Three-dimensional structures of enzyme-substrate complexes of the hydroxynitrile lyase from Hevea brasiliensis.

The 3D structures of complexes between the hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL) and several substrate and/or inhibitor molecules, including trichloracetaldehyde, hexafluoracetone, acetone, and rhodanide, were determined by X-ray crystallography. The complex with trichloracetaldehyde showed a covalent linkage between the protein and the inhibitor, which had apparently resulted from nucleophilic attack of the catalytic Ser80-Ogamma. All other complexes showed the substrate or inhibitor molecule merely hydrogen bonded to the protein. In addition, the native crystal structure of Hb-HNL was redetermined at cryo-temperature and at room temperature, eliminating previous uncertainties concerning residual electron density within the active site, and leading to the observation of two conserved water molecules. One of them was found to be conserved in all complex structures and appears to have mainly structural significance. The other water molecule is conserved in all structures except for the complex with rhodanide; it is hydrogen bonded to the imidazole of the catalytic His235 and appears to affect the Hb-HNL catalyzed reaction. The observed 3D structural data suggest implications for the enzyme mechanism. It appears that the enzyme-catalyzed cyanohydrin formation is unlikely to proceed via a hemiacetal or hemiketal intermediate covalently attached to the enzyme, despite the observation of such an intermediate for the complex with trichloracetaldehyde. Instead, the data are consistent with a mechanism where the incoming substrate is activated by hydrogen bonding with its carbonyl oxygen to the Ser80 and Thr11 hydroxy groups. A hydrogen cyanide molecule subsequently replaces a water molecule and is deprotonated presumably by the His235 base. Deprotonation is facilitated by the proximity of the positive charge of the Lys236 side chain.     

Protonation state and substrate binding to B2 metallo-beta-lactamase CphA from Aeromonas hydrofila

The zinc enzymes metallo beta-lactamases counteract the beneficial action of beta-lactam antibiotics against bacterial infections, by hydrolyzing their beta-lactam rings. To understand structure/function relationships on a representative member of this class, the B2 M beta L CphA, we have investigated the H-bond pattern at the Zn enzymatic active site and substrate binding mode by molecular simulation methods. Extensive QM calculations at the DFT-BLYP level on eleven models of the protein active site, along with MD simulations of the protein in aqueous solution, allow us to propose two plausible protonation states for the unbound enzyme, which are probably in equilibrium. Docking procedures along with MD simulations and QM calculations suggest that in the complex between the enzyme and its substrate (biapenem), the latter is stable in only one of the two protonation states, in addition it exhibits two different binding modes, of which only one agrees with previous proposals. In both cases, the substrate is polarized as in aqueous solution. We conclude that addressing mechanistic issues on this class of enzymes requires a careful procedure to assign protonation states and substrate docking modes.     

In silico analyses of substrate interactions with human serum paraoxonase 1

Human paraoxonase (HuPON1) is a serum enzyme that exhibits a broad spectrum of hydrolytic activities, including the hydrolysis of various organophosphates, esters, and recently identified lactone substrates. Despite intensive site-directed mutagenesis and other biological studies, the structural basis for the specificity of substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The results suggest that the active site of HuPON1 is characterized by two distinct binding regions: the hydrophobic binding site for arylesters/lactones, and the paraoxon binding site for phosphotriesters. The unique binding modes proposed for each type of substrate reveal a number of key residues governing substrate specificity. The polymorphic residue R/Q192 interacts with the leaving group of paraoxon, suggesting it plays an important role in the proper positioning of this substrate in the active site. MD simulations of the optimal binding complexes show that residue Y71 undergoes an "open-closed" conformational change upon ligand binding, and forms strong interactions with substrates. Further binding free energy calculations and residual decomposition give a more refined molecular view of the energetics and origin of HuPON1/substrate interactions. These studies provide a theoretical model of substrate binding and specificity associated with wild type and mutant forms of HuPON1, which can be applied in the rational design of HuPON1 variants as bioscavengers with enhanced catalytic activity.     

Effects of cholesterol side-chain groups and adrenodoxin binding on the vibrational modes of carbon monoxide bound to cytochrome P-450scc: implications of the productive and nonproductive substrate bindings.

Effects of the bindings of cholesterol and its hydroxylated analogues on the Fe-CO stretching and the C-O stretching vibrations of cytochrome P-450scc-CO complex were examined by resonance Raman and FT-IR spectroscopies to reveal the spatial relationship between the steroid side-chain groups and the heme-bound C-O moiety at the active center. These C-O and Fe-CO vibrations exhibited considerable variations depending on the steroids used; however, analyses on the nu Fe-CO vs nu C-O plot for cytochrome P-450scc indicated the absence of the negative correlation between these two vibrations, which is common among various Fe(2+)-porphyrin-CO complexes having imidazole ligands. Rather, we noticed the existence of two groups depending on substrates, the one exhibiting C-O infrared absorption bands in the region from 1930 to 1940 cm-1 and higher enzymatic turnover numbers in the reconstituted enzymatic systems and the other exhibiting C-O infrared absorption bands in the region above 1945 cm-1 and lower enzymatic turnover numbers. Thus, the former substrate group is likely to be fitted into the substrate binding site in the efficient "productive substrate binding" structure, whereas the latter group may be bound to the enzyme in the structure not suitable for the efficient enzymatic reaction ("nonproductive substrate binding" conformation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Factor Xa active site substrate specificity with substrate phage display and computational molecular modeling

Structural origin of substrate-enzyme recognition remains incompletely understood. In the model enzyme system of serine protease, canonical anti-parallel beta-structure substrate-enzyme complex is the predominant hypothesis for the substrate-enzyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experimentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-enzyme recognition modes emerged from the experimental data as necessary to account for the sequence-dependent specificity of the model enzyme. Computational molecular models were constructed, with both energetics and pharmacophore criteria, for the substrate-enzyme complexes of several of the representative substrate peptide sequences. In contrast to the canonical conformation hypothesis, the binding modes of the substrates to the model enzyme varied according to the substrate peptide sequence, indicating that an ensemble of binding modes underlay the observed specificity of the model serine protease.     

Entamoeba invadens: Cloning and molecular characterization of chitinases

Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern.     

Studies on the allosteric nature of acetate kinase from Bacillus stearothermophilus.

Fructose 1,6-bisphosphate (FBP) stimulates the reaction of Bacillus stearothermophilus acetate kinase (AK). FBP changes the reaction curve for ATP from a sigmoidal type to a Michaelis-Menten one. The binding of FBP to AK was studied by an equilibrium dialysis method and by measuring changes in fluorescence. The extent of binding of FBP to the enzyme paralleled its activation. In addition, the binding constant for FBP increased in the presence of substrate, ATP. These results suggest that FBP is an allosteric activator of B. stearothermophilus AK. Only two moles of FBP bound to this tetrameric enzyme. No cooperativity was found for the binding of FBP. These observations support the previous conclusion, that a set of two subunits in the tetramer is a unit of the enzymatic function. A model is presented to interpret the sigmoidal kinetics for ATP, the absence of cooperativity for FBP binding, and the allosteric activation by FBP of this enzyme. The kinetic properties of the enzyme can be explained quantitatively by this model.     

Monoamine Oxidases A and B as Components of a Membrane Complex

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane-bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high-speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A₂, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.     

Structure of a closed form of human malic enzyme and implications for catalytic mechanism

Malic enzymes are widely distributed in nature and have many biological functions. The crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme in a quaternary complex with NAD+, Mn++ and oxalate has been determined at 2.2 A resolution. The structures of the quaternary complex with NAD+, Mg++, tartronate or ketomalonate have been determined at 2.6 A resolution. The structures show the enzyme in a closed form in these complexes and reveal the binding modes of the cation and the inhibitors. The divalent cation is coordinated in an octahedral fashion by six ligating oxygens, two from the substrate/inhibitor, three from Glu 255, Asp 256 and Asp 279 of the enzyme, and one from a water molecule. The structural information has significant implications for the catalytic mechanism of malic enzymes and identifies Tyr 112 and Lys 183 as possible catalytic residues. Changes in tetramer organization of the enzyme are also observed in these complexes, which might be relevant for its cooperative behavior and allosteric control.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Chemical modification of the essential arginine in malate dehydrogenases

Chemical modification of arginine in malate dehydrogenases from pig heart mitochondria and from Bacillus subtilis was done using 4-hydroxy-3-nitrophenylglyoxal. Incorporation of 2 reagent molecules per subunit was observed concomitantly with complete loss of enzymatic activity. Partial protection was obtained with a substrate analogue and by formation of abortive ternary complexes, whereas coenzyme alone did not inhibit the inactivation. Modified inactive enzymes formed binary complexes with coenzyme as well as the ternary complex with NAD/sulfite. The substrate analogue 8-hydroxy-1,3,6-pyrenetrisulfonate was bound with reduced affinity, however. Because of the known stoichiometry of two reagent molecules per arginine we conclude that one arginine essential for substrate binding was modified in both enzymes.     

Kinetic fluorescence measurement of fluorescein di-beta-D-galactoside hydrolysis by beta-galactosidase: intermediate channeling in stepwise catalysis by a free single enzyme

Kinetic fluorescence measurements were employed to quantitative to stepwise hydrolysis of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase and the intermediate fluorescein mono-beta-D-galactoside (FMG) channeling. The kinetic parameters, Michaelis-Menten constant Km and enzymatic catalysis rate k2, for FDG hydrolysis to FMG by beta-galactosidase were obtained as 18.0 microM and 1.9 mumol.(min-mg)-1, respectively. The FMG intermediate is hydrolyzed via two modes: (1) FMG that is in free solution binding to the enzyme substrate binding site in competition with FDG and then being hydrolyzed (binding mode); (2) FMG being directly hydrolyzed into the final products of fluorescein and galactose before the FMG can diffuse away from the enzyme active site (channeling mode). The extent of the FMG channeling mode was found to depend on the FDG hydrolysis rate but to be independent of the free enzyme concentration. A channeling factor, defined as the ratio of the real FMG hydrolysis rate with both binding and channeling modes over that which would be observed with an exclusive binding mode, was used to quantitate the effect of the intermediate channeling. The FMG channeling factor was determined to be close to 1 at low FDG concentration (about 5.1 microM), where the slow FDG hydrolysis rate gives an ineffective channeling and where the FMG is then hydrolyzed mainly via the binding mode. However, the channeling factor dramatically increases at higher FDG concentrations (greater than Km), strongly indicating that the effective FMG channeling mode, resulting from the considerable FDG hydrolysis rate at high FDG concentrations, becomes a primary pathway to channel a steady system hydrolysis with a high rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

A unified model for signal transduction reactions in cellular membranes.

An analytical solution is obtained for the steady-state reaction rate of an intracellular enzyme, recruited to the plasma membrane by active receptors, acting upon a membrane-associated substrate. Influenced by physical and chemical effects, such interactions are encountered in numerous signal-transduction pathways. The generalized modeling framework is the first to combine reaction and diffusion limitations in enzyme action, the finite mean lifetime of receptor-enzyme complexes, reactions in the bulk membrane, and constitutive and receptor-mediated substrate insertion. The theory is compared with other analytical and numerical approaches, and it is used to model two different signaling pathway types. For two-state mechanisms, such as activation of the Ras GTPase, the diffusion-limited activation rate constant increases with enhanced substrate inactivation, dissociation of receptor-enzyme complexes, or crowding of neighboring complexes. The latter effect is only significant when nearly all of the substrate is in the activated state. For regulated supply and turnover pathways, such as phospholipase C-mediated lipid hydrolysis, an additional influence is receptor-mediated substrate delivery. When substrate consumption is rapid, this process significantly enhances the effective enzymatic rate constant, regardless of whether enzyme action is diffusion limited. Under these conditions, however, enhanced substrate delivery can result in a decrease in the average substrate concentration.     

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.     

Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii evidence for the formation of a precatalytic complex with 2-oxoglutarate.

The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nM, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mM, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mM substrate inhibition is observed. From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10(-6) to 10(-5) M), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10(-4) to 10(-3) M) the relative contribution of the 2-oxo group in the binding increases. A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site. The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.     

Computer modeling studies on the subsite interactions of ribonuclease T1.

The modes of binding of pGp,ApG,CpG and UpG to the enzyme ribonuclease T1 were determined by computer modeling. Essentially two binding modes are possible for all the four ligands--one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5'-phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5'-phosphate moieties occupy the same sites on the enzyme as 5'-GMP when bound to RNase T1. The ribose moiety of pGp adopts a C3'-endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5'-GMP in the RNase T1-5'-GMP complex. Based on these results, a mechanism for the release of the product subsequent to cleavage of the substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5'-side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the 1N subsite for adenine.     

The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.     

26 kDa endochitinase from barley seeds: Real-time monitoring of the enzymatic reaction and substrate binding experiments using electrospray ionization mass spectrometry

A 26 kDa endochitinase from barley seeds was enzymatically characterized exclusively by electrospray ionization mass spectrometry (ESI-MS). At first, oligosaccharide hydrolysis catalyzed by the barley chitinase was monitored in real-time by ESI-MS. The reaction time-course obtained by ESI-MS monitoring was found to be consistent with the data obtained earlier by HPLC, and the quantitative profile was successfully simulated by kinetic modeling of the enzymatic hydrolysis. It is obvious that the real-time monitoring method by ESI-MS allows a faster and cheaper determination of the chitinase activity with unlabeled substrate. Further, the enzymatic activity of the E67Q mutant of the barley chitinase was analyzed and the role of Glu67 was discussed comparing the mass spectra of enzyme protein obtained in native and in denatured conditions. Then it was determined that the observed loss of the enzymatic activity in E67Q is definitely caused by a point mutation of Glu67 but not due to partial unfolding of the mutated enzyme. Finally, association constants of enzyme–oligosaccharide complexes were calculated from Scatchard plots obtained by mass spectra. The binding free energy values obtained for E67Q were found to be comparable to those previously obtained in liquid phase, but less dependent upon the chain length of the oligosaccharides. To our knowledge, this study is the first enzymatic characterization of chitinase exclusively by such an innovative ESI-MS system.     

Exploring the mechanism of binding of UDP-galactopyranose to UDP-galactopyranose mutase by STD-NMR spectroscopy and molecular modeling

UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. In this study, reliable structural binding modes of the natural substrate, UDP-Galp, and inhibitor, UDP, in the UGM active site were provided with the combined use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations. UDP-Galp and UDP exhibited similar binding epitopes recognized by UGM. However, the relative binding affinities of the ligands changed dramatically upon reduction of UGM, as explored by competitive STD-NMR experiments. UDP-Galp competes with UDP for binding to UGM, especially when UGM is in its reduced state. Docking studies for predicting the binding mode within the active site of the two monomers in UGM explored the possibility that the mobile loop might act as a gateway for substrate binding, and the structure of the binding cleft in monomer A might be a closer approximation of the substrate-bound active site than monomer B. Important information regarding the critical interactions of UGM with UDP-Galp has been obtained.     

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods-namely, charge optimization and protein design-were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT-RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a 10-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease.