV treatment. However it appears that HIV-PRs develop a resistance to the inhibitor. <ref> PMID:12543689 </ref>

3ggu (also PR<sub>DRV5</sub>) is a mutated clinically derived PR that shows phenotypical resistance to darunavir. [[Darunavir]] is a human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) which has inhibiting effects on many HIV type 1 PR variants that show resistance to earlier-generation-PIs. <ref name="Molecular"> PMID:19535439 </ref>

== '''Darunavir: HIV-Protease Inhibitor''' ==

[[Darunavir]] (also known as TMC114) is a HIV-PR Inhibitor. The wild-type HIV as well as a large panel of PI-resistant recombinant viruses derived from clinical samples show high susceptibility to [[darunavir]]. <ref> PMID:15917527 </ref>
One reason for the high potency of [[darunavir]] is the strong binding of the PI to the main-chains of the protease active-site amino acids. <ref> PMID:14506019 </ref> A second reason might be the ability of the PI to fit closely into the substrate envelope. <ref>PMID:15479840</ref>
[[Darunavir]] shows a high genetical barrier to resistance development. In fact studies indicate that the development of resistance needs more specific mutations and develops more slowly. <ref> PMID:15917527 </ref>
[[Darunavir]] resistance-associated mutations are V11I, V32I, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, I47V, I50V, I54L, I54M, G73S, L76V, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and 
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene> ([[HIV Protease Resistance]]). Those mutations occurred in patients who have a high number of PI resistance-associated mutations. <ref> PMID:17416261 </ref>

== '''Structure''' ==
=== General structure of HIV-1 proteases ===

[[HIV-1 protease]]s are essential for HIV to survive. They are small enzymes, which are made out of two identical, 99 amino acids long, protein chains.
These chains form a tunnel, which is covered by so called (flexible) "flaps". The flaps's function is to "wrap around" the substrate-protein and holding it close to the tunnel and therefore to the active site.
The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> is located in the tunnel. The <scene name='Sandbox_Reserved_712/Activesite/2'>active site</scene> uses water molecules to break the subrate-protein chain. Its most important residues are two aspartate amino acids.
Inhibitors occupy similar positions as the natural substrate-protein chains.
<ref>HIV-1 Protease, June 2000 Molecule of the Month by David Goodsell  [http://dx.doi.org/10.2210/rcsb_pdb/mom_2000_6 DOI: 10.2210/rcsb_pdb/mom_2000_6]</ref>

=== X-ray structure analysis of 3ggu ===
[[Image:Positions_of_the_mutations_in_PR_variants_used_for_structural_studies.jpg|left|320px|thumb| Fig.1 Positions of the mutations in PR variants used for structural studies. <ref name="Molecular" />]]

[[Image: Structural_changes_in_PRdrv5_mutant.jpg|right|200px|thumb| Fig.2 Structural changes in PR<sub>DRV5</sub> mutant relative to wild-type PR. <ref name="Molecular" />]]
[[Image: Detailed_view_of_darunavir-enzyme_interactions.jpg|right|200px|thumb| Fig.3 Detailed view of the darunavir-enzyme interactions. <ref name="Molecular" />]]
The crystal structure was determined in complex with [[darunavir]] with 1.8-Å resolutions. The crystal is formed out of one PR dimer in the asymmetric unit with two inhibitor molecules bound in alternative orientations.
Surface residues <scene name='Sandbox_Reserved_712/R45/1'>R45</scene> and <scene name='Sandbox_Reserved_712/R55/1'>R55</scene> have disordered side chains, but the other amino acid residue changes could be modeled into well-defined electron density maps.

PR<sub>DRV5</sub> contains darunavir mutations <scene name='Sandbox_Reserved_712/V82t/1'>V82T</scene> and 
<scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> (see Fig.1, Part B, indicated in bold print) that are directly involved in substrate-darunavir-interactions (change of S2/S2' subsites).

The other 18 mutations are outside the binding cleft, but some are still in direct contact with the binding residues (e.g. <scene name='Sandbox_Reserved_712/L10i/1'>L10I</scene>, K20M, <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I54v/1'>I54L/V</scene> and <scene name='Sandbox_Reserved_712/L90m/1'>L90M</scene>).

<scene name='Sandbox_Reserved_712/Mutations_flap/1'>Mutations</scene> <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/M36l/1'>M36L</scene>, <scene name='Sandbox_Reserved_712/N37t/1'>N37T</scene>, 
<scene name='Sandbox_Reserved_712/P39s/1'>P39S</scene>, <scene name='Sandbox_Reserved_712/K45r/1'>K45R</scene>, M46I, <scene name='Sandbox_Reserved_712/I54v/1'>I54V</scene> and <scene name='Sandbox_Reserved_712/K55r/1'>K55R</scene> cause structural changes in the flap region and the flap hinge.
The pictures on the right (Fig.2 and Fig.3) show the regions (indicated in blue) that undergo structural changes caused by the mutations.
To see the full images, with changes in PR<sub>DRV1</sub> and comparative structure of wild-type, PR<sub>DRV1</sub> and PR<sub>DRV5</sub> follow the links: 
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f4/ Structural changes in PR<sub>DRV</sub> mutants relative to wild-type PR] and
[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/figure/f5/ Detailed view of the darunavir-enzyme interactions]

It was discovered that the inhibitor substituents can adjust their positions depending on changes of the substrate binding pockets. Among them the P2' aminophenyl moiety undergoes the biggest changes. <ref name="Molecular" />

== '''Phenotypic susceptibility  and enzymatic analysis''' ==
	 
Samples PR<sub>DRV1</sub> to PR<sub>DRV6</sub> have been cloned and expressed in ''E. coli'', purified and characterized in vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of specific PIs.

PR<sub>DRV5</sub> (<scene name='Sandbox_Reserved_712/3ggu/1'>3ggu</scene>) shows twenty mutated amino acids. The PR mutations that are associated to the darunavir resistance ([[HIV Protease Resistance]]) are <scene name='Sandbox_Reserved_712/L33f/1'>L33F</scene>, <scene name='Sandbox_Reserved_712/I84v/2'>I84V</scene> and 
<scene name='Sandbox_Reserved_712/L89v/1'>L89V</scene>.
	 
These mutations lead to a change in the susceptibility to the PI. In the case of 3ggu we observe a 32-fold susceptibility to [[darunavir]]. In comparison to [[amprenavir]], which is a structural related PI of [[darunavir]], it only shows a 24-fold susceptibility. The key-mutations that are responsible for the darunavir resistance are V32I, I54L and I54M. Those were not found in PR<sub>DRV5</sub> which explains the smaller phenotypic changes in the susceptibility to [[darunavir]]. (Complete Table: [http://jvi.asm.org.scd-rproxy.u-strasbg.fr/content/83/17/8810/T4.expansion.html/ Genotypes and phenotype changes analyzed with recombinant virus assay]) Nevertheless, determining the inhibition constants by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor, we can observe an increase of the K<sub>i</sub> value for all the samples in comparison to the wild-type virus. PR<sub>DRV5</sub> - which only has a specific activity of 5% of the wild-type value - also shows a smaller difference in k<sub>i</sub> value for [[darunavir]] in comparison to the other used samples. (Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t6/ K<sub>i</sub> values for the inhibitors of PR mutants]) <ref name="Molecular"> PMID:19535439 </ref>
	 	
[[Image:Relative_vitality_values_for_recombinant_PRs_and_PRIs.jpg | thumb | 220px | left | Fig.4 Relative vitality values. <ref name="Molecular"/>]]

The relative vitality values are defined as v = (K<sub>i</sub>k<sub>cat</sub>/K<sub>m</sub>)<sub>MUT</sub>/(K<sub>i</sub>k<sub>cat</sub>/K<sub>m</sub>)<sub>WT</sub>. It describes the relative ability of a PR species to hydrolyze its substrate when the inhibitor is present. This means the higher the vitality the more does the mutated PR support the viral replication. <ref name="Kinetic"> PMID:7626598 </ref>

The relative vitality is related to the phenotypic changes in the susceptibility to [[darunavir]]. As one can see in the diagram, the more darunavir-associated mutations there are, the higher is the relative vitality (PR<sub>DRV4</sub> > PR<sub>DRV1</sub> > PR<sub>DRV2</sub> > PR<sub>DRV6</sub>). Due to the fact that PR<sub>DRV5</sub> does not have the key mutations, it has a low vitality value for [[darunavir]] and the structural related [[amprenavir]] in comparison to the other samples. The [[lopinavir]] pattern looks different than the overall pattern of [[darunavir]] and [[amprenavir]], because it has a different structure and resistance profile than the others.(Fig.4) <ref name="Molecular"> PMID:19535439 </ref> 

Despite the many mutations the k<sub>cat</sub> values were still between 30% and 50% of the wild-type value. In contrast the K<sub>m</sub> values of the mutants were (mostly) four- to eightfold higher than the wild-type PR.
(Complete Table: [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738195/table/t5/ Enzyme characteristics of PR variants analyzed in  this study]) <ref name="Molecular" />

== '''Applications''' ==

HIV (human immunodeficiency virus) which causes AIDS (Acquired immunodeficiency syndrome) is one of the most threating viruses today. 
The high mutation rate of the virus leads to the fast development of drug resistance. The phenotypic characterization, enzyme kinetics and X-ray structural analysis of recombinant viruses offer a way to get a better understanding of the drug resistance.<ref name="Molecular" /> The knowledge we gain through that kind of experiments could help to develop new drugs for HIV-positive patients in the future.

== '''External Resources''' ==

[http://www.rcsb.org/pdb/explore.do?structureId=3GGU PDB file of 3ggu]

[http://www.rcsb.org/pdb/results/results.do?startAt=0&resultsperpage=50&qrid=77F7037F&tabtoshow=Current Human immunodeficiency virus type 1 (BRU ISOLATE)]

[http://www.rcsb.org/pdb/static.do?p=education_discussion/educational_resources/hiv-animation.html/ An interactiv image that explains all the compontents of a virus]

== '''References''' ==
<references/>

== '''Contributors''' ==
Julia Baaske, Angelika Wackerl