Sandbox 645: Difference between revisions

← Older edit

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

Ashraf Hasasneh, Tahreer Mutair, Charles Short

Revision as of 19:43, 27 November 2012 view source Charles Short (talk \| contribs) 89 edits No edit summary ← Older edit		Latest revision as of 19:56, 27 November 2012 view source Charles Short (talk \| contribs) 89 edits No edit summary
Line 16:		Line 16:
	In the mid 1980's, the structure of HIV-1 Protease was hypothesized by Lawrence Pearl and William Taylor to consist of a single domain of the eukaryotic aspartic protease and to function in dimeric form.<ref>PMID: 3306411</ref> As NMR was not in wide use at this time, x-ray crystallography was implored but required a large quantity of large crystals. This was problematic as heavy atom derivatives had to be used to overcome the phase problems with this new structure and a considerable amount of protein was needed. Eventually these problems were overcome and the first images of HIV-1 protease where announced by the Merck group, who were most successful at large-scale protein purification and crystallization.		In the mid 1980's, the structure of HIV-1 Protease was hypothesized by Lawrence Pearl and William Taylor to consist of a single domain of the eukaryotic aspartic protease and to function in dimeric form.<ref>PMID: 3306411</ref> As NMR was not in wide use at this time, x-ray crystallography was implored but required a large quantity of large crystals. This was problematic as heavy atom derivatives had to be used to overcome the phase problems with this new structure and a considerable amount of protein was needed. Eventually these problems were overcome and the first images of HIV-1 protease where announced by the Merck group, who were most successful at large-scale protein purification and crystallization.

	Unlike most members of the aspartyl protease class, which generally exist as two domain monomers, HIV protease is a dimmer with two identical <scene name='Sandbox_645/Monomer/2'>subunits</scene> that are comprised of 99 amino acids. These two subunits come together to form a very narrow 7.46 angstrom diameter <scene name='User:David_Canner/Sandbox_HIV/Narrow_tunnel/1'>tunnel</scene> ~~for the ligand to navigate~~; enclosing the active site in the middle. Instead of one flap (a long flexible β-hairpin loop from the N-terminal domain) closing over the active site like in pepsins, the homodimeric retroviral enzyme has two poorly ordered flaps.<ref>PMID:20593466</ref> The flaps contribute to the substrate binding site by containing two aspartyl residues on the hydrophobic interior side. When the active site is unligated, <scene name='Sandbox_645/Ile_water/1'>Ile50</scene> is hydrogen bonded to a water molecule at the turn of each flap. The polyprotein is capable of making it to the active site not by squeezing through the narrow tunnel but rather by having the flaps <scene name='User:David_Canner/Sandbox_HIV/Hiv_tunnel_morph/3'>open</scene>. The water molecules hydrogen bonded to the Ile50 seem to play a pivotal role in the opening of the flaps as well as increasing the substrate-enzyme binding affinity. Each subunit donates the catalytic triad, Asp-Thr-Gly, to the highly conserved active site, which also closely resembles that of monomeric proteases like pepsin. The <scene name='User:David_Canner/Sandbox_HIV/Catalytic_triad/3'>active site</scene> residues are Asp25, Thr26 and Gly27 on each of the subunits. The active site is <scene name='Sandbox_645/Protease_conservation/1'>highly conserved</scene> in the retroviral aspartyl protease just as in the eukaryotic aspartyl proteases. As will be seen in the mechanism, the two <scene name='User:David_Canner/Sandbox_HIV/Catalytic_asp/1'>aspartate residues</scene> will hydrolytically cleave the polyprotein that comes into the active site.		Unlike most members of the aspartyl protease class, which generally exist as two domain monomers, HIV protease is a dimmer with two identical <scene name='Sandbox_645/Monomer/2'>subunits</scene> that are comprised of 99 amino acids. These two subunits come together to form a very narrow 7.46 angstrom diameter <scene name='User:David_Canner/Sandbox_HIV/Narrow_tunnel/1'>tunnel</scene>; enclosing the active site in the middle. Instead of one flap (a long flexible β-hairpin loop from the N-terminal domain) closing over the active site like in pepsins, the homodimeric retroviral enzyme has two poorly ordered flaps.<ref>PMID:20593466</ref> The flaps contribute to the substrate binding site by containing two aspartyl residues on the hydrophobic interior side. When the active site is unligated, <scene name='Sandbox_645/Ile_water/1'>Ile50</scene> is hydrogen bonded to a water molecule at the turn of each flap. The polyprotein is capable of making it to the active site not by squeezing through the narrow tunnel but rather by having the flaps <scene name='User:David_Canner/Sandbox_HIV/Hiv_tunnel_morph/3'>open</scene>. The water molecules hydrogen bonded to the Ile50 seem to play a pivotal role in the opening of the flaps as well as increasing the substrate-enzyme binding affinity. Each subunit donates the catalytic triad, Asp-Thr-Gly, to the highly conserved active site, which also closely resembles that of monomeric proteases like pepsin. The <scene name='User:David_Canner/Sandbox_HIV/Catalytic_triad/3'>active site</scene> residues are Asp25, Thr26 and Gly27 on each of the subunits. The active site is <scene name='Sandbox_645/Protease_conservation/1'>highly conserved</scene> in the retroviral aspartyl protease just as in the eukaryotic aspartyl proteases. As will be seen in the mechanism, the two <scene name='User:David_Canner/Sandbox_HIV/Catalytic_asp/1'>aspartate residues</scene> will hydrolytically cleave the polyprotein that comes into the active site.