Structural Biology
For the AIDS virus to mature, HIV protease must process the gag and gag-pol viral polyproteins specifically at nine non-homologous sites (Chou et al., 1996; Henderson et al., 1988; Kohl et al., 1988; Robins & Plattner, 1993). Thereby, the protease has been a prime target in the massive structure-based drug design effort to combat AIDS (Wlodawer & Erickson, 1993). As a result of this effort, nearly over 200 crystal structures of complexes are now available (http://www.ncifcrf.gov/HIVdb). The homodimeric HIV protease (Wlodawer et al., 1989) undergoes a large conformational change upon binding substrates or inhibitors. From peptidestudies, the enzyme optimally binds at least eight residues prior to the cleavage event. The active site of this aspartyl protease is at the dimer interface, with each monomer contributing a single aspartic acid residue.
Since, HIV protease recognizes various cleavage sites in the gag and gag-pol polyproteins, it is one of the examples of which one molecularsurface can recognize a variety of other seemingly non-homologous surfaces. How this symmetric enzyme recognizes its various substrates is still not clearly understood. Most of the ligands of these HIV-protease and inhibitor (esp. protein and peptide inhibitor) complexes are relatively small and short, they primarily cover only small part of the active site (i.e. P3-P3' subsites). There are four regions in each monomer where the protein structures deviate from each other due to ligands. These are P1-P1' subsites (Ile15-Leu19 or Ile15'-Leu19'), P2-P2' subsites (Met35-Lys45 or Met35'-Lys45'), P3-P3' subsites (Gly49-Gly52 or Gly49'-Gly52') and P3-P3' subsites (Pro79-Val82 or Pro79'-Val82').
vs. Renin Complexed With Polyhydroxymonoamide Inhibitor Bila 980 (Tong, L. et al, 1995)
Secondary Structure Elements in the HIV-1 Protease
Name Residues beta-strand a 1-4 beta-strand b 9-15 beta-strand c 18-27 beta-strand d 30-35 beta-strand a' 43-49 beta-strand b' 52-66 beta-strand c' 69-78 beta-strand d' 83-85 helix-h' 86-94 beta-strand q 95-99
In this enzyme report of HIV protease, how it adjusts conformation to recognize its asymmetric substrates is elucidated by some examples of the protease-inhibitor substrate complex (PDB ID: 1A8G, 2HVP, 9HVP and 1F7A, etc). One example (1F7A) has particular asymmetric (in both size and charge distribution) peptide as a ligand, with the sequence of Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser, which corresponds to the catalytic site that releases the capsid protein (CA-p2) from polyprotein. In this example, the catalytic Asp residue has been changed to Asn, so the substrate is therefore not cleaved due to loss of enzyme activity. Four regions in the protein backbone between the monomers rearrange in different manners to bind this substrate, also, side-chains and water molecules adopt a variety of positions for optimal binding. These adjustments function in an interdependent manner to accommodate the residues in the substrate. Interdependence of the conformational changes allows the protease to exhibit its wide range of substrate specificity, and also suggest the mechanism by which HIV protease achieves substrate specificity. This pattern provides a detailed example of protein-protein recognition in which molecules undergo conformational changes to recognize each other with high specificity.To accommodate ligand asymmetry, the two protease-monomers adopt different conformations by burying large surface area at the protease-substrate interface. The specificity for the CA-p2 substrate peptide is mainly hydrophobic, as most of the hydrogen bonds are made with the backbone of the peptide substrate. Two water molecules bridge the two monomers through the loops Gly49-Gly52 (Gly49'-Gly52') and Pro79'-Val82' (Pro79-Val82).Although the crystal structure of HIV-1 protease has been solved many times in the presence of peptidomimetics and inhibitors, the complex described here (1F7A) is considered as the closest representation of an actual HIV protease-substrate complex. Unlike other complexes, the length of the peptide (ten residues) is long enough to cover the entire binding epitope that has been determined by enzyme kinetics (Rasnick, 1997). Also, mutational studies on HIV-1 protease (Loeb et al., 1989) indicate that the activity of the enzyme is compromised by mutations that occur in the regions of the three-dimensional structure that are rigid. However, many of the residues in the four variable peak regions are associated with drug-resistant mutations (Erickson & Burt, 1996). Peak 1 is the hinge between the terminal domain and the active site. Peak 2 is the hinge before the flap. Peak 3 is the tip of the flap containing Ile50 that borders the P1, P2 and P3 substrate subsites. Peak 4 is the P1-loop that stabilizes the P1 and P3 substrate subsite. Two water molecules mediate P3 and P4 regions, also bridge between the monomers in many of the complex structures. Wat7 and Wat70 molecules are present in many complexes and another examples have at least one of them. Therefore, these water molecules likely facilitate the cooperative structural adaptation between the substrate subsites.
The shape complementarity (Lawrence & Colman, 1993) between the interacting surfaces of the peptide and the protein is very tight. Such a high level of complementarity is attained by way of protein changing its conformation to accommodate the substrate. Water molecules within the complex play an integral role in assisting adaptation. In addition to the water molecule mediating the interaction with Arg, other water molecules mediate interactions between the peptide backbone and the protein. Throughout the complex, symmetry-related parts of the protein form hydrogen bondsto different parts of the peptide. In the center of the complex, Wat3 bridges Ile50N and Ile50'N with the peptide at the carbonyls of P2 and P1', a pattern which is conserved across many of the protease- ligand complexes (Baldwin et al., 1995). Moving away from the cleavage site, coordinate water molecules that are also bound to the peptide. In the first monomer, Wat11 bridges to the carbonyl oxygen of ArgP3, whereas in the second monomer, Wat1 bridges to the carbonyl oxygen of GluP2' and Arg8'NH2 (of the protease). This pattern was also observed in the protease-KNI,272 complex (Baldwin et al., 1995). Moving further away from the cleavage site, Asp29 OD2 in the first monomer makes a direct hydrogen bond to ArgP3N, whereas in the second monomer Wat51 coordinates the hydrogen bond to MetP4N. In the final pair, the carbonyl of Met46 of the first monomer hydrogen bonds through Wat27 to LysP5O but in the second monomer binds directly to SerP5'N. Even this analysis of relatively few water-mediated interactions between the protein and the peptide clearly shows the asymmetry of the complex formation.
Asymmetry between the monomersThe protease clearly presents the asymmetric substrate with different types of binding surfaces to accommodate the variations in the substrate. The adaptability can be seen by directly comparing the interactions made by the equivalent residues of the two monomers with the substrate. Each monomer participates in five water-mediated hydrogen bonds to the peptide. The first monomer, however, makes only six direct hydrogen bonds with the peptide, whereas the second one makes ten.
The protease residues surrounding the substrate binding sites have been extensively studied and they often mutate to confer drug resistance (Erickson & Burt, 1996). Nevertheless, the adaptability of these subsites has not been sufficiently examined. The residues in each of the subsite pockets can be divided into two sets: (i) those residues whose positions adjust in response to the binding of a substrate but do not make direct interactions with the substrate and (ii) those residues that do make direct van der Waals contacts. Each residue of the substrate is in contact with at least five protease residues and between one and three water molecules; the residues with the most contacts are the P2 and P2' residues that are surrounded by eight and seven residues, respectively. To attain such a high level of shape complementarity with an asymmetric substrate, the van der Waals interactions in the subsites on opposite sides of the cleavage site, i.e. P2/P2', arise from different residues.
Index of the Enzyme Report: