How many polypeptide chains comprise the hiv protease

The active site is situated within the core and consists of three residues from each of the monomers. This pair of Asp—Thr—Gly triads is a form of active site common to aspartic proteases Toh et al. The catalytic triads are located at residues 25—27 and, due to symmetry, residues — The active site lies beneath the flaps, the tips of which are indicated in Figure 1.

These are located at the widest section of the protein. The flap tips are a glycine-rich area of the protein and thus have enhanced conformational flexibility Hong et al.

Structure of HIV-1 protease. The primary structure of chain A, labelled with residue numbers, is given beneath with the same shading. The terminal region is shown in black, the core in dark grey and the flaps in light grey; the inhibitor is shown in the same way as in a. The active site and the two isoleucine residues Ile50 and Ile at the very tips of the flaps are shown as black sticks.

A large number of structures of HIV-1 protease, with various mutations in the sequence and with various inhibitors bound, are available in the PDB. We have selected a set of structures Supplementary Fig. In order to prepare the crystal structures for rigidity analysis, all the crystal water molecules are removed, leaving the dimer unit of HIV-1 protease along with the inhibitor with which it was crystallized.

It has previously been suggested that the presence of buried water is important as it coordinates the protease flap residues Wlodawer and Vondrasek, However, we note that this should not affect the rigidity analysis presented by F irst , as molecular dynamics simulations Mamonova et al. The R educe software Word et al. For a set of eight test cases, we have examined the effect of running R educe before deletion of the crystal waters, and no significant difference in our results was observed.

The structures have a range of crystallization conditions, inhibitors and mutations. For each structure that was crystallized with a ligand, a copy is made with its inhibitor manually deleted, as in the molecular dynamics study by Hornak et al. We take it that this inhibitor-free structure is part of the ensemble of conformations explored by the inhibitor-free protein during its natural flexible motion Hornak et al. An underconstrained region is flexible, in the sense that dihedral angles can vary and so atoms can move without violating distance constraints Thorpe et al.

An isostatic region has an exact balance of constraints and degrees of freedom; the removal of any constraint would make the region flexible. In overconstrained regions, it is possible to remove a constraint without altering the rigidity of the region—there are constraints to spare.

A division such as this is called a rigid cluster decomposition RCD. The results of an RCD obviously depend on the set of bonds that are included to rigidify the structure.

In F irst , the strong bonding forces such as covalent bonds and hydrophobic interactions are always included. Long-range electrostatic forces and van der Waals forces do not contribute to the distance constraints between atoms because they are generally weak at this level and not highly directional Jacobs and Thorpe, Hydrogen bonds and salt bridges are included in the constraint network on a selective basis.

In F irst , the energy of each potential hydrogen bond in the structure is calculated using the Mayo potential Dahiyat et al. Thus, E cut determines the bond network, which in turn determines the RCD. This dependence on hydrogen bond geometry means that the atomic coordinates need to be precise, hence our selection of high-resolution structures.

An RCD can be visualized by appropriate labelling of the 3D structure of a protein. For easier comparison between different structures, it is also useful to consider the mainchain rigidity of the protein.

Large rigid clusters are represented by coloured blocks, while flexible regions are represented by a thin black line. Some key features of the RCD are clearly visible; a single rigid cluster can include residues from several non-contiguous sections of the primary sequence, and indeed in this dimeric protein a single large rigid cluster can include residues from both chains A and B.

The 1D representation shows clearly the change from a largely rigid structure to a largely flexible structure with a small shift in E cut. Rigidity dilution was carried out on the structures as crystallized a , c and after deletion of the inhibitor b , d , indicated by the suffix U. The areas surrounding the 50th and the th residue of each structure, which correspond to the flap tips are indicated by labelled arrows.

The highest E cut at which residue 50 is flexible is E flap and is highlighted in blue on the vertical axis. The resulting loss in rigidity is visualized by plotting a new 1D representation of the RCD whenever main chain rigidity changes. This highlights where in the structure most of the rigidity resides, and where rigidity is most readily lost.

We note that the manner in which these plots are constructed means that the vertical axis is non-linear with E cut Wells et al. Figure 2 consists of four dilution plots describing the rigidity of two crystal structures, 3LZU and 2HS1, before and after the deletion of an inhibitor. The structures 2HS1 and 3LZU were both selected because they were crystallized with the inhibitor darunavir, an antiviral drug that was approved by the FDA in June and was designed to form strong interactions with many different mutated structures of the protease Ghosh et al.

The dilution plots of the structures as they were crystallized are given in Figure 2 a and c. In both cases, the protein loses the majority of its rigidity abruptly, as the 1D profiles change from being mostly rigid to being mostly flexible with a single step in E cut.

The flap regions around residues 50 and are visible in the rigidity dilution as they become flexible at higher E cut than the main body of the protein. The helical regions near the terminal residues 86—94 are also visible as their rigidity persists after the main body of the protein has become flexible.

Previous studies Tan and Rader, ; Wells et al. Supplementary Table S1 and Fig. Plots for the same structures with the inhibitors deleted are given in Figure 2 b and d. Deletion of the inhibitor does not appear to alter the basic pattern of rigidity loss during the dilution. However, the flap regions become flexible at even higher E cut.

The active site of the enzyme is not distinctive in the rigidity analysis of either the inhibited or uninhibited structures, forming part of the largest rigid cluster until the protein becomes mostly flexible. The principal effect of the inhibitor upon the rigidity profile of the protein is to rigidify the flap tip region rather than the active site.

For quantitative analysis, it is typical to extract significant energy cutoff values from the RCD plots. Previous studies such as those on rigidity in the context of thermostability Rader, ; Radestocke and Gohlke, , have considered measures such as the folding core energy. This is defined to be the lowest line in the dilution plot where at least three consecutive residues are mutually rigid with at least three other consecutive residues of another secondary structural element Hespenheide et al.

In this instance we are interested in the function rather than the folding or melting point of the protein and so, in order to quantify the influence of inhibitor binding on the overall rigidity of the protein and on the flexibility of the flap regions, we define two significant values of E cut for our analysis.

We also note that for HIV-1 protease the folding core energy and E body are similar, typically separated in the dilution plots by just one line. Two aspartate amino acids, shown with asterisks, do all of the work, attacking the protein chain at the very middle.

Four drugs that attack HIV-1 protease are currently being used to treat people infected with the virus. In the illustration, the enzyme is displayed as a ribbon that follows the two protein chains and the drugs are shown as spacefilling models. The view is from the top--notice how the flaps cover the top of the drug molecules. Notice how similar these drugs are. They all have carbon-rich groups arrayed along either side, interacting with the sides of the active site tunnel.

They each have two oxygen atoms at the center, pointing towards us in the illustration, that interact with a special water molecule that is normally trapped under the flaps not shown here.

The drugs all mimic a protein chain, binding to the enzyme like protein chains do. But they are more stable than a protein chain. HIV-1 protease cannot cleave them, so they stay lodged in the active site, blocking the normal function of the enzyme. This illustration was created with RasMol. You can create similar pictures of these drug complexes by clicking on the PDB accession codes above, and then picking one of the options for 3D viewing.

About Molecule of the Month. Nowadays, in vitro and in silico studies of protease inhibition constitute an advanced field in biological researches.

In this article, we tried to simulate protease-substrate complexes in different states; a native state and states with whiskers deleted from one and two subunits. Our results showed that whisker truncation of protease subunits causes the dimer structure to decrease in compactness, disrupts substrate-binding site interactions and changes in flap status simultaneously. Based on our findings we claim that whisker truncation even when applied to a single subunit, threats dimer association which probably leads to enzyme inactivation.

We may postulate that inserting a gene to express truncated protease inside infected cells can interfere with protease dimerization. The resulted proteases would presumably have a combination of native and truncated subunits in their structures which exert no enzyme activities as evidenced by the present work. Our finding may create a new field of research in HIV gene therapy for protease inhibition, circumventing problems of drug resistance.

The replication cycle of human immunodeficiency virus type 1 HIV-1 requires three necessary enzymes. These are reverse transcriptase, integrase and protease. The protease, E. The C-shaped subunits of the protein join each other via non covalent interactions to form a dimeric structure of C2 symmetry. The interactions formed between N and C terminal residues, 1—5 and 95—99 referred to as whiskers, stabilize this dimeric assembly[ 4 , 5 ]. The basic role of the enzyme in HIV-1 infection is the selective and proficient cleavage of peptide bonds in viral glycoproteins of gag and pol genes during viral maturation processes[ 6 — 9 ].

Each flap is composed of two anti-parallel beta sheets connected by a beta turn structure. It is proposed that flaps act as a gate to control substrate or inhibitor access to the active site[ 11 — 13 ]. Being of functional importance, the residues located at the protease active site include about 40 percent hydrophobic residues[ 10 ]. These hydrophobic residues enable conformational changes during HIV-1 protease catalysis via exchanging Van der Waals contacts, maintaining structurally significant hydrogen bonds involved in the flap opening[ 16 ].

The dissociation of the dimeric enzyme to its subunits results in complete loss of its enzymatic activity. Therefore, the peptide sequence at the contact points of the dimers is highly conserved. It has been shown that complementary structures binding to interface residues prevent protease dimerization but decrease enzyme activity to one third of magnitude[ 4 , 17 ].

On the other hand, it was shown that truncating only four residues from N terminus of subunits results in formation of monomeric species of extremely decreased activity[ 20 , 21 ]. The whisker truncation removes hydrogen bonds which are normally formed between N and C terminal groups, hence threatening the dimer stability[ 22 ].

Given its critical role in viral maturation, the HIV-1 protease is considered to be an important target for drug designing to control HIV consequences[ 6 , 13 — 15 , 18 — 20 , 23 ]. Inhibitors could be directed to compete with substrates for the enzyme active site or even to interfere with enzyme dimerization.

None of these two inhibitory mechanisms are dealt with in the present work, instead, we aimed to simulate three complexes including native, single and double subunit truncated proteins as well as enzyme substrates.

The main scope of this work was to study the effect of whisker truncation on the enzyme structure and flap opening status providing details on substrate and enzyme binding site interaction. Obtained by X-Ray diffraction method and refined at the resolutions of 2. The same structure was used to obtain single truncated STP and double truncated DTP proteases by deleting four residues from N and C terminals whiskers of one or two subunits respectively.

Each enzyme-substrate complex was placed in the center of a rectangular box having dimensions of 4. The boxes were filled with SPCE water molecules using genbox command of gromacs package so that to cover the simulated proteins with water shell of 1. System neutralization was done by adding equivalent number of negative chloride ions. LINCS algorithm was used to apply constraint on bonds lengths. The systems were then subjected to a short molecular dynamic with all-bonds restrains for a period of ps before performing a full molecular dynamics without any restrains[ 26 ].

Berendsen, Thermostat and Barostat, were used for temperature and pressure coupling respectively and Particle Mesh Ewald PME method for electrostatic interactions. The time steps of 1 femtosecond were applied to all simulations. The protein binding site residues of protease were extracted using ArgusLab 4. SPSS, Inc. The similar pattern of progression in RMSD of all three complexes and the states of equilibrium indicates similar structural alterations.

The counts of hydrogen bonds formed between each of A and B subunits and the solvent are shown in Figure 2 a. The apparent decrease in number of hydrogen bonds formed between subunit A and the solvent in STC was because of reduced length of A chain as a result of truncation of four residues from each end.

However, comparing hydrogen bonds of native A chain in native complex with that of truncated one in DTC indicates that formation of hydrogen bonds is different in both A and B chains. In general, Figure 2 a shows that HIV-1 protease truncation increases protein hydration in its dimeric form. This means that upon truncating residues from N and C terminal, protease becomes more hydrated. Rotate the molecule until you find the N-terminal residue of this polypeptide. Now rotate the molecule so that you can identify the bound inhibitor; you can recognize the inhibitor easily because it contains quinoline and decahydroquinoline moieties that is not found in proteins.

The inhibitor is structurally similar to the normal substrate of the HIV protease, and it binds at a location known as the active site. As you just saw, macromolecular structures can be quite complex, and sometimes a simplified representation of the molecule is more useful than a detailed atomic representation.

One simplified way to present protein structures is by their secondary structures. To render the secondary structure of this protein follow these steps:.