HIV-1 Protease
Created by Shiry Guirguis
HIV-1 Protease (PR) in its biological form is a dimer composed of two identical
subunits (1). Since its
active site contains aspartic acid, HIV-1 Protease is categorized as a catalytic aspartate protease (2). This dimer is especially stable due to non-covalent interactions, stabilization from the active site, and hydrophobic packing of side chains (16). Dissociation of the two monomers would lead to the complete loss of function of the enzyme (13).
The general structure of HIV-1 Protease is a dimer of two 99-amino acid monomer subunits. Each subunit has one α-helix and nine β-sheets, with sheets 2-9 forming a "jelly-roll" β-barrell (15). Additionally, each monomer has approximately eight random coils. There is a distorted helix which resembles a wide loop between residues 36-42. There are two functionally important β-sheets: the NH2 terminal, composed of residues 1-4 and the outer part of a β-sheet, and the COOH-terminal between residues 95-99 (14). The terminal domain of HIV-1 Protease is made up of beta sheets 1-4 and 95-99 in each monomer, the random coil residues 4-9, and the alpha-helix residues 86-94 in each monomer (16).
The active site triad is made up of
Asp-25,
Thr-26, and
Gly-27 and is stabilized by Hydrogen bonding. Asp-25 hydrogen bonds to the backbone nitrogen Gly-27 (this is often referred to as the fireman's grip)(16). Ile-50 forms a hydrogen bond with the substrate by using a water molecule in the active site which allows for the active conformation of the enzyme (16). The catalytic triad is located in the
core domain of HIV-1 Protease which includes residues 10-32 and 63-85 and is valuable for its role in active site stabilization (16). The
"flap" domain is located in the Beta-hairpin region between residues 46-56 and functions in surrounding the active site and plays a role in specific ligand binding interactions (16). In HIV-1 Proteases which lack ligands (free enzymes), the positions of the beta-hairpin flaps are conformationally different (open/closed) through the continuous collapse and reformation of the flap ends (18). The flap domain has
Glycine-rich ends which allow it to be mobile and flexible, qualities necessary in order to bind and release products easily (16).
The polyproteins which compose Human Immunodeficiency Virus (HIV) are cleaved at specific sites by the
HIV-1 Protease in order to form functioning and infectious viruses (2). This formation of mature viruses is achieved by cleaving the proteins Gag and GagPol, which encode for the structural proteins of the virus and for its viral enzymes (4). Without HIV-1 Protease, these polyproteins would not be cleaved into functional products and ultimately would not produce viruses from a host cell, and HIV would be uninfectious (1). Due to the crucial role of HIV-1 Protease on the success of HIV, much work has been done to find effective PR
active site inhibitors. The active site of HIV-1 Protease is categorized with the retropepsin endopeptidases (16). This active site tends to be in an extended area where the protease can bind the substrate (3). This extended active site allows the substrate to come in contact with HIV-1 Protease in multiple specific regions. The chemical drug inhibitor present in this specific HIV-1 Protease complex is a substrate-based hydroxyethylamine
inhibitor (17). This inhibitor has the amino acid sequence Serine-Leucine-Asparagine-Phenylalanine-
Psi(CH(OH)-CH2N)-Proline-Isoleucine-Methyl L-Valinate (17). This inhibitor attaches to the ligand of HIV-1 Protease, an acetyl group (17) (C2H4O). The
cleavage site is between an aromatic amino acid (Phenylalanine, Tyrosine, or Tryptophan) and Proline or a cleavage between two hydrophobic amino acids (3). There are no human enzymes besides HIV-1 Protease which cleave between an aromatic amino acid and Proline (16). The enzymatic ability of HIV-1 Protease to catalyze is dependent on the mechanical fluctuation and conformational changes of the whole protein (15).
There are 95 water molecules present in the
crystallized structure of HIV-1 Protease and its inhibitor. In general, the nucleotide sequence of HIV-1 Protease is highly subject to change due to the large degree of error which results from HIV-reverse transcriptase; one of the few highly conserved sequences is found in the
catalytic triad active site previously mentioned and which is made up of Aspartic Acid, Threonine, and Glycine (16). HIV-1 Protease is able to maintain a high amount of selectivity for function while using a mainly non-conserved sequence (16). This ability is perhaps the reason why HIV-1 Protease is so difficult to render inactive. A remarkable amount of research has been done to create HIV-1 Protease drug inhibitors due to its indispensable role in activating the life cycle of HIV. The drugs developed as Protease inhibitors, such as HIV-1 Protease with a hydrolase inhibitor share the ability to bind to the active site of HIV-1 Protease in order to inhibit binding of natural substrates to the active site (16). However, continued use of these inhibitory drugs has resulted in the formation of resistant generations of HIV-1 Proteases (16). Many of these drugs are developed to enhance the stability of the enzyme-inhibitor unit and to imitate hydrogen bonding trends in the enzyme-substrate unit (16).
Two proteins were chosen from among those with similar tertiary structure (9). A protein with a similar tertiary structure is the Rous Sarcoma Virus (RSV) protease. The Rous Sarcoma Virus Protease, just as the HIV-1 Protease, is an aspartic protease dimer (6). This enzyme functions to cleave polyproteins of the Rous Sarcoma Virus, a virus which induces cancer-like growth in the connective tissue of chickens (7). A functionally active mutant of the RSV protease has been created via site-directed mutagenesis which is capable of performing the same cleavages performed by HIV-1 Protease on a set of HIV polyproteins, which indicates strong structural and functional similarities between the two proteins (5). Another protein with a similar tertiary structure is the Equine Infectious Anemia Virus (EIAV) Protease (8). Both the Equine Infectious Anemia Virus and the Human Immunodeficiency Virus are classified as lentiviruses, which are a type of retrovirus which uses reverse transcriptase to form DNA from RNA and can replicate in non-dividing cells (10). EIAV is a retroviral disease which is transmitted to horses most commonly through the bite of an insect and it is characterized by anemia, weakness, and sometimes sudden death (12). EIAV proteases play an important role in the early stages of viral infection and they function in processing nucleocapsid proteins inside intact viral capsids; understanding more about its structure has proved to be an important model for HIV studies and may lead to a better understanding of early-stage HIV inhibition (11).