HIV protease is a dimeric enzyme. The structure you just analyzed was only one monomer of the dimer. Many proteins function as dimers or higher oligomers. HIV protease only can cleave the peptide bond if it is correctly assembled into a dimer.
To download the structure of the dimeric enzyme, search the PDB Data Bank for the code '7HVP' and download the file. The structure contains a non-hydrolyzable peptide inhibitor different from the previous inhibitor. Open 7HVP.pdb with SYBYL after deleting the previous monomer.
One approach that medicinal chemists often take when designing inhibitors is to mimick the natural substrate but change the key part such that the enzyme cannot act on the modified molecule. Two sequences that are cleaved in the middle (-*-) by HIV protease are Ser-Gln-Asn-Tyr-*-Pro-Ile-Val-Gln and Thr-Leu-Asn-Phe-*-Pro-Ile-Ser-Pro. Recalling what you know of model proteases, such as chymotrypsin, think what kind of peptide analog you would make so that it would bind well to the enzyme but would be resistant to the cleavage.
Now analyze the bound peptide. The PDB website reveals that the peptide is chain C, so you may want to undisplay protein chains A and B for a moment. Notice that the non-hydrolyzable peptide analog is N-acetylated in the N-terminus, and esterified in the C-terminus. Answer the following questions:
Assignments (HIV Protease continues):Display the protein along with the inhibitor. As frequently is the case, the structure on the screen is quite complex. Even experienced researchers find it difficult to analyze such structures without some visual signposts. A simple, yet powerful trick is to use unique colors to highlight important parts of the molecule. For example, you can easily visualize the bound inhibitor if all its atoms are colored green. To select and color atoms of the inhibitor, follow the instructions below:
In the 'View' menu, under Display Style ->Color select 'Atoms... '
Click on the button marked Substructures, select ( these are at the very bottom; you can use SHIFT to select a range) C/ACE0, C/SER1, C/LEU2, C/ASN3, C/PHE4, C/CH25, C/PRO6, C/ILE7, C/VAL8, C/OME9, and click OK
Another window pops up; select GREEN as the color of the inhibitor peptide and hit OK
Examine the active site pocket closely. See how the inhibitor fits into the active site. Identify at least one amino acid of the protein that is very close to the inhibitor. You can do this as follows:
In the 'View' menu, under Display Style ->Label select 'Substructure'
Click on any atom an amino acid in the HIV protease that is near the inhibitor. The selected amino acid is marked with green squares.
Hit OK. The name and the sequence number of this amino acid are displayed. Record these in your notebook
Medicinal chemists can design new HIV drugs by carefully studying how active site residues of the HIV protease interact with bound inhibitors. HIV, however, is capable of changing its active site residues so that drugs will not bind to the mutant protein. This causes drug-resistant strains to appear quite quickly, and new drugs must be developed to ward off the the mutant virus. Drug resistance is common in many diseases, but particularly serious for HIV infections.
The scene is still quite busy and we can simplify the picture further by showing only the cartoon of the protein backbone. We again will use the Ribbon/Tube model of the backbone:
In the 'View' menu, under 'Biopolymer Display', select 'Ribbon/Tube '
Highlight Chains A and B by dragging cursor over the sequence window and hit OK:
Another window pops up; answer OK to color the protein according to the secondary structure.
In the 'View' select 'Undisplay Atoms...'
Click on the button marked Substructures, select (these are at the very bottom!)
ACE0, SER1, LEU2, ASN3, PHE4, CH25, PRO6, ILE7, VAL8, OME9, and click OK
Click Invert to select everything but the inhibitor peptide, and hit OK.
Can you identify the dimeric structure of the protein? Also, note that there is a defect in the rendering of this dimer: two yellow arrowheads are not connected! This is because the structure of highly flexible parts of the protein often cannot be determined from X-ray diffraction data.
Studies have shown that the activity of HIV protease depends on the active site aspartic acid (Asp25) residue. To selectively show this residue, follow these steps:
Assignments (HIV Protease continued):In 'View', select 'Display Atoms...'
Click on the button marked Substructures
Select A/ASP25 and B/ASP25 under the Residues column. (There are two Asp25 residues, as this is a dimer!)
Click OK and OK to finish selection
The structure and function of HIV protease are further described in a Chime-based tutorial from University of Virginia in Charlottesville. Please note that you need a Chime plug-in to see this structure. The plug-in is not installed on the Linux computers. The characterization of hydroxyethylamine analogues that eventually lead to the successful development of ritonavir is discussed in the J. Med. Chem., 1990, 33, pp 1285-1288.