Molecular recognition of macrocyclic peptidomimetic inhibitors by HIV-1 protease.

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.     

Dynamics in the active site of β-secretase: a network analysis of atomistic simulations

The aspartic protease β-secretase (BACE) catalyzes the hydrolysis of the amyloid precursor protein (APP) which leads to amyloid-β aggregation and, ultimately, the perilous Alzheimer's disease. The conformational dynamics and free energy surfaces of BACE at three steps of the catalytic cycle are studied here by explicit solvent molecular dynamics simulations (multiple runs for a total of 2.2 μs). The overall plasticity of BACE is essentially identical for the three states of the substrate: the octapeptide reactant, gem-diol intermediate, and cleavage products. In contrast, the network of hydrogen bonds in the active site is more stable in the complex of BACE with the gem-diol intermediate than the other two states of the substrate. The spontaneous release of the C-terminal (P1'-P4') fragment of the product follows a single-exponential time dependence with a time constant of 50 ns and does not require the opening of the flap. The fast dissociation of the C-terminal fragment is consistent with the transmembrane location and orientation of APP and its further processing by γ-secretase. On the other hand, the N-terminal (P4-P1) fragment of the product does not exit the BACE active site within the simulation time scale of 80 ns. A unified network analysis of the complexes of BACE with the three states of the substrate provides an estimation of the activation free energy associated with the structural rearrangements that involve only noncovalent interactions. The estimated rearrangement barriers are not negligible (up to 3 kcal/mol) but are significantly smaller than the barrier of the peptide bond hydrolysis reaction.     

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.     

Thermal effects on an enzymatically latent conformation of coagulation factor VIIa.

Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891-18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease structure of the N-terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which the N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive conformation is reflected in the relative accessibility of the active site and the N-terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilization of the active conformation; and (c) activity measurements of a cross-linked factor VIIa-tissue factor complex; this showed that cross-linking stabilized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.     

Closing of the flaps of HIV-1 protease induced by substrate binding: a model of a flap closing mechanism in retroviral aspartic proteases

The active site of aspartic proteases is covered by one or more flaps, which control access to the active site and play a significant role in the binding of the substrate. An extensive conformational change of the flaps takes place upon binding of substrate to the active site. A long molecular dynamics simulation was performed on the complex consisting of a peptide (CA-p2) from a natural substrate cleavage site of the gag/pol polyprotein placed in the active site of HIV-1 protease (PR) with an open flap conformation. During the simulation, the substrate induced the closing of the flaps into the closed conformation in an asymmetrical way through a hydrophobic intermediate state cluster. The nature of the residues of HIV-1 PR identified to be important in the flap closing mechanism is conserved across known structures of retroviral aspartic proteases family. The flap closing mechanism described in HIV-1 PR is proposed to be a general model for flap closing in retroviral aspartic proteases.     

Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.     

Tobacco etch virus proteinase: crystal structure of the active enzyme and its inactive mutant

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 A resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217-221 of the enzyme are involved in formation of the binding pockets S3-S6. This indicates that the autolysis of the peptide bond Met218-Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in considerable decrease in the enzymatic activity.     

In vitro characterization of a purified NS2/3 protease variant of hepatitis C virus

The cleavage of the hepatitis C virus polyprotein between the nonstructural proteins NS2 and NS3 is mediated by the NS2/3 protease, whereas the NS3 protease is responsible for the cleavage of the downstream proteins. Purification and in vitro characterization of the NS2/3 protease has been hampered by its hydrophobic nature. NS2/3 protease activity could only be detected in cells or in in vitro translation assays with the addition of microsomal membranes or detergent. To facilitate purification of this poorly characterized protease, we truncated the N-terminal hydrophobic domain, resulting in an active enzyme with improved biophysical properties. We define a minimal catalytic region of NS2/3 protease retaining autocleavage activity that spans residues 904-1206 and includes the C-terminal half of NS2 and the N-terminal NS3 protease domain. The NS2/3 (904-1206) variant was purified from Escherichia coli inclusion bodies and refolded by gel filtration chromatography. The purified inactive form of NS2/3 (904-1206) was activated by the addition of glycerol and detergent to induce autocleavage at the predicted site between Leu(1026) and Ala(1027). NS2/3 (904-1206) activity was dependent on zinc ions and could be inhibited by NS4A peptides, peptides that span the cleavage site, or an N-terminal peptidic cleavage product. This NS2/3 variant will facilitate the development of an assay suitable for identifying inhibitors of HCV replication.     

Probing the S1/S1' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.     

Mutations that alter the activity of the Rous sarcoma virus protease.

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.     

Ligand modifications to reduce the relative resistance of multi-drug resistant HIV-1 protease

Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection and viral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug design for the treatment of HIV-1 infected patients. To examine the role of the P1 and P1′positions of the substrate in inhibitory efficacy of multi-drug resistant HIV-1 protease 769 (MDR 769), we performed a series of structure–function studies. Using the original CA/p2 cleavage site sequence, we generated heptapeptides containing one reduced peptide bond with an L to F and A to F double mutation at P1 and P1′ (F-r-F), and an A to F at P1′ (L-r-F) resulting in P1/P1′ modified ligands. Here, we present an analysis of co-crystal structures of CA/p2 F-r-F, and CA/p2 L-r-F in complex with MDR 769. To examine conformational changes in the complex structure, molecular dynamic (MD) simulations were performed with MDR769–ligand complexes. MD trajectories show the isobutyl group of both the lopinavir analog and the CA/p2 L-r-F substrate cause a conformational change of in the active site of MDR 769. IC₅₀ measurements suggest the non identical P1/P1′ ligands (CA/p2 L-r-F and lopinavir analog) are more effective against MDR proteases as opposed to identical P1/P1′ligands. Our results suggest that a non identical P1/P1′composition may be more favorable for the inhibition of MDR 769 as they induce conformational changes in the active site of the enzyme resulting in disruption of the two-fold symmetry of the protease, thus, stabilizing the inhibitor in the active site.     

C-terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity

HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4-40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.     

X-ray structure of HIV-1 protease in situ product complex

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.     

Catalytic Water Co-Existing with a Product Peptide in the Active Site of HIV-1 Protease Revealed by X-Ray Structure Analysis

Background

It is known that HIV-1 protease is an important target for design of antiviral compounds in the treatment of Acquired Immuno Deficiency Syndrome (AIDS). In this context, understanding the catalytic mechanism of the enzyme is of crucial importance as transition state structure directs inhibitor design. Most mechanistic proposals invoke nucleophilic attack on the scissile peptide bond by a water molecule. But such a water molecule coexisting with any ligand in the active site has not been found so far in the crystal structures.

Principal Findings

We report here the first observation of the coexistence in the active site, of a water molecule WAT1, along with the carboxyl terminal product (Q product) peptide. The product peptide has been generated in situ through cleavage of the full-length substrate. The N-terminal product (P product) has diffused out and is replaced by a set of water molecules while the Q product is still held in the active site through hydrogen bonds. The position of WAT1, which hydrogen bonds to both the catalytic aspartates, is different from when there is no substrate bound in the active site. We propose WAT1 to be the position from where catalytic water attacks the scissile peptide bond. Comparison of structures of HIV-1 protease complexed with the same oligopeptide substrate, but at pH 2.0 and at pH 7.0 shows interesting changes in the conformation and hydrogen bonding interactions from the catalytic aspartates.

Conclusions/Significance

The structure is suggestive of the repositioning, during substrate binding, of the catalytic water for activation and subsequent nucleophilic attack. The structure could be a snap shot of the enzyme active site primed for the next round of catalysis. This structure further suggests that to achieve the goal of designing inhibitors mimicking the transition-state, the hydrogen-bonding pattern between WAT1 and the enzyme should be replicated.     

Rationale for More Diverse Inhibitors in Competition with Substrates in HIV-1 Protease

The structural fluctuations of HIV-1 protease in interaction with its substrates versus inhibitors were analyzed using the anisotropic network model. The directions of fluctuations in the most cooperative functional modes differ mainly around the dynamically key regions, i.e., the hinge axes, which appear to be more flexible in substrate complexes. The flexibility of HIV-1 protease is likely optimized for the substrates' turnover, resulting in substrate complexes being dynamic. In contrast, in an inhibitor complex, the inhibitor should bind and lock down to inactivate the active site. Protease and ligands are not independent. Substrates are also more flexible than inhibitors and have the potential to meet the dynamic distributions that are inherent in the protease. This may suggest a rationale and guidelines for designing inhibitors that can better fit the ensemble of binding sites that are dynamically accessible to the protease.     

How does a symmetric dimer recognize an asymmetric substrate? A substrate complex of HIV-1 protease

The crystal structure of an actual HIV-1 protease-substrate complex is presented at 2.0 A resolution (R-value of 19.7 % (R(free) 23.3 %)) between an inactive variant (D25N) of HIV-1 protease and a long substrate peptide, Lys-Ala-Arg-Val-Leu-Ala-Glu-Ala-Met-Ser, which covers a full binding epitope of capsid(CA)-p2, cleavage site. The substrate peptide is asymmetric in both size and charge distribution. To accommodate this asymmetry the two protease monomers adopt different conformations burying a total of 1038 A(2) of surface area at the protease-substrate interface. The specificity for the CA-p2 substrate peptide is mainly hydrophobic, as most of the hydrogen bonds are made with the backbone of the peptide substrate. Two water molecules bridge the two monomers through the loops Gly49-Gly52 (Gly49'-Gly52') and Pro79'-Val82' (Pro79-Val82). When other complexes are compared, the mobility of these loops is correlated with the content of the P1 and P1' sites. Interdependence of the conformational changes allows the protease to exhibit its wide range of substrate specificity.     

Crystal structure of isoaspartyl aminopeptidase in complex with L-aspartate

The crystal structure of Escherichia coli isoaspartyl aminopeptidase/asparaginase (EcAIII), an enzyme belonging to the N-terminal nucleophile (Ntn)-hydrolases family, has been determined at 1.9-A resolution for a complex obtained by cocrystallization with l-aspartate, which is a product of both enzymatic reactions catalyzed by EcAIII. The enzyme is a dimer of heterodimers, (alphabeta)(2). The (alphabeta) heterodimer, which arises by autoproteolytic cleavage of the immature protein, exhibits an alphabetabetaalpha-sandwich fold, typical for Ntn-hydrolases. The asymmetric unit contains one copy of the EcAIII.Asp complex, with clearly visible l-aspartate ligands, one bound in each of the two active sites of the enzyme. The l-aspartate ligand is located near Thr(179), the N-terminal residue of subunit beta liberated in the autoproteolytic event. Structural comparisons with the free form of EcAIII reveal that there are no major rearrangements of the active site upon aspartate binding. Although the ligand binding mode is similar to that observed in an l-aspartate complex of the related enzyme human aspartylglucosaminidase, the architecture of the EcAIII active site sheds light on the question of substrate specificity and explains why EcAIII is not able to hydrolyze glycosylated asparagine substrates.     

Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases delta-selinene synthase and gamma-humulene synthase

Two recombinant sesquiterpene synthases from grand fir, delta-selinene synthase and gamma-humulene synthase, each produce more than 30 sesquiterpene olefins from the acyclic precursor farnesyl diphosphate. These enzymes contain a pair of DDxxD motifs, on opposite lips of the presumptive active site, which are thought to be involved in substrate binding and could promote multiple orientations of the substrate alkyl chain from which multiple families of cyclic olefins could derive. Mutagenesis of the first aspartate of either DDxxD motif resulted in depressed k(cat), with lesser effect on K(m), for delta-selinene synthase and afforded a much simpler product spectrum composed largely of monocyclic olefins. Identical alterations in gamma-humulene synthase produced similar kinetic effects with a simplified product spectrum of mostly acyclic and monocyclic olefins. Although impaired in product diversity, none of the mutant synthases lost entirely the capacity to generate complex structures. These results confirm the catalytic significance of the DDxxD motifs and imply that they also influence permitted modes of cyclization. Deletion of an N-terminal arginine pair in delta-selinene synthase (an element potentially involved in substrate isomerization) altered kinetics without substantially altering product outcome. Finally, mutation of an active-site tyrosine residue thought to play a role in proton exchange had little influence; however, substitution of a nearby active site aspartate dramatically altered kinetics and product outcome.     

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.     

Multiple-site trimethylation of ribosomal protein L11 by the PrmA methyltransferase

Ribosomal protein L11 is a universally conserved component of the large subunit, and plays a significant role during initiation, elongation, and termination of protein synthesis. In Escherichia coli, the lysine methyltransferase PrmA trimethylates the N-terminal alpha-amino group and the epsilon-amino groups of Lys3 and Lys39. Here, we report four PrmA-L11 complex structures in different orientations with respect to the PrmA active site. Two structures capture the L11 N-terminal alpha-amino group in the active site in a trimethylated post-catalytic state and in a dimethylated state with bound S-adenosyl-L-homocysteine. Two other structures show L11 in a catalytic orientation to modify Lys39 and in a noncatalytic orientation. The comparison of complex structures in different orientations with a minimal substrate recognition complex shows that the binding mode remains conserved in all L11 orientations, and that substrate orientation is brought about by the unusual interdomain flexibility of PrmA.