Structurally HIV-pr is a homodimeric aspartyl protease [Fitzgerald and Springer 1991, Hong et al. 1998, Baca et al. 1993] whose activity is indispensable to the virus [Kohl et al. 1988, Seelmeier et al. 1988, McQuade et al. 1990]. It contains two identical chains of length 99 amino acids each with a molecular weight of ~ 22 Kd. The protease in its monomeric form is inactive and exhibits C2 symmetry in its unliganded state. The residues of HIV-pr are numbered as 1–99 and 100–198 for each monomer (chain A and B, respectively). Flap (residues 43–58 and 142-157), flap elbow (residues 35–42 and 134- 141), fulcrum (residues 11–22 and 110-121), cantilever (residues 59–75 and 158-174),
and ligand binding regions, the active site for both the chains-A & B are represented in the Figure 1.7(a) and the HIV-pr cleavage sites are represented in Figure 1.7(b).
(a)
(b)
Figure 1.7: (a) Structure of HIV-1 protease and (b) the Gag and Pol sequence cleaved at the blue and pink regions shown. (Adapted from MWCHO)
MA, CA, NC, p6 are the structural proteins that form the viral shell and capsid; RT is the reverse transcriptase and IN is the integrase enzymes. Note that HIV-pr itself is also a
part of the POL polypeptide chain [Debouck et al. 1999]. The most important region of HIV-pr is the active site or ligand binding region, which is characterized by the highly conserved catalytic triad sequence Asp-Thr- Gly in the dimer interface with contribution of each monomer as Asp25(25’)-Thr26(26’)-Gly27(27’) [Davies 1990]. The Asps are essential to the protease both catalytically and structurally while the Thr & Gly are buried in the active site. The active site is capped by two identical flexible glycine rich ß- hairpins, or flaps that restrict access to the active site. The mobile flap, residues 43-58, in either chains contains three characteristic regions: side chains that extend outward (Pro44, Met46, Phe53, Lys55, Arg57), hydrophobic chains extending inward (Lys43, Lys45, Ile47, Ile54, Val56, Asp58), and a glycine rich region (Gly48, Gly49, Gly51, Gly52) with Ile50 remains at the tip of the turn. The tips of the flaps, notably the glycine rich region, are highly flexible and this is thought to be necessary for substrate binding and product release. The tips curl into the cleft resulting in an open space wide enough for a substrate to enter. Schiffer et al. suggested that, upon curling, the electronegative active site is exposed and a neutral or positively charged substrate could potentially be guided into a conformation optimal for binding [Scott et al. 2000]. It is already notified that the curling of each monomer does not occur in a symmetric fashion given the symmetric axis. The active site wall region is present just below to the flap region, which contains the P1-loop (residues 78-81) in either chain. However the residues 78-80 do not have direct interactions to substrates or inhibitors. They have an indirect role in ligand binding through their contact with the flap region. X-ray diffraction studies [Spinelli et al. 1991, Lapatto et al. 1989, Wlodawer et al. 1989] showed that in the apo-form of HIV- pr, the flaps are packed onto each other loosely in a semi-open conformation, and the overall structure of the protease is less compact compared to the crystal structure of the inhibitor bound protease where the flaps are packed tightly, i.e., closed-structure [Louis et
al.1998, Jeyabalan et al. 2000].
For HIV-pr, there are a large number of published works, both computational and experimental, exploring its structural and mechanistic aspects. Mutations in HIV-pr are of two types, one at or near the active site and the others are far from the active site. Near the active site mutations may reduce the affinity of the enzyme for its substrate and
inhibitors by change in their direct interactions. However, non-active site mutations usually called as compensatory mutations, can act through long-range electrostatic interactions affecting the ligand binding in the active site region [Ohtaka et al. 2003]. In accordance to the NMR and computational studies, certain mutations change the conformational dynamics especially the flap dynamics of HIV-pr. This in turn changes the binding affinity of the ligands by alteration of cavity size leading to various drug resistant mutant forms of the protease [Rose et al. 1998]. Some mutations may confer both direct and indirect effect [Bandyopadhyay and Meher 2006]. Mutations have been identified in at least 50 positions within the HIV-pr [Gulnik et al. 2000, Kozalet al. 1996, Shafer et al. 1999, Boden et al. 1998, Hertogs et al. 2000]. Lists of mutations that occur on the HIV-pr backbone are shown in the Figure 1.8. A molecular level understanding of drug resistance requires the knowledge of both direct and indirect effects of mutation.
Figure 1.8: The structural distribution of the most common mutations associated with drug resistance in the HIV-1 protease. Mutations can occur anywhere in the protease structure. Mutations within the binding cavity are very conservative and operate by distorting the shape of the cavity. Conformationally constrained inhibitors have difficulties in adapting to the altered geometry and lose significant binding affinity.
(Adapted from Overcoming HIV-1 resistance to protease inhibitors. Drug Discovery Today. Disease Mechanisms | Infectious diseases Vol. 3, No. 2 2006)
For every protein/enzyme, its structure and dynamics play crucial role in the functional activities. For HIV-pr, the proper folding of the two monomers to form a catalytically competent dimer and the conformational dynamics especially the flap dynamics has special impact on its catalytic efficiency. From that point of view, it is necessary to gain knowledge about how the protein folds to its optimum function and how the dynamics of the protein help in the catalytic processes. A brief discussion regarding the flap dynamics and the folding dimerization of HIV-pr along with a general review of the works carried out by several groups is presented here.
1.6.1. Flap dynamics:
The flexibility of the flap tips is known as flap dynamics. It opens and closes the flaps determining the cavity size, which changes with several mutations leading to various drug resistant mutant forms of the protease by lowering the affinity for drugs.
Understanding the mystery behind flap dynamics is an important step in designing highly potent new drugs against HIV with lesser drug resistance. Thus several earlier computational studies intended at understanding flap dynamics. In recent past, MD has been used to study the dynamics of flaps: how the protease flaps move to allow substrate access to the active site and drug escape from the active site, how is the arrangement of the flaps during liganded and unliganded form and how mutations induce the conformational flexibility of the flap region [Collins et al. 1995, Scott et al. 2000, Piana et al. 2002, Freedberg et al. 2002, Perryman et al. 2004, Meagher et al. 2005, Bandyopadhyay and Meher 2006, Hornak et al. 2006, Perryman et al. 2006, Toth et al.
2006, Lauria et al. 2007, Seibold et al. 2007, Sadiq et al. 2007, Tozzini et al. 2007, Singh and Senapati 2008].
There has been a vast major of works performed by many groups in order to understand the drug resistance behavior of HIV-pr in a variety of ways. Many groups has explored on the different mutation positions and their effects on HIV-pr conformation, while others have concentrated on dimerization and folding of HIV-pr. However, there has been inadequate argument regarding the role of different simulation protocols and physiological conditions on the conformational dynamics or flap dynamics of HIV-pr.