Eur J Biochem 271, 4451–4461 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04384.x Role of hydroxyl group and R/S configuration of isostere in binding properties of HIV-1 protease inhibitors ´ ˇ ´ ´ ´ ´ ´ˇ ´ ´ Hana Petrokova1, Jarmila Duskova1, Jan Dohnalek1, Tereza Skalova1, Eva Vondrackova-Buchtelova1, ˇ ˇ´ ´ ´ˇ ˇ ˇ Milan Soucek2, Jan Konvalinka2, Jirı Brynda3, Milan Fabry3, Juraj Sedlacek3 and Jindrich Hasek1 Institute of Macromolecular Chemistry, 2Institute of Organic Chemistry and Biochemistry and 3Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Praha, Czech Republic The crystal structure of the complex between human immunodeficiency virus type (HIV-1) protease and a peptidomimetic inhibitor of ethyleneamine type has been ˚ refined to R factor of 0.178 with diffraction limit 2.5 A The peptidomimetic inhibitor Boc-Phe-Y[CH2CH2NH]-PheGlu-Phe-NH2 (denoted here as OE) contains the ethyleneamine replacement of the scissile peptide bond The inhibitor lacks the hydroxyl group which is believed to mimic tetrahedral transition state of proteolytic reaction and thus is suspected to be necessary for good properties of peptidomimetic HIV-1 protease inhibitors Despite the missing hydroxyl group the inhibition constant of OE is 1.53 nM and it remains in the nanomolar range also towards several available mutants of HIV-1 protease The inhibitor was found in the active site of protease in an extended conformation with a unique hydrogen bond pattern different from hydroxyethylene and hydroxyethylamine inhibitors The isostere nitrogen forms a hydrogen bond to one catalytic aspartate only The other aspartate forms two weak hydrogen bridges to the ethylene group of the isostere A comparison with other inhibitors of this series containing isostere hydroxyl group in R or S configuration shows different ways of accommodation of inhibitor in the active site Special attention is devoted to intermolecular contacts between neighbouring dimers responsible for mutual protein adhesion and for a special conformation of Met46 and Phe53 side chains not expected for free protein in water solution The HIV-1 protease, the aspartic protease that cleaves specific peptide bonds in precursor gag-pol proteins to form the mature proteins, is essential for production of infectious HIV particles Its inhibition is an efficient method of treatment of the acquired immunodeficiency syndrome (AIDS) and related diseases [1,2] No matter what is the inhibitor type (symmetrical or unsymmetrical), the HIV protease covers any inhibitor under its flaps (Fig 1) forming thus a characteristic long binding tunnel with the cleavage site in the middle One structural feature present in most tight-binding aspartic protease inhibitors is a critical hydroxyl group that replaces the catalytical water molecule in the active site This hydroxyl group forms hydrogen bonds to the catalytically active aspartates [3] in HIV protease complexes with all hydroxyethylamine inhibitors being by far the most frequently studied structures in the Protein Data Bank [4] and the HIV Protease Database (http://srdata.nist.gov/hivdb) and therefore it has been supposed necessary for tight-binding of aspartic protease inhibitors This paper presents the structure of native HIV-1 protease in a complex with inhibitor Boc-PheY[CH2CH2NH]-Phe-Glu-Phe-NH2 (denoted here as OE) The inhibition constant of OE remains low (1.53 nM) in spite of the fact that the critical hydroxyl group is completely missing in this inhibitor and it remains in the nanomolar range also for several available mutants of HIV1 protease (e.g 4.1 nM for the A71V/V82T/I84V mutant arising after Indinavir treatment) [5,6] The structure of OE complex has been solved in the frame of a systematic structure study of a group of inhibitors with very similar chemistry They differ only in the presence of the hydroxyl group in the isostere and in its configuration (R or S) which is considered crucial for tight binding of hydroxyethylamine inhibitors The fact that the inhibition constant of the respective inhibitors (denoted here as OE, RE and SE) does not differ much deserves closer attention The inhibitor OE (without OH group) has only a slightly lower inhibition efficiency than similar inhibitors possessing hydroxyl group in S or R configuration (Ki,OE ¼ 1.5 nM, Ki,RE ¼ 0.12 nM, Ki,SE ¼ 0.15 nM) [5,7–9] Our recent studies of the hydroxyethylamine inhibitor complexes [7–9] revealed that the binding tunnel of protease, abundant in hydrogen bond donors and acceptors, can bind the inhibitors in several possible ways Most of the ´ Correspondence to H Petrokova, Institute of Macromolecular ´ Chemistry, Academy of Sciences of the Czech Republic, Heyrovskeho ´ nam 2, 162 06 Praha Fax: +420 296809 410, Tel.: +420 296809 205, E-mail: petrokova@imc.cas.cz Abbreviations: OE, Boc-Phe-Y[CH2CH2NH]-Phe-Glu-Phe-NH2; RE, Boc-Phe-Y[(R)-CH(OH)CH2NH]-Phe-Glu-Phe-NH2; SE, Boc-Phe-Y[(S)-CH(OH)CH2NH]-Phe-Glu-Phe-NH2 Enzyme: retropepsin (EC 3.4.23.16) Note: The crystallographic data of the complex HIV-1 protease with OE inhibitor have been deposited with the Protein Data Bank and are available under access code 1m0b (Received 21 June 2004, revised September 2004, accepted 29 September 2004) Keywords: ethyleneamine inhibitor; HIV-1 protease; peptidomimetic inhibitor; X-ray structure Ó FEBS 2004 ´ 4452 H Petrokova et al (Eur J Biochem 271) BM29 beamline equipped with a MAR 345 detector Data were collected from a single crystal (0.06 · 0.06 · 0.7 mm) at a temperature of 100 K The oscillation range was 1.5° and each frame was exposed for 30 s The distance of the crystal to detector plate was ˚ 100 mm The diffraction data extended to 2.0 A Intensities were integrated, scaled and merged using the HKL software [12] Data were reduced in the P61 space group ˚ The unit cell dimensions were a ¼ b ¼ 62.7 A, c ¼ ˚ 82.2 A The details of X-ray diffraction data collection are described in Table Refinement Fig Front view of the structure of native HIV-1 protease complexed with OE inhibitor The inhibitor (stick model) sits over the catalytic aspartates (ball-and-stick model) and is completely covered by protease flaps belonging to two monomers of protease related by an approximate two-fold symmetry axis The exact C2 symmetry is perturbed by asymmetry of the inhibitor and also by contacts between neighbouring protease subunits hydroxyethylene inhibitors are bound to the HIV)1 protease by interaction of the hydroxyl group of isostere with both aspartates Asp25, Asp125 A slight shift (about ˚ 0.5 A) of the isostere group in the case of hydroxyethylamine inhibitors SE or SQ [7,8] causes that the catalytic aspartates bind mainly to the isostere NH group leaving only one contact to the isostere hydroxyl group The NH group thus partly substitutes the role of the hydroxyl group in hydroxyethylene inhibitors In this paper, we present another unusual binding mode of the ethyleneamine inhibitor OE where only its isostere NH group makes a contact to one catalytic aspartate We suggest that the binding mode where the role of the hydroxyl group is completely overtaken by the isostere NH group can be generalized for the whole class of ethyleneamine inhibitors Materials and methods Refinement was carried out using the CNS program package [13] Parameters for nonstandard parts of inhibitor were set in agreement with several structures found in the CCDC database [14] The rigid body refinement was performed with the starting model of the protease dimer taken from the Protein Data Bank [4] (PDB code 1aaq) Several cycles of CNS refinement (positional and individual B factor optimization) and rebuilding using the graphics program O [15] were carried out The noncrystallographic symmetry was applied during the refinement to both the protease and the inhibitor at the initial stages of refinement with the ˚ weight of 300 kcalỈmol)1ỈA)2 Later, it was partially Table Statistics of diffracted intensity measurement Complex of native HIV-1 protease with inhibitor OE Rsym ¼ S|I ) |/ S All reflections ˚ Diffraction limits (A) No of observed reflections No of unique reflections Rsym Completeness (%) I/rI Mosaicity (deg.) The highest shell 25–2.45 42257 6765 0.091 99.2 14.5 0.495 2.51–2.45 3548 865 0.495 98.5 3.14 – Crystallization and crystal parameters A solution of HIV protease at concentration mgỈmL)1 in 50 mM sodium acetate buffer (pH 5.8) containing 0.5% (v/v) 2-mercaptoethanol was mixed with the inhibitor [12] dissolved in dimethyl sulfoxide at 11 mM in the volume ratio 20 : and left at °C for at least 30 prior to crystallization; this gave the final fourfold molar excess of inhibitor [11] Co-crystallization by hanging drop diffusion technique against mL reservoir of 0.2–0.6 M NaCl in 0.1 M Na citrate buffer, pH 4.5–5.5 followed Rod-shaped hexagonal crystals appeared overnight and continued to grow over the next days Before flash-freezing, the crystals were soaked for 30 s in the mother liquor containing 20% glycerol Data collection and processing X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF) in Grenoble at Table Parameters describing the quality of refined model of the native HIV-1 protease complexed with inhibitor OE Parameter No of non-H protein atoms No of non-H inhibitor atoms No of refined water oxygens R factor (all reflections) R factor (working set of reflections) R free (5% of randomly selected reflexions) ˚ rmsd from ideal bond lengths (A) rmsd from ideal bond angles (°) % of cases in the most favored regions of Ramachandran plot % of cases in disallowed regions of Ramachandran plot ˚ average B for main chain atoms (A2) ˚ average B for side chain atoms (A2) 1516 50 159 0.18 0.18 0.24 0.011 1.8 92.4 30.1 31.5 Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur J Biochem 271) 4453 0.4 0.35 0.3 0.25 0.2 0.15 0.1 Fig Root-mean-square distances between the corresponding atoms of individual residues after the least-squares alignment of Ca atoms of two subunits A and B of the complex of HIV-1 protease with OE inhibitor 0.05 11 21 31 41 51 61 71 91 81 Residue number B factor 80 chain A 60 40 20 0 11 21 31 41 51 61 71 81 91 20 40 chain B Fig The complex of HIV-1 protease with OE inhibitor B factors averaged over each residue Top, chain A; bottom, chain B ˚ liberated to 50 kcalỈmol)1ỈA)2 Cross validation was performed with the use of the Rfree factor calculated from 5% of the reflections The inhibitor was uniquely identified and modeled in the 2Fo-Fc map in its expected position in two opposite orientations at R ¼ 0.256 and Rfree ¼ 0.294 After the structure refinement to R ¼ 0.235 and Rfree ¼ 0.272, a total of 159 water molecules were gradually included The criteria for accepting waters in refinement were as follows: the presence of the Fo-Fc electron density peak at r level, the 2Fo-Fc peak at r level, and at least one hydrogen-bonding ˚ partner within the distance 2.2–3.6 A The resulting structure (Fig 1) has R ¼ 0.178 and Rfree ¼ 0.242 Parameters describing the quality of the final structure were checked by program PROCHECK [16] and are given in Table The symmetry of the complex and rigidity of different parts of the protease can be seen in Figs 2,3 The figures were produced using programs MOLSCRIPT [17] and RASTER3D [18] or WLVIEWERPRO (http://molsim.vei.co.uk/ weblab) 60 80 Residue number B factor Results Structure of the complex of native HIV-1 protease with OE inhibitor The structure of the HIV-1 protease in the complex with inhibitor OE (Fig 1) has been finally refined to R ¼ 0.178 and Rfree ¼ 0.242 The protein is a homodimeric molecule, made up of two 99-residue polypeptide chains: chain A, Pro1-Phe99 and chain B, Pro101-Phe199 The inhibitor is bound as an extended chain in a tunnel running under flaps across the dimer interface The flaps consist of the aminoacid residues 46–55 and 146–155 from both polypeptide chains and completely close the inhibitor in the active site channel The tips of flaps bind together by hydrogen bond (NH Gly51…C ¼ O Ile150 or NH Gly151…C ¼ O Ile50) The nitrogen atoms of Ile50 and Ile150 in flaps interact with the inhibitor via hydrogen bonding through a single water molecule (W401) typically present in structures of HIV-1 protease with peptidomimetic inhibitors Ó FEBS 2004 ´ 4454 H Petrokova et al (Eur J Biochem 271) Fig Stereoview of two alternative positions of the inhibitor OE in the electron density 2Fo-Fc at r level Both orientations were refined with occupation factors 0.5 In ball-and-stick model the active site Asp25–Gly27 and Asp125–Gly127 are highlighted O O Ile50 N N Ile150 W401 3.3 3.1 W402 2.4 O O O N 3.5 O O 2.6 2.5 N 3.2 2.7 Gly127 Gly48 3.6 N O O O N O O N 3.3 O 3.5 OH 2.8 O Asp125 Fig Network of hydrogen bonds formed by the inhibitor OE in the native HIV-1 protease ˚ Distances are given in A Statistics: one intramolecular hydrogen bond CO…NH, seven hydrogen bonds to protease main-chain NH, three to side-chain carboxyls, three to main-chain carbonyls and seven to water molecules 3.3 OH W424 O N O W507 2.9 N 3.0 W464 Asp25 3.0 O Gly27 N N N O Asp30 3.3 N 3.1 O N O O O Asp29 Inhibitor binding The inhibitor OE binds into the protease binding tunnel in an extended conformation in two opposite directions (Fig 4) The flaps are locked over the inhibitor by water molecule W401 forming hydrogen bonds to NH groups of Ile50, Ile150 and to carbonyls of the inhibitor in positions P1¢ and P2 (Fig 5) The side chains of the tertbutyloxycarbonyl (P2), phenylalanine (P1), phenylalanine (P1¢), glutamine (P2¢) and phenylalaninamide (P3¢) bind in the respective S2, S1, S1¢, S2¢ and S3¢ pockets of the HIV-1 protease (Fig 6) The inhibitor was modelled into a well defined Fo-Fc electron density after the protein was refined to R ¼ 0.25 and Rfree ¼ 0.29 The disordered inhibitor was modelled in the active site of protease in two opposite orientations (Fig 4), here referred as I and Y chains (residue numbers in PDB file 301A-306A and 301B-306B, respectively) The average rmsd of inhibitors in opposite orientations (I and Y) ˚ was 0.15 A The scheme of hydrogen bonds (Fig 5) shows that all proton donors and acceptors of the inhibitor are involved in hydrogen bonding Five hydrogen bonds of the total 18 S1' S3' S2 I150 I47 G48 V32 D25 I150 I84 T80 G149 V82 I84 F153 R8 G127 A28 O O O N G48 I50 G27 L123 V182 P181 O N N G49 S1 D129 P81 G149 D30 G148 N O N O O OH N G148 O D25 A128 O D129 I147 V132 D130 S2' Fig Review of hydrophobic interactions (short C-C contacts up to ˚ 4.1 A) of the inhibitor OE side chains with binding pockets of HIV-1 protease Ó FEBS 2004 hydrogen bonds connect directly the inhibitor main chain with the main chain of protease (NH…CO to Gly23, Gly123, Gly48, CO…NH to Gly48, Asp29) Two hydrogen bonds connect the side chain of the inhibitor (Glu) with the protein main chain (CO…NH to Asp29, Asp30) Three hydrogen bonds connect inhibitor with protease side chains (NH…Asp125, COOH…Asp30, NH2…Asp30) Three structurally important water molecules W401, W402 and W464 bridge directly the inhibitor with protease and form five hydrogen bonds to inhibitor P2 (CO…W402), P1 (NH…W402 and CO…W401), P01 (CO…W401), P02 (CO…W464) The whole inhibitor is almost completely buried in the binding tunnel of protease Only the inhibitor ends seem to be exposed to solvent Two water molecules W424 and W507 were found in the difference map forming two hydrogen bridges to the N terminal group of inhibitor Unusual conformation of isostere in the inhibitor OE enables also a weak intramolecular hydrogen bond connecting the Boc carbonyl with the NH group at P1¢ position ˚ (O…N at 3.6 A) The inhibitor refined in the opposite orientation binds in a similar way The inhibitor Glu at P2¢ is totally buried in the protease S2¢ site and makes six hydrogen bonds to Asp30 and Asp29 The importance of this residue and the strength of its binding to protease are supported also by the fact that it has the lowest B-factors of the whole inhibitor The water molecule W401 that hydrogen bonds to the main chain NH group of both flaps as well as to the inhibitor carbonyls is observed in most HIV protease complexes with peptidomimetic inhibitors It was clearly seen at the difference map though it has a considerably high ˚ B factor (64 A2) High displacement factor is a result of probable disorder of W401 caused by two orientations of inhibitor and asymmetrical binding of water with respect to the noncrystallographic C2 axis The positions of two inhibitor carbonyls (one from Boc group at P2 position and another from Phe at P1¢) that are bridged by W401 are not symmetrical with respect to the noncrystallographic C2 axis and thus the alternative positions of the bridging water W401 are different However, only the average position of W401 was refined in our structure model In spite of numerous hydrogen bonds, hydrophobic forces seem to be a dominating interaction between protease and inhibitor As an indicator of these hydrophobic interactions, distances between carbon atoms of inhibitor and the protease ˚ ˚ were calculated using cutoffs of 3.6 A and 4.1 A [19] All hydrophobic contacts and hydrogen bonds that are involved in the inhibitor OE binding are summarized in Table Conformation of isostere Two different conformations of the isostere of inhibitor fitting well the electron density were modeled (Fig 7) In the first conformation, the NH group of Phe in position P1¢ makes only one hydrogen bond to one of the catalytic aspartates and also forms a weak intramolecular hydrogen bond to the carbonyl group of the Boc residue In the second conformation, the isostere NH group binds almost symmetrically between the Asp25 and Asp125 gaining thus additional hydrogen bond to the protease However, the CNS refinement run with both conformations resulted in the same result very similar to the first conformation Therefore, the inhibitor was refined in form that corresponds to the first conformation with only one HIV-1 Protease Inhibitors (Eur J Biochem 271) 4455 Table Summary of all contacts and hydrogen bonds for inhibitor OE complexed in the native HIV-1 protease Three water molecules involved in protein inhibitor interaction are included The upper table (six rows) concerns the I orientation of inhibitor, the lower table (six rows) concerns the Y orientation of inhibitor Contacts to water molecules (W) are given after the + sign Short contacts in the C-C column are supposed to be repulsive, the short contacts (hydrogen bonds) in columns 3–5 contribute to good binding ability of inhibitor to HIV protease Hydrophobic contacts Hydrogen bonds C-C up to ˚ 4.1 A C-O/N (+W) up to ˚ 3.6 A O/N-O/N up to ˚ 3.6 A O/N-W up to ˚ 3.6 A I/Boc I/Po0 I/Phe I/Glu I/Phe+Nh2 Total I 14 18 10 59 7+2 4+1 9 33 + 1 12 1 Y/Boc Y/Po0 Y/Phe Y/Glu Y/Phe+Nh2 Total Y 13 17 10 11 60 7+2 6+1 27 + 1 12 1 Fig Two conformations of isostere in the inhibitor OE with very similar energy can be placed in the 2Fo-Fc electron density of the refined structure of the complex of HIV-1 protease with OE inhibitor Torsion angles of the preferred conformation of the OE isostere are listed in Table Only one orientation of the disordered inhibitor OE is shown in this figure hydrogen bond to catalytic aspartates not observed with other inhibitors However, it seems that the isostere conformation is probably not fixed and that we have to admit possible concerted conformational changes at this site Because both conformations of the isostere group fit well into the 2Fo-Fc electron density map, we assume that the inhibitor can change its conformation inside the cavity and that this positively contributes to the inhibitor binding Two fold noncrystallographic symmetry of HIV protease The two HIV protease subunits are related by an approximate two-fold noncrystallographic axis Figure shows the deviations between the corresponding atoms of chain A and chain B (averaged for each residue) after the least-squares ´ 4456 H Petrokova et al (Eur J Biochem 271) alignment of Ca atoms According to expectation, the main differences were found in the tips of flaps where the two-fold noncrystallographic symmetry is not possible because the hydrogen bonds connecting tips of flaps cannot be present with its two-fold image in the same molecule at once Therefore, the alternative positions were modeled at the main chains for Ile50-Gly51 and Ile150-Gly151 The leastsquares fit of all Ca in superimposed monomers of protease gives the best overlap by rotation of 178° with the rmsd ˚ ˚ 0.07 A for all 758 atom pairs and 0.06 A when the residues 49–53 and 149–153 from the tips of flaps are excluded ˚ B-factors The highest B factors (above 50 A2) indicate high conformational instability in loops Leu38-Lys44 and Leu138-Lys144, symmetrically in hinges of both flaps, whereas the flap ends seem to be well stabilized by interactions with inhibitor, namely those mediated by water molecule locked over the inhibitor carbonyls (Fig 3) The map of electron density shows the highest orientational disorder in Arg41, Arg141, Lys43 and Lys143 side chains (zero occupation factors in the PDB file) Adhesion between proteins and protease activity in the crystal form Adhesion between protein molecules plays an important role in their function in biological systems [8] Structure changes of the protein surface when exposed to solvent or when involved in adhesion with neighbouring protein molecule have undoubtedly important biological implications Here, the HIV-1 protease molecules stick together by middle parts of flaps and form a special helical arrangement with the 61 symmetry and with active sites directed into the wide solvent tunnels passing through the whole crystal enabling thus an easy exchange of solvent even in the active sites of proteases This explains experimentally verified exchange of inhibitors in the protease single crystal without an extensive destruction of the crystal [20] In our structure, one of the preferred interactions between two neighbouring HIV-1 protease dimers is localized at the top of flaps The Phe53 from one protease dimer and the Phe153 from the other Ó FEBS 2004 symmetrically related dimer form a convenient parallel stacking of phenyl rings joining thus these two dimers together (Fig 8A) These p–p interactions appear on both sides of each protease dimer and thus form an infinite helical arrangement of the protease complexes in the P61 space group This molecular arrangement is supported by Met46 (Met146) which forms S…H-C short contacts to Phe53 (Phe153) from the same molecule (Fig 8B) The time-averaged view of the protease complex shows that all residues involved in contact – Phe53, Phe153, Met46, Met146 were found in two distinct conformations with an occupation factor of 0.5 (confirmed by refinement) These alternative conformations form two favorable parallel stackings leading to two quite different water channels – called here closed and open solvent channel The inner virtual diameter of the closed solvent channel is ˚ 8.7 A The inner surface of the channel is formed by 12 Cc atoms of six phenyl pairs (Phe53 and Phe153) per one helix turn (Fig 9A) The phenyl pairs are held together by p–p interactions and the Sd atoms of Met46 and Met146 form close interactions with these phenyls Six of these residue quartets form a steep spiral ridge inside the solvent channel The virtual diameter of the open solvent channel is ˚ 12.4 A The surface of the channel is formed by 12 sulfur Sd atoms of Met46 and Met146 per one turn of helix (Fig 9B) In the open solvent channel the methionines are turned into the solvent and not form significant contacts to protein Thus, the solvent tunnel cross-section is not rigid because different conformations of Phe53, Phe153, Met46 and Met146 lead to different tunnel diameters and also to different hydrophobicity of the solvent tunnel surface The fact that inter–protein interaction can influence the inhibition process may be important for interpretation of the protease function Alternative conformations The HIV protease complexes are not rigid The crystal structure of HIV-1 PR with OE described here is a mixture of many conformation states In addition to residues Leu38-Lys44 and Leu138-Lys144 localized in flexible flap hinges (see the chapter on Fig Dimers of the complex of HIV-1 protease with OE inhibitor are linked together by p–p interactions of phenyl rings of Phe53 and Phe153 of neighbouring molecules to form a helix along the crystallographic c axis This p–p interactions are supported by CH…S hydrogen bonds between the Phe53A … Met46A and Phe153A … Met146A (A) Stacking of neighbour molecules and positions of inhibitors in subsequent protease dimers forming the helix (B) The detail of interacting residues Phenylalanines are disordered : in two conformations A and B leading to parallel stacking of phenyl rings in each conformation Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur J Biochem 271) 4457 Fig Solvent tunnels passing through the crystal of HIV-1 protease complexed with OE inhibitor along the crystallographic axis c The residues Met46, Phe53, Met146 and Phe153 participating in the solvent tunnel formation have two different conformations: A, narrow tunnel (A) and B, wide tunnel (B) The inhibitors OE of six complexes forming one turn of the helix are also shown in one orientation in thick stick representation and ˚ coloured according to atom types In the case of Ônarrow tunnelÕ, the diameter of the effective view through the solvent channel is 8.7 A (determined by distance of projections of two opposite Cc atoms of Phe53 and Phe153) In the case of Ôwide tunnelÕ, the diameter of the effective view through the ˚ solvent channel is 12.4 A (measured as a distance of projections of opposite Sd atoms of Met46 and Met146) B-factors), other eight amino-acid residues have been found each in two distinct alternative conformations: Met46, Met146, Ile50, Ile150, Gly51, Ile151, Phe53 and Phe153 All of them have clear interpretation Two conformations of Met46 are associated with corresponding alternative conformations of Phe53 so that either Met46(A) and Phe53(A) or Met46(B) and Phe53(B) can be present simultaneously in the structure and analogously in the second chain of the protease dimer The residues Met46 and Phe53 placed on opposite sides of one flap seem to be of principal importance for intermolecular arrangement of molecules in crystal Figure shows that the Phe53 rings are in both orientations stacked by p–p interaction with Phe153 rings of the neighbouring protein dimer in crystal The alternative positions of phenylalanine rings are formed in concert with the alternative positions of Sd and Cc of Met46 and Met146 (from neighbouring protease dimer) Residues Ile50 and Ile150, Gly51 and Gly151 are found at the tips of the flaps making a hydrogen bond to each other These hydrogen bonds are mediated by NH groups of one chain with carbonyl groups of the other chain Because the main chains of the two loops have the same orientation of C ends at the place of contact, the noncrystallographic two-fold symmetry cannot be realized in this location Therefore, two possible orientations were modelled in this structure, which differ in the flipped peptide bond between residues Ile50-Ile51 The side chains of neighbouring Ile50 and Ile150 already fitted well the electron density in a single position Comparison of inhibitors OE, SE and RE complexing the native HIV-1 protease The availability of three experimental structure determinations of very similar inhibitors OE, RE and SE complexed with the native protease gives a detailed insight into the nature of interaction inhibitor – protease The chemical structure of OE, RE, SE inhibitors can be seen from the scheme in Table The inhibitors RE, SE differ in absolute configuration of the CHOH group of the isostere and their inhibition constants are surprisingly the same Ki,SE ¼ 0.15 nM, Ki,RE ¼ 0.12 nM [1] The isostere carboxyl group replaced in OE by CH2 makes the inhibition constant somewhat lower Ki,OE ¼ 1.53 nM [1]; however, the resistance of OE to protease mutations seems to be better The experimental structure of HIV-1 protease with ´ inhibitor SE was determined by Dohnalek [8] R ¼ 0.18, ˚ diffraction limit 3.1 A, PDB code 1fqx The structure with ´ the RE inhibitor was determined by Dusˇ kova (unpublished ˚ results) with R ¼ 0.173, diffraction limit 2.0 A The overall layout of the inhibitor in the protease binding site is very similar for all the compared inhibitors The side chains of all the inhibitors are placed similarly in their pockets, though, not identically (Fig 10) Conformation of inhibitor backbones The compared inhibitors differ mainly in their isostere areas The OE inhibitor possesses the nonscissile isostere without any hydroxyl group, whereas the hydroxyethylamine inhibitors SE and RE have the hydroxyl group of isostere in the S or R configuration, respectively The isostere of the OE inhibitor has quite different conformation compared with the RE and SE inhibitors The inhibitor OE lacks the hydroxyl group which fixes a unique conformation by strong hydrogen bonds to Asp25 and/or Gly27 allowing thus in principle more possible conformations of the isostere group Possible variations in torsion angles in the inhibitor are cooperative This means that any change in one torsion angle should be compensated by change of other torsion angles in the opposite direction to keep all the side chains in the whole Ó FEBS 2004 ´ 4458 H Petrokova et al (Eur J Biochem 271) Table Torsion and dihedral angles (degrees) describing conformation of isostere in inhibitors OE, RE and SE in complex with HIV-1 protease Diagram shows schematically the structure of inhbitors and measured torsion angles X ¼ H; (R) OH and (S) OH for OE, RE and SE inhibitors, respectively Torsion angle Dihedral angle Inhibitor N1-CA1-C1-C2 CA1-C1-C2-N2 C1-C2-N2–CA2 C2-N2–CA2-C3 N1-CA1-C1 CA2-C3-N2 OE RE SE 112 56 60 -89 -175 -173 165 -148 -142 -21 77 67 141 148 148 Fig 10 Stereoview of the position of inhibitor OE (yellow carbons), RE (green carbons) and SE (violet carbons) in the binding tunnel of native HIV protease Conformation of different inhibitors is not identical Note a different orientation of OH groups in isosteres of RE and SE The S configuration forces the OH groups to the inconvenient orientation for COH.O hydrogen bonds with aspartates from one side whereas the R configuration makes the same from the opposite side The conformation of isostere in the case of OE seems to be more conformationally flexible The preferred conformation of OE inhibitor is supported by the intramolecular hydrogen bond CO…HN (P2 – P1¢) However, more conformation states of OE isostere fit the same map of electron density (Fig 9) Rotations of benzyl groups in the protein pockets P1, P1¢ and P3¢ compensate the stress imposed by different geometries of the H-bond network inhibitor approximately in the same position Thus, in spite of the fact that the individual torsion angles in the isostere differ largely (Table 5), the Ôoverall dihedral anglesÕ (i.e the dihedral angles determining the mutual orientation of the side chains in S1 and S1¢ sites) are almost identical (141, 148 and 148° for inhibitors OE, SE and RE, respectively) The NH group of Phe in P1¢ position of the OE inhibitor is rotated with respect to SE, RE preserving one hydrogen bond to the catalytic Asp25 only The intramolecular hydrogen bond NH…O between ester oxygen of Boc and NH of Phe in P1¢ position probably stabilizes this unusual conformation of the OE inhibitor in the active site The aspartate that does not bind to the isostere NH seems to make weak hydrogen bonds to both CH2 groups of the isostere The changes of torsion angles in the isostere also resulted in shifts of Ca of Phe in P1¢ position of the OE ˚ inhibitor by 0.58 and 0.46 A in comparison with SE and RE complexes, respectively, in both cases in the direction away Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur J Biochem 271) 4459 Table Review of interactions of native HIV-1 protease with inhibitors OE, RE, SE analysed residue by residue Comparison of protease residues which are in contact with individual side chains of inhibitors OE, RE and SE All contacts up to 0.41 nm are accounted for Number of contacts to certain residue is given S2 OE RE SE – 2 2 – 2 2 – – 16 A28 D29 D30 V32 I47 G48 I84 I150 S1¢ S1 17 17 OE G27 G48 G49 I50 L123 P181 V182 RE SE – 1 5 – – – 17 16 12 S2¢ OE L23 D25 T80 P81 V82 I84 G127 G149 I150 RE SE – 1 7 26 – 1 – 31 – – 19 from the catalytic aspartates at the bottom of the active site tunnel The shift of inhibitor OE main chain upwards propagates to P2¢ Glu However, the Oe1 and Oe2 of inhibitors RE and OE form already similar hydrogen bond pattern to the Asp29 and Asp30 in both cases Thus, even if the backbones of these inhibitors not follow the same line, the hydrogen bond pattern remains similar, showing high flexibility and adaptation of not only the inhibitor to the protease but also of the protease to the inhibitor Comparison of inhibitors side chains Boc groups at P2 of SE and RE inhibitors lie in the same position Only in the case of inhibitor OE, the Boc carbonyl is significantly rotated to form an intramolecular hydrogen ˚ bond (with O…N distance 3.6 A) to NH group in P1¢ position This results also in significant shifts of the tert˚ butyl group by 0.3 and 0.6 A when compared with SE and RE inhibitors Phenylalanine residues in position P1 The orientation of benzyl group in P1 position of the SE inhibitor differs from those in OE and RE Both, Fig 10 and Table show that the rotation of SE benzyl group leads to a lower number of contacts to protease – compare 12 contacts in SE with 17 and 16 contacts in OE and RE, respectively (contact is ˚ defined here as an interatomic distance lower than 4.1 A) Phenylalanine residues in position P1¢ The Ca of OE is shifted higher to the flaps than Ca of SE and RE inhibitors ˚ (about 0.5 A in both cases) Although phenyl rings of OE and RE have very similar positions and orientation and have similar contact patterns (26 and 31 contacts, respectively), the P1¢ phenyl ring of SE is turned down to make less contacts (19) to protease, namely to the flap residues in comparison with OE and RE inhibitors Glutamate residues in position P2¢ In comparison with RE and SE, the Ca at P02 in OE is shifted upwards to the flaps and also closer towards the symmetry axis of protease However, the Glu Oe1 and Oe2 in OE and RE overlap and make the same network of hydrogen bonds to the Asp29 S3¢ OE I50 A128 D129 D130 V132 I147 G148 RE SE – 14 – – 2 11 – – 31 18 23 OE R8 D129 G148 G149 F153 RE SE 1 13 – 1 11 – 22 20 13 ˚ and Asp30 with bond-length differences up to 0.3 A The v3 torsion angle (C-C-C-OH) of Glu is for all inhibitors significantly different, namely in the case of RE (165°, )54° and )179° for OE, RE and SE, respectively) This results in different hydrogen bonds between the Glu at P2¢ and protein binding site, although their numbers (6, 7, 7, see Table 6) remain unchanged Phenylalanine residues in position P3¢ Benzyl groups of Phe at P3¢ are partially exposed to the solvent in all cases However, their positions and orientations are significantly different for the compared inhibitors After the Ca superposition, the phenyl rings of different inhibitors were found rotated to each other The angles between plains of P3¢ phenyl rings are 98° for OE and SE inhibitors, and 38° for OE and RE inhibitors The S3¢ groups of OE and RE inhibitors having a similar orientation phenyl rings form a similar number of close contacts to protease (22 and 20 short C-C contacts for OE, RE, respectively), whereas in the case of SE inhibitor, there are 13 short C-C contacts to the protease only (Table 6) Discussion The experimentally determined structure of a complex of HIV-1 protease with OE, RE and SE inhibitors allowed us to answer the following general questions: (a) why the presence of hydroxyl group in the isostere of an inhibitor (often referred to as necessary replacement of the catalytic water molecule in reaction intermediate) is not necessary for good inhibition properties of inhibitor (b) why the R or S configuration at the carboxyl of the isosteric group has no influence on the inhibition constant, and (c) what is the influence of small chemical changes in the inhibitor molecule on its conformation in the binding tunnel of HIV protease In answer to the first question, it was shown, that the hydroxyl binding to catalytic aspartates considered originally as the main condition for good inhibition properties of substrate-mimicking inhibitors can be easily replaced by that of the isostere NH group (if not present as this is the case of OE) Some energy loss caused by a less dense hydrogen bond network in place of the missing hydroxyl is probably Ó FEBS 2004 ´ 4460 H Petrokova et al (Eur J Biochem 271) ˚ Table Comparison of hydroden bonds (2.4–3.6 A) of inhibitors OE, RE and SE towards their HIV-1 proteases Stated numbers are distances ˚ between inhibitor and protease atoms in A Reside of Atoms of Inhibitor Protease Boc Inhibitor complex Inhibitor Protease OE RE SE OH2 3.3 3.5 3.5 201 Wat 401 O2 202 202 202 202 202 Asp Asp Asp Gly Gly 25 25 125 127 127 OR, OR, OR, OR, N Phe Phe Phe Phe Phe 203 203 203 203 203 Asp Asp Asp Asp Wat 25 25 125 125 401 N N N N O OD1 OD2 OD2 OD1 OH2 Glu Glu Glu Glu Glu Glu Glu Glu Glu 204 204 204 204 204 204 204 204 204 Gly Asp Asp Asp Asp Asp Asp Asp Asp 27 29 29 29 30 30 30 30 30 N O O OE2 OE1 OE1 OE1 OE2 OE2 O N OD2 N O OD2 N OD2 N Phe Phe Nhh Nhh 205 205 206 206 Gly Gly Asp Asp 48 48 29 30 N O Nhh Nhh O N OD2 OD2 Po0, Po0, Po0, Po0, Po0, Pr0, Pr0, Pr0, Pr0, Pr0, Ps0 Ps0 Ps0 Ps0 Ps0 compensated by increased flexibility of the central part of the OE inhibitor leading to a favorable entropy contribution In answer to the second question, the negligible difference between inhibition constants of RE and SE can be explained by the fact that the strain imposed on inhibitor during docking into the binding site of protease does not allow correct orientation of the hydroxyl group to catalytic aspartates either in S or in the R configuration The resulting orientation of the hydroxyl group deviates from the most convenient direction similarly, but in opposite directions In other words, inhibitor remains halfway as far as correct orientation in both cases is concerned In answer to the final question, the X-ray structures discussed in this paper showed that some even small changes in inhibitor chemistry can have a large influence on conformation of inhibitor main chain Thus, different inhibitors differ not only in the binding affinity, but also in the degree of freedom of the inhibitor inside the binding tunnel Some special inhibitors (such as the OE discussed here) can find more conformations in the binding tunnel of the HIV protease with comparable interaction energies This can form a significant contribution to the stability of the complex through the entropy term in the average energy of the system Structure determination by X-ray crystallography shows why theoretical predictions of inhibitor properties have been relatively unreliable so far [21] Even small changes in the inhibitor chemistry, no matter whether they have a high or OS OS OS OS OD1 OD2 OD2 O O 2.9 2.8 3.0 3.4 3.3 3.3 3.5 2.6 3.6 2.8 3.5 2.8 3.0 3.3 2.9 2.7 2.7 2.5 2.4 3.2 3.0 3.3 2.7 3.0 3.6 3.2 2.8 2.7 3.6 3.1 3.3 3.1 2.6 3.0 3.6 3.5 2.9 3.0 3.6 3.5 3.2 2.8 3.4 3.5 3.1 3.0 3.0 2.8 3.2 small effect on inhibition constant, result often in similarly small differences in the overall geometry of the inhibitor inside the binding tunnel Relatively small shifts of Ca atoms ˚ (about 0.5 A) and rotations of side chains can significantly re-form a network of hydrogen bonds, which is, of course, compensated by a change of the inner torsion energy of chains destabilizing the complex Several water molecules always taking part in the formation of hydrogen bond network increase the number of possible configurations and conformation states of the complex Calculation of energy differences between the states containing different number of atoms and their configurations is a difficult task because this requires an exact mutual scaling of all the energy contributions including the entropy and hydrophobic effects A good check for selection of a good inhibitor conformation among many others theoretically possible is to verify which of them fit at least approximately the experimental map of electron density that can be nowadays easily calculated for any structure deposited in the PDB database with its experimentally measured intensities Acknowledgements The work was supported by the Grant Agency of the Czech Republic (projects 203/98/K023, 204/00/P091, 203/00/D117) and the Grant Agency of the Academy of Sciences of the Czech Republic (projects KJB4050312, A4050811, AVOZ4050913) The authors thank the beamline BM29 staff at ESRF in Grenoble for providing beam time Ó FEBS 2004 HIV-1 Protease Inhibitors (Eur J Biochem 271) 4461 References from 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this paper, we present another unusual binding mode of the ethyleneamine inhibitor OE where only its isostere NH group makes