Adaptability and flexibility of HIV-1 protease Mukesh Kumar and Madhusoodan V. Hosur Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, India Even though more than 200 three-dimensional structures of HIV-1 protease complexed to a variety of inhibitors are available in the Protein Data Bank; very few struc- tures of unliganded protein have been determined. We have recently solved structures of unliganded HIV-1 protease tethered dimer mutants to resolutions of 1.9 A ˚ and 2.1 A ˚ , and have found that the flaps assume closed- flap conformation even in the absence of any bound lig- and. We report comparison of the unliganded closed-flap structure with structures of HIV-1 protease inhibitor complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV-1 protease in the crystal. These studies reveal that the least flexible region present in the active site of HIV-1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexi- bility and adaptability of proteins in general. Keywords: adaptability; flexibility; HIV-1 protease; inhibi- tors; structure. The significance of human immunodeficiency virus type 1 (HIV-1) protease in the life cycle of HIV has made it a prime therapeutic target for the development of anti-HIV drugs. This has resulted in the determination of a large number of structures of identical or closely related sequences with different ligands by X-ray, NMR and theoretical molecular modelling approaches. To date a total of 213 such structures are available from the Protein Data Bank (PDB) [1] and HIV-protease database (http://home2.ncifcrf.gov/HIVdb). In spite of such a large number of studies on a single system consisting of closely related sequences, we still lack the proper understanding to tackle the problem of drug resistance mutations, a typical characteristic associated with HIV infection. This inability to understand the behaviour of the protease demands the development of more sophisti- cated tools for the study of protein structure. There is an inevitable need to improve our knowledge of the inherent flexibility and adaptability in the three-dimensional struc- ture of proteins. These two features of proteins are as important as the structure itself and are very often responsible for the functional characteristics of a particular structure under different ‘natural’ and ‘stressed’ environ- ments. As reported by Zoete et al.[2],X-raystructures through the B-factors, molecular dynamics simulations and normal mode analyses give a fair idea about the fluctuations of different residues/regions of proteins about their mean positions. This mobility may be described as the flexibility of the region. In contrast, adaptability of a residue is the ability of that residue to alter its mean position itself in response to changes in its chemical environment. Information about residue adaptabilities can be obtained only by detailed comparison of the protein structures in the presence and absence of the environmental stress, which may be in the form of a point mutation or a bound ligand. We determined earlier the structure of the double mutant C95M/C1095A in an unliganded state. By comparison with the isomorphous structure of unliganded C95M single mutant, we analysed [3] the effect of C95A mutation on the structure of the protease itself. In addition to the changes on the dimer interface, we observed that the catalytic aspartates Asp25/ 1025 and the catalytic water move to make this catalytic site more accessible to the substrate (Fig. 1). This structural change in the active site was subsequently shown to be associated with increased autolysis rates in protease carrying the C95A mutation [3]. To analyse further the behaviour of these catalytic aspartates, we have extended our comparison to structures of C95A mutant protease complexed with different inhibitors. We find that the polypeptide chain segment 23–26, which includes the catalytic aspartates, is structurally most adaptable even though it is least flexible. This observation is significant and can contribute to the fact that HIV-1 protease enzyme has the unique property of cleaving protein substrates at eight different amino acid sequences [4]. Materials and methods ‘Closed-flap’ structure of unliganded HIV-1 protease The X-ray structures of C95M single mutant- and C95M/ C1095A double mutant-tethered HIV-1 protease have been solved to 1.9 A ˚ and 2.1 A ˚ resolution, respectively, and deposited in the PDB (files 1G6L and 1LV1). Residues in the first monomer have been numbered 1–99 and those in the second monomer 1001–1099. Residues in the linker are numbered 101–105. Electron density was not visible for the linker peptide suggesting that the linker was highly flexible. Correspondence to M. V. Hosur, Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, India-400085. Fax: +91 22 5515050, Tel.: +91 22 5593614, E-mail: hosur@magnum.barc.ernet.in Abbreviations: HIV-1, human immunodeficiency virus type 1; Rmsd, root mean square deviation. Enzyme: HIV-1 protease (EC: 3.4.23.16). (Received 2 August 2002, revised 14 November 2002, accepted 27 January 2003) Eur. J. Biochem. 270, 1231–1239 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03483.x These two structures revealed for the first time the closed- flap conformation of HIV-1 protease even in the absence of any ligand bound in the active site [3,5]. Comparison of such a structure with the closed-flap ligand-bound structure is expected to be more rational and useful. Inhibitor complex structures containing C95A HIV-1 protease Eight structures deposited in the PDB (files 1DAZ, 1A94, 1HWR, 1HVR, 1QBS, 1DMP, 1QBR and 1QBT) are of complexes between different types of inhibitor molecules and HIV-1 protease containing the C95A mutation. In two of these structures (1DAZ and 1A94), the protein is complexed with a peptide inhibitor [6,7] and in the remaining six (1HWR, 1HVR, 1QBS, 1DMP, 1QBR and 1QBT) the enzyme is complexed with a cyclic urea-based inhibitor [8–13]. All the cyclic urea-based inhibitors contain a central seven-membered ring having a urea moiety, and a diol group. P1/P1¢ substituents are attached to C3/C6 atoms of the central ring and P2/P2¢ substituents are attached to urea nitrogen atoms of the ring as shown in Fig. 2A. The seven membered ring sits in the active site cavity as a bridge between the flaps and the catalytic aspartates. While the urea moiety makes hydrogen bonds with main chain NH of the flap residue Ile50 from both the monomers, the diol group makes hydrogen bonds with the two catalytic residues Asp25 and Asp1025. The P2 moities are all different among the six inhibitors listed in Fig. 2B. All of the superposition and structure comparisons were performed using software O [14] and molecular images were made using MOLRAY [15]. All structure alignments were based on superposition of Ca atoms of all residues of the entire HIV-1 protease dimer. With the whole structures so superposed, the root mean square deviations (Rmsd values) quoted for any polypeptide segment is then calcu- lated for the Ca atoms of the amino acid residues in that polypeptide segment. Error-scaled difference distance mat- rix plots were generated using the program ESCET [16]. The errors in atomic positions for error-scaling were estimated using ‘diffraction-component precision index’ plus linear B-scaling [17]. Results Structural features of C95A mutation in HIV-1 protease Fig. 3 shows a B-factor vs. residue number plot for C95M/ C1095A HIV-1 protease. It may be seen in the figure that the residues 23–26 in the first monomer and 1023–1026 in the second monomer located in the active site of protease have the lowest B-factors suggesting that these residues are relatively ‘rigid’ entities in the protein structure. The electron density in this region is also very well defined indicating that these residues have very well-defined conformation. Karplus and his colleagues have drawn a similar conclusion recently, after analysing 73 crystal structures of HIV-1 protease complexed to a variety of inhibitors. Comparison of the C95M/C1095A double mutant with the C95M single mutant HIV-1 protease structure by superposition of all 198 Ca atom pairs showed that the main chain of the residues 23–26/1023–1026 has moved towards the flap after C1095A mutation. Interestingly, this movement includes movement of catalytic aspartates Asp25/1025 along with the bound catalytic water (Fig. 1) in a direction that would enable more access to the catalytic centre for the incoming substrate. Eventually this resulted in increased autolysis after C1095A mutation [3]. Therefore, this subtle movement in the catalytic centre is of prime importance from the point of view of enzymatic activity. These atomic movements were also verified by means of difference distance matrix plots, which have the advantage of not depending on any particular frame of superposition. These plots are shown in Fig. 4 for one subunit. In Fig. 4A the upper right triangle shows the normal difference distance matrix plot while the lower left triangle shows error-scaled difference distance matrix plot for the structures 1LV1 vs. 1G6L. It may be seen that the region 23–26 is involved in significant atomic Fig. 1. Superposition of catalytic aspartates of C95M HIV-1 protease (shown by green carbons) and C95M/C1095A HIV-1 protease (shown by yellow carbons). 1232 M. Kumar and M. V. Hosur (Eur. J. Biochem. 270) Ó FEBS 2003 movement. The error-scaled difference distance matrix plot also shows that this segment in 1LV1 has moved towards the flaps and along the direction joining the active site aspartates to the tips of the flaps. Structural effect of inhibitor binding on HIV-1 protease The closed-flap structure of the unliganded C1095A mutant enables, through structural comparisons, accurate Fig. 2. Basic structure of P2 analogues of cyclic urea inhibitors (A) and properties and structures of all inhibitors (B). Ó FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur. J. Biochem. 270) 1233 identification of the changes, if any, brought about by inhibitor binding. The unliganded and all six of the urea inhibitor complex structures belong to the same space group (Fig. 2B) further enhancing the meaningfulness of these comparisons. Table 1 shows the Rmsd values for pair-wise superposition of the unliganded C95M/C1095A structure with each of the six cyclic urea-complexed structures listed in Fig. 2B. Fig. 3. Average B-factors for different residues of C95M/C1095A HIV-1 protease. Fig. 4. Difference distance matrix plot. The upper right triangle shows the normal difference distance matrix plot and the lower left triangle shows the error-scaled difference distance matrix plot. (A) 1LV1 vs. 1G6L. (A) 1LV1 vs. 1QBS. 1234 M. Kumar and M. V. Hosur (Eur. J. Biochem. 270) Ó FEBS 2003 The corresponding Rmsd for superposition onto the 1DAZ structure, which belongs to the orthorhombic crystal system, is 0.45 A ˚ . Thus, in spite of different crystal environments, the overall conformation of HIV-1 protease is very similar, with Rmsd values ranging from 0.26 to 0.45 A ˚ . However, there are subtle differences character- istic of the bound ligand. Fig. 5 shows the superposition of peptide complex structure 1DAZ with unliganded structure 1LV1. Interestingly, the catalytic aspartates Asp25/1025 in the two structures (1LV1 and 1DAZ) are very much super imposable, and the Rmsd for residues 23–26/1023–1026 is 0.11 A ˚ . Similar is the case with another peptide–inhibitor complex (PDB code 1A94) suggesting that the linear chain of peptide inhibitors does not cause much alteration in the position of catalytic aspartates. However, when the cyclic urea complex structures are superimposed on to the unliganded structure 1LV1, the position of the main chain of the catalytic residues Asp25/1025 is significantly different (see Fig. 7). The shift of the Ca atoms of Asp25/1025 ) when averaged over all six comparisons ) is 0.37 A ˚ , and has apparently been induced by the inhibitor to relieve what would otherwise be bad steric contacts (2.7 A ˚ and 2.81 A ˚ )betweenthe cyclic urea ring of the inhibitor and the aspartate side chains, as shown in Fig. 6. This movement of catalytic Asp25 is also revealed by the error-scaled difference distance matrix plot (Fig. 4B) for comparison of 1LV1 and 1QBS structures. The residues 23–26 in 1QBS have moved away from the flap-tips, and to the direction joining active-site aspartates to flap-tips, in agreement with results of molecular superposition (Fig. 7). It thus appears that the position of polypeptide chain segment 23–26/1023–1026 is influenced by both mutational stress C95A (Fig. 1) below the active site cavity and also by steric stress from inhibitor binding in the active site cavity (Fig. 7). Thus, of utmost interest is the fact that what is considered the most rigid polypeptide segment in the active site of HIV-1 protease can alter its position when required. Structural rearrangement in the form of movements of the side-chain atoms in the S1 pocket on Table 1. Rmsd values for pair-wise superposition of unliganded C95M/C1095A structure with each of the six cyclic urea-complexed structures shown in Fig. 2B. 1LV1 1HWR 1DMP 1QBS 1HVR 1QBR 1QBT 1LV1 0.00 0.39 0.37 0.34 0.32 0.35 0.35 1HWR 0.00 0.38 0.39 0.41 0.41 0.41 1DMP 0.00 0.32 0.36 0.33 0.34 1QBS 0.00 0.31 0.33 0.34 1HVR 0.00 0.37 0.37 1QBR 0.00 0.26 1QBT 0.00 Fig. 5. Superposition of peptide inhibitor-bound C95A HIV-1 protease in PDB file 1DAZ (shown by green carbons) on unliganded HIV-1 protease structure in PDB file 1LV1 (shown by yellow carbons). The water molecule is present only in the unliganded structure. Ó FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur. J. Biochem. 270) 1235 inhibitor binding has been recently observed crystallo- graphically [18]. However, the movements of the main chain containing catalytic aspartates Asp25/1025 of the active site has been reported for the first time here. Thus the main chain containing these catalytic aspartates, although having small B-factors, is very much adaptable to the external stress induced by the cyclic urea scaffold. This observation suggests that it is important to distinguish such adaptability from the concept of general flexibility arising out of multiple conformations of the side chains and thermal motions of the atoms therein. In the six cyclic urea complex only the substituents at the P2/P2¢ site are different as shown in Fig. 2B. While changes induced by cyclic urea inhibitors in the S1/S1¢ pocket are very similar (Fig. 7), changes induced in the S2/S2¢ pocket depend on the substituents at the P2/P2¢ site. These substituents point directly towards the polypeptide segment 27–32 of the S2/S2¢ subsite. The allyl substituent at this site in the case of XK216 is small and therefore there is not much change in the conformation of these residues (Fig. 8). However, when a larger hydrophobic moiety like the naphthyl group, as in case of XK263, is present at the P2/P2¢ Fig. 6. Superposition of cyclic urea bound HIV-1 protease in PDB file 1HWR (shown by green carbons) on unliganded HIV-1 protease (shown by yellow carbons). The relieved steric contacts are marked. Fig. 7. Superposition of cyclic urea inhibitor-bound HIV-1 protease (shown by green carbons) on the unliganded structure (shown by yellow carbons). 1236 M. Kumar and M. V. Hosur (Eur. J. Biochem. 270) Ó FEBS 2003 site, the main chain as well as the side chain of the hydrophillic residue Asp30 moves away as shown in Fig. 9. Further, when a hydrophilic group such as NH 2 replaces one of the benzene rings of the naphthyl moiety above, as in the case of DMP450, the aspartic acid residue Asp30 moves toward the inhibitor and makes a hydrogen bond with this new substituent (Fig. 10). More bulky groups at this site, as in SD146, causes steric hindrance near Asp30 inducing the side chain of Asp30 to move away (Fig. 11). Discussion and conclusion Structure alignments are very often used to derive functional information on proteins. Distant evolutionary relationships are also detected exclusively by structure alignments. Structural superpositions using complete proteins or domains thereof, are a powerful method of identifying ligand- or mutation-induced conformational changes. The inferences drawn from such comparisons are reliable especially when the structures superimposed are variants of the same protein crystallizing in isomorphous space groups, as is the case here. Comparison of unliganded and ligand-bound HIV-1 protease is important for obtaining information about adaptability of residues in the active site. The flaps in unliganded HIV-1 protease assume two very different conformations, open and closed, whereas the flaps always assume a closed conformation in ligand-bound structures. When ligand-bound and open-flap unliganded HIV-1 protease structures are superimposed the Rmsd is greater than 1.0 A ˚ , with changes being distributed through- out the protein. This might suggest that concerted changes distributed throughout the protein are needed to bring the flaps into a closed conformation. Therefore, comparison of closed-flap unliganded structure with ligand-bound struc- tures would accurately reflect changes induced by ligand binding alone. The uliganded structure we have determined is of closed-flap conformation. Comparison of the unligan- ded C95A mutant structure of HIV-1 protease with that complexed to cyclic urea-based inhibitors reveals that the residues which are known to be less flexible and are characterized by a small B-factor are not necessarily less Fig. 8. Superposition of XK216 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons). Fig. 9. Superposition of XK263 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons). Ó FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur. J. Biochem. 270) 1237 adaptable to environmental changes. Residues 23–26 in both monomers of HIV-1 protease are considered to be very ‘robust’ and ‘rigid’ [2]. The electron density of these segments is also very well defined and they have a small B-factor, implying less flexibility of this segment. However the present study shows that these residues are very adaptable to internal or external stresses. This property may have been built into HIV-1 protease to cater to the functional requirement that the enzyme cleave substrates of eight different sequences [4]. Cys95 at the dimer interface is far away from the active site. Also the B-factors of residues 91–99 are significantly more than those of segment 23–26 (Fig. 3). Even so the C95A mutation does not cause much change in the structure near the mutation site [3]. Instead it affects the catalytic site residues 23–26 thereby suggesting that the residues 23–26 are more adaptable than residues around 95. Hence it is important to distinguish this type of adaptability from the concept of flexibility. Zoete et al.[2] have analysed the X-ray structures of 73 HIV-1 protease complexes to look at the overall structural variability of protease. Interestingly, they found that the pattern of structural variability of different residues in all of these structures is very much the same as the pattern of B-factors of these residues in any single structure. Thus the analysis of an ensemble of structures gives information about the inherent flexibility of a protein. However, the information about adaptability is lost. Only analysing and comparing structures on a one to one basis would provide information about adaptability. Here in this paper we are trying to distinguish between the concept of flexibility and that of adaptability in protein structures. Very often, these two terms are used inter- changeably in the literature. However with the growing amount of structural information available about proteins, there arises a need to analyse and understand the structural features in a more comprehensive and accurate way. One must have a well-defined tool to understand different properties of protein structure. We thus feel that the word ‘flexibility’ with reference to protein structure should be reserved to describe the conformational variability of a Fig. 11. Superposition of SD146 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons). Fig. 10. Superposition of DMP450 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons). 1238 M. Kumar and M. V. Hosur (Eur. J. Biochem. 270) Ó FEBS 2003 residue as well as to include the effects of thermal motions of atoms therein. On the other hand, the word ‘adaptability’ should be used to describe the ability of a residue/region to adjust and accommodate itself in response to a stress/ change in its environment. This stress could be either internal, as in case of mutation of a nearby residue, or external, as in case of presence of an inhibitor or other nonprotein molecule in its surrounding. This distinction is important: we see in the present analysis that a less flexible region need not necessarily be less adaptable. Acknowledgements We thank the National Facility for Macromolecular Crystallography, BARC for providing all the X-ray and biochemistry equipment used in this investigation. We are thankful to S. K. 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Basic structure of P2 analogues of cyclic urea inhibitors (A) and properties and structures of all inhibitors (B). Ó FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur. J. Biochem flexi- bility and adaptability of proteins in general. Keywords: adaptability; flexibility; HIV-1 protease; inhibi- tors; structure. The significance of human immunodeficiency virus type 1 (HIV-1) protease. methods ‘Closed-flap’ structure of unliganded HIV-1 protease The X-ray structures of C95M single mutant- and C95M/ C1095A double mutant-tethered HIV-1 protease have been solved to 1.9 A ˚ and 2.1 A ˚ resolution,