Potentiation of Ligand Binding through Cooperative Effects in Monoamine Oxidase B

Abstract

Crystallographic and biochemical studies have been employed to identify the binding site and mechanism for potentiation of imidazoline binding in human monoamine oxidase B (MAO B). 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) inhibits recombinant human MAO B with a K_i of 8.3 ± 0.6 μm, whereas tranylcypromine-inhibited MAO B binds 2-BFI with a K_d of 9 ± 2 nm, representing an increase in binding energy Δ(ΔG) of −3.9 kcal/mol. Crystal structures show the imidazoline ligand bound in a site that is distinct from the substrate-binding cavity. Contributions to account for the increase in binding affinity upon tranylcypromine inhibition include a conformational change in the side chain of Gln²⁰⁶ and a “closed conformation” of the side chain of Ile¹⁹⁹, forming a hydrophobic “sandwich” with the side chain of Ile³¹⁶ on each face of the benzofuran ring of 2-BFI. Data with the I199A mutant of human MAO B and failure to observe a similar binding potentiation with rat MAO B, where Ile³¹⁶ is replaced with a Val residue, support an allosteric mechanism where the increased binding affinity of 2-BFI results from a cooperative increase in H-bond strength through formation of a more hydrophobic milieu. These insights should prove valuable in the design of high affinity and specific reversible MAO B inhibitors.

Introduction

Imidazoline compounds have attracted considerable attention in the literature since their original discovery as antihypertensive agents (1). Three separate pharmacological targets for this class of compounds have been identified and termed the I₁-, I₂-, and I₃-binding sites. I₁ receptors are found in brain and in several peripheral tissues, the I₃ site is located in pancreatic β-cells involved in glucose-dependent insulin secretion, and the I₂ site is known to be subclassed into I_2a and I_2b sites based on their respective sensitivities to the guanidine diuretic, amiloride. A number of studies have identified the outer membrane mitochondrial enzymes, monoamine oxidases A and B (MAO A⁵ and MAO B), as possessing I₂-binding sites. These studies range from photoaffinity labeling (2, 3) to I₂-binding site population analysis in MAO (A or B)-deficient transgenic mice (4, 5). However, a great deal of uncertainty exists in the literature in understanding the molecular nature of these interactions. Inhibition studies show that a number of I₂ ligands function as reversible inhibitors of MAO A and of MAO B either competitively or non-competitively. The K_i values determined for reversible inhibition are usually found to be in the micromolar range (6, 7). However, radiolabeled I₂ ligand binding studies with membrane preparations of MAO B show nanomolar K_d values that are in a concentration range below that where pharmacological responses are observed (8, 9). A number of investigators have found that the level of MAO B in preparations exhibiting high affinity sites is only 5–10% of the total enzyme present (9–11). It is not known whether this high affinity binding also results in enzyme inhibition. To date, no molecular explanation has been found to resolve these observations except to propose that the nanomolar binding site is separate from the active site and is possessed by a subpopulation of enzyme occurring by an unknown mechanism. No evidence exists for any altered enzyme forms (alternate splicing or post-translational modification(s)), which might account for the observed substoichiometric levels of high affinity binding sites on MAO B.

The Eli Lilly group (11–13) observed that inhibition of human MAO B by tranylcypromine increases the level of high affinity I₂-binding sites from 5–10% to ∼90% of the total enzyme. This potentiation is observed with MAO B in human platelets, in membrane preparations from human cortex, and from medulla preparations as well as with membrane particles of human recombinant MAO B (but not with human MAO A) expressed in insect cells (13). These data suggest that inhibition of MAO B by tranylcypromine alters the enzyme to a form that now contains a site with high affinity for I₂ ligands. This behavior is reported to occur with human MAO B but not with rat MAO B although the two enzymes are ∼90% identical in sequence. Therefore, with these unknown questions regarding the generation of natural high affinity I₂ sites on MAO B, a structural study of the influence of tranylcypromine-inhibited purified human MAO B on its interaction with 2-(2-benzofuranyl)-2-imidazoline (2-BFI) (see structure in Fig. 1a) offers a model system to provide molecular insights. Further studies on this system are of interest not only to probe the nature of I₂ sites on MAO B but also to investigate the molecular interactions responsible for this synergistic behavior, which could be of importance in the development of highly specific reversible MAO B inhibitors.

FIGURE 1.

Structural framework of I₂ ligand binding to human MAO B. a, structure of 2-BFI. b, overall structure of the MAO B monomer in complex with 2-BFI (carbons in magenta), which binds in the entrance cavity space, and tranylcypromine (carbons in gray), which is covalently bound to the FAD flavin ring (carbons in yellow). Oxygens are in red, and nitrogens are in blue. The protein backbone is shown as green ribbon. c, superposition of the 2-BFI-tranylcypromine-MAO B ternary complex (carbons in green) onto the tranylcypromine-MAO B binary complex (carbons in gray). Carbons of 2-BFI are in magenta. Hydrogen bonds are shown as dashed lines.

Our laboratories as well as others have expressed and purified recombinant forms of both human and rat MAO A and MAO B (14–17) and have determined crystal structures of the human enzymes (18–20) in complex with several different inhibitors. Therefore, the necessary systems to explore the I₂-binding site structure in human MAO B are available. The questions addressed in this study include the following. 1) Do any differences exist for the site localization for I₂ ligand binding in human MAO B in its resting form (low affinity site) and in its tranylcypromine-inhibited form (high affinity site)? 2) Can a high affinity imidazoline site be identified and demonstrated with purified enzyme samples inhibited with tranylcypromine? 3) Does purified, recombinant human MAO B exhibit imidazoline ligand binding behavior comparable with that observed in membrane preparations from human tissue sources? The I₂ ligand, 2-BFI (Fig. 1a), is used exclusively because this I₂ ligand has been extensively investigated as one of the more potent I₂ ligands and has been shown to exhibit high affinity binding to human MAO B after tranylcypromine inhibition (11–13).

The results presented in this study describe a unique binding behavior to the well known drug target, MAO B, which may have relevance for future specific inhibitor design. The I₂ site for 2-BFI is identified as the “entrance cavity” of the bipartite active site of human MAO B. The closed conformation of the Ile¹⁹⁹ “gate” side chain and an altered conformation of the Gln²⁰⁶ side chain are induced by the covalent modification of the flavin cofactor by tranylcypromine and result in the creation of the high affinity binding site for 2-BFI. These results are discussed with consideration of previous studies of membrane preparations of MAO B found in the literature.

EXPERIMENTAL PROCEDURES

Chemicals and Reagents

All reagents were purchased from Sigma-Aldrich and from Tocris and used without further purification. Farnesylamine was a generous gift provided by Dr. Neil Castagnoli (Virginia Polytechnic Institute and State University). Recombinant human MAO B and mutant forms were expressed in Pichia pastoris and purified as described previously (14–17). I199A MAO B mutant enzyme was prepared by gene mutations using the Stratagene QuikChange XL site-directed mutagenesis kit and confirmed by gene sequence analysis. Crystals of MAO B and the I199A mutant were grown using established protocols (18); in particular, the enzyme was incubated with the covalent inhibitor (either tranylcypromine or rasagiline), gel-filtered to remove the excess inhibitor, and then incubated with 2 mm 2-BFI prior to crystallization. Data collections were performed at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, and high resolution structural analysis was performed as described previously (18). Diffraction data statistics for all structures presented are listed in Table 1.

TABLE 1.

Crystallographic data collection and refinement statistics

	Wild type					I199A, 2-BFI + tranylcypromine
	Tranylcypromine	2-BFI + tranylcypromine	2-BFI	2-BFI + rasagiline	2-BFI + isatin
PDB entry	2XFU	2XCG	2XFN	2XFQ	2XFP	2XFO
Unit cella (Å)	a = 130.6	a = 131.9	a = 131.5	a = 131.5	a = 131.1	a = 130.6
	b = 222.5	b = 223.7	b = 222.4	b = 223.4	b = 222.6	b = 221.9
	c = 86.2	c = 86.7	c = 86.4	c = 86.6	c = 86.4	c = 86.0
Resolution (Å)	2.2	1.9	1.6	2.2	1.7	2.1
Rsymb,c (%)	13.0 (47.2)	9.9 (34.8)	7.5 (22.2)	8.6 (20.6)	8.6 (44.0)	17.0 (58.1)
Completenessc (%)	99.9 (99.9)	100.0 (100.0)	99.0 (100.0)	99.1 (99.5)	96.2 (97.9)	95.5 (97.6)
Unique reflections	64,040	100,933	164,059	64,575	141,445	69,410
Redundancyc	3.7 (3.9)	6.2 (6.2)	3.0 (3.0)	4.1 (4.0)	3.5 (3.3)	2.9 (2.8)
I/σc	4.9 (2.1)	15.6 (5.2)	9.8 (4.1)	11.3 (6.7)	9.9 (2.5)	5.4 (1.8)
Average B value for 2-BFI/inhibitor (Å2)	Not present/37.3	10.6/14.8	21.1/Not present	20.4/16.1	11.9/19.4	25.9/32.8
Rcrystd (%)	18.8	15.5	16.6	16.0	16.1	18.9
Rfreed (%)	23.9	18.9	19.0	21.2	19.2	24.4
r.m.s.e bond length (Å)	0.010	0.020	0.018	0.022	0.030	0.020
r.m.s.e bond angle (°)	1.3	1.5	1.7	1.7	2.3	1.8

^a The space group is C222.

^b R_sym = Σ|I_i − 〈I〉|/ΣI_i, where I_i is the intensity of i^th observation and 〈I〉 is the mean intensity of the reflection.

^c Values in parentheses are for reflections in the highest resolution shell.

^d R_cryst = Σ|F_obs − F_calc|/ΣF_obs where F_obs and F_calc are the observed and calculated structure factor amplitudes, respectively. R_cryst and R_free were calculated using the working and test set, respectively.

^e r.m.s., root mean square.

Competition Assays

K_i values for 2-BFI inhibition of human MAO A, rat and human MAO B, and the I199A MAO B mutant were determined by measuring the initial rates of substrate oxidation (6–8 different concentrations) in the presence of varying concentrations of inhibitor (4–8 different concentrations). All samples were incubated for 5 min at 25 °C prior to the addition of enzyme. All assays were performed using the horseradish peroxidase-Amplex Red-coupled assay (21) in which the increase in H₂O₂ product concentration with time is determined spectrally (ϵ₅₀₀ = 54,000 m⁻¹ cm⁻¹). Standard MAO A and MAO B activity assays were performed using p-trifluoromethyl benzylamine and farnesylamine, respectively, at 25 °C in a 50 mm phosphate buffer (pH 7.5) and 0.5% (w/v) RTX-100 at air saturation.

All steady state kinetic data were fit to the appropriate form of the Michaelis-Menten equation and analyzed using GraphPad Prism software, version 5.0. The mode of inhibition was determined using global fit analysis of the velocity versus concentration curves in the presence and absence of 2-BFI to fit equations for competitive, mixed, non-competitive, and uncompetitive inhibition; the fit providing the highest r² value was selected for determination of the inhibition constants.

Fluorescence Binding Assays

An independent approach for determination of 2-BFI binding took advantage of the fluorescence emission of the benzofuran ring of 2-BFI, which exhibits an emission maximum at 400 nm. Changes in emission intensity of 2-BFI were monitored in the 350–600 nm spectral region using an Aminco-Bowman Series 2 luminescence spectrophotometer with a 1-cm path length quartz cuvette at 25 °C. Binding was monitored by the decrease in 2-BFI fluorescence emission intensity on binding to the enzyme. The emission changes were corrected for residual fluorescence contribution due to the enzyme, and the data were plotted as a change in fluorescence versus concentration of MAO and fit to a single site binding equation accounting for ligand depletion (22) for determination of the K_d value.

where F₀ = 0.5 (the degree of fluorescence quenching on binding of 2-BFI to MAO-B), X = [MAO B], and Y = the measured reduction in fluorescence at 400 nm.

The tranylcypromine-inhibited MAO samples were prepared by titrating MAO solutions in 50 mm potassium phosphate at pH 7.2 in 50% glycerol (v/v) with minimal amount of tranylcypromine (in an aqueous solution) until complete loss of activity was observed. These buffer conditions are found to prevent precipitation of the protein on inhibition by tranylcypromine. The inhibited samples were stored in the dark overnight at 4 °C (tranylcypromine-inhibited MAO samples that were not incubated overnight did not demonstrate high affinity binding, indicating that slow conformational changes are required). For the high affinity site titrations, 10 nm 2-BFI was dissolved in a buffer solution of 50 mm potassium phosphate at pH 7.4, which had been filtered through a 0.2-μm filter to remove any particulates. This solution was titrated with 10 μm tranylcypromine-inhibited MAO B in microliter portions. A control was set up by titrating MAO B into the buffer listed above in the absence of 2-BFI. The emission spectra were then subtracted from the 2-BFI binding titration to remove any background fluorescence originating from MAO. Binding was determined from the Δfluorescence intensity at λ₃₉₉. To probe the low affinity site binding (micromolar K_d), slightly different conditions were employed. Because higher MAO B concentrations are required, these titrations required a detergent component. 2-BFI (20 μm) was dissolved in a buffer solution of 50 mm potassium phosphate at pH 7.4, 20% glycerol (v/v), and 0.8% octylglucoside (w/v). This solution (3 ml) was titrated with 40 μm MAO B in 10–100-μl aliquots. Control titrations were performed to be able to subtract any interfering enzyme fluorescence.

RESULTS

Structure of the 2-BFI Complex with Tranylcypromine-inhibited MAO B

To identify the site for 2-BFI binding to tranylcypromine-inhibited MAO B, the 1.9 Å x-ray structure was determined for the inhibited enzyme co-crystallized in the presence of 2-BFI as shown in Fig. 1. The structure shows that 2-BFI is bound in the entrance cavity of the bipartite cavity of the enzyme with H-bonds between the carbonyl of Pro¹⁰², the phenolic OH group of Tyr³²⁶, and the imidazole moiety of the bound ligand. The benzofuran ring is inserted into an aliphatic cleft formed by the side chains of Ile¹⁹⁹ and Ile³¹⁶ (Fig. 1, b and c). As a result of 2-BFI binding, the loop spanning from amino acids 100 to 103 is moved toward the interior of the protein, bringing Pro¹⁰² in to the proper position for H-bonding to 2-BFI.

This higher resolution structure of the ternary 2-BFI-tranylcypromine enzyme complex provides new structural insight that revises our previous structure of the binary tranylcypromine-enzyme complex (18). The ring-opened form of tranylcypromine is confirmed to be covalently bound to the C(4a) position on the flavin but at the benzyl carbon rather than at the carbon β to the aromatic ring, as suggested previously (Fig. 2, a and b). Updated coordinates of the MAO B structure in complex with tranylcypromine have been deposited (Protein Data Bank (PDB) code 2XFU, which replaces PDB code 1OJB).

FIGURE 2.

Tranylcypromine binding to human MAO B. a, weighted 2F_o − F_c electron density map (1.5 σ contour level, 1.9 Å resolution, Table 1) for the FAD cofactor and tranylcypromine in the 2-BFI-tranylcypromine-MAO B ternary complex. The electron density clearly indicates the presence of a covalent adduct between the inhibitor and the C4a atom of the flavin. Carbons are in gray, nitrogens are in blue, and oxygens are in red. b, structure of the tranylcypromine-FAD covalent adduct.

Binding of 2-BFI to MAO B

With native enzyme, steady state kinetic data show reversible inhibition of human MAO B activity, which fits competitive behavior with a K_i value of 8.3 ± 0.6 μm (Table 2). Farnesylamine was used as a substrate because previous structural data show bound farnesol to traverse both entrance and substrate cavities (23) so that any inhibition observed by 2-BFI would occur by its binding to only the free form of the enzyme. (A more comprehensive kinetic analysis of 2-BFI inhibition will be reported elsewhere.) Inhibition studies with purified preparations of human MAO A and with rat MAO B show similar or higher K_i values and exhibit patterns of competitive inhibition (human MAO A, K_i = 17 ± 1 μm; rat MAO B, K_i = 67 ± 5 μm) (Table 2).

TABLE 2.

Inhibition data for 2-BFI with purified MAO

MAO preparation	Substrate/type of reversible inhibition	Ki
		μm
Human WT MAO B	Farnesylamine/competitive	8.3 ± 0.6
Human MAO B I199A	p-CF3-Benzylaminea/non-competitive	58 ± 6
Rat WT MAO B	Farnesylamine/competitive	67 ± 5
Human WT MAO A	p-CF3-Benzylaminea/competitive	17 ± 1

^a Farnesylamine could not be used as a substrate for this mutant enzyme due to the high K_m value exhibited. The same behavior with this substrate analogue is also exhibited by human MAO A.

To probe the binding of 2-BFI to the tranylcypromine-inhibited forms of the enzymes, an alternative approach had to be taken because these inhibited enzymes exhibit no catalytic activity. In addition, if high affinity binding is to be observed, highly sensitive techniques had to be employed. The benzofuran ring of 2-BFI is expected to exhibit intrinsic fluorescence, which is readily observed with an emission maximum at 400 nm. (Fig. 3a). On the addition of tranylcypromine-inhibited human MAO B, the fluorescence intensity decreases as a result of binding. The total level of fluorescence quenching on saturation approaches 50%. 2-BFI binds to tranylcypromine-inhibited human MAO B with a K_d value of 9 ± 2 nm (Fig. 3,a, right panel, and b). In contrast, no high affinity binding to native human MAO B is observed, and only a partial fluorescence quenching of 2-BFI (20 μm) is observed (Fig. 3a, left panel) on the addition of MAO B. The addition of higher concentrations of MAO B resulted in protein precipitation; thus, a complete titration could not be done. Analysis of the titration data (Fig. 3c) indicates that 2-BFI binds with an estimated K_d value in the micromolar range, in agreement with the K_i value determined by inhibition studies (Fig. 3c). Most importantly, fluorescence titrations with purified preparations show no high affinity binding of 2-BFI after inhibition by tranylcypromine for human MAO A, rat MAO B, and the I199A mutant of human MAO B.

FIGURE 3.

Interaction of 2-BFI with human MAO B. a, left panel, quenching of 2-BFI (20 μm) fluorescence on the addition of aliquots of human MAO B. Right panel, quenching of 2-BFI (10 nm) fluorescence on the addition of aliquots of tranylcypromine-inhibited human MAO B (protein concentration 0–717 nm). b, binding analysis of 2-BFI to tranylcypromine-inhibited MAO B (TCP-MAO B, protein concentration 0–30 nm). The solid line represents a fit to a single site binding with a K_d = 9 ± 2 nm. Assays were performed at 25 °C in 50 mm potassium P_i (pH 7.4). c, binding analysis of 2-BFI to native human MAO B (Hwt MAO B). The solid line denotes the pseudo-linear relation expected with MAO B concentrations below the K_d value. Assays were performed at 25 °C in 50 mm potassium P_i (pH 7.4) with 20% (v/v) glycerol and 0.8% (w/v) octylglucoside. Fluorescence intensities are represented in arbitrary units.

Structures of Complexes of MAO B with 2-BFI

The ∼10³-fold increase in binding affinity for 2-BFI on tranylcypromine inhibition specifically observed in human MAO B corresponds to ∼−3.9 kcal/mol. Comparative structural approaches were used to determine the source of this potentiation (Fig. 4, a–e). Structural elucidation of native MAO B with 2-BFI in the absence of tranylcypromine showed that 2-BFI is bound in the entrance cavity (Figs. 1b and 4b). Two differences are apparent relative to the structure determined after tranylcypromine inhibition (compare Fig. 4, a and b). The first is that the conformation of the side chain of Ile¹⁹⁹ is in the open conformation for tranylcypromine-uninhibited enzyme, whereas it is in the closed conformation in the tranylcypromine-inhibited form. In addition, the conformation of Gln²⁰⁶ is altered in the tranylcypromine-inhibited form to a position where it can form an H-bond with the phenolic OH of Tyr³²⁶. This conformation of Gln²⁰⁶ is only observed to date in enzyme structures after inhibition by tranylcypromine. It has not been observed to occur with any of the other MAO B structures determined with either covalent or non-covalent inhibitors (24).

FIGURE 4.

Close-up view of 2-BFI binding in different MAO B complexes. Oxygens are in red, nitrogens are in blue, protein carbons are in green, 2-BFI carbons are in magenta, and flavin carbons are in yellow. Waters are shown as red spheres. a, ternary complex of MAO B with 2-BFI and tranylcypromine (gray carbons). b, binary complex with 2-BFI. c, ternary complex with 2-BFI and rasagiline (gray carbons). d, ternary complex with 2-BFI and isatin (gray carbons). e, ternary complex of the I199A MAO B mutant bound to 2-BFI and tranylcypromine (gray carbons).

Because the conformation of the Ile¹⁹⁹ gate residue is suggested to be involved in the increase in 2-BFI binding affinity, the I199A MAO B mutant was crystallized. 2-BFI binds to the mutant enzyme weakly with a K_i of 58 ± 6 μm exhibiting non-competitive inhibition behavior (Table 2). Fluorescence titrations with the tranylcypromine-inhibited mutant enzyme show no high affinity binding of 2-BFI as found for wild-type protein. Co-crystallization of tranylcypromine-inhibited I199A MAO B with 2-BFI and structural elucidation (Fig. 4e) shows no structural alterations when compared with wild-type enzyme with the exception that the Ile¹⁹⁹ side chain is now absent and replaced by the methyl group of the Ala (in effect, opening the gate between the two cavities). The side chain of Ile¹⁹⁹ is no longer available for hydrophobic interactions with the benzofuran ring of 2-BFI. These results demonstrate the importance of the “gating” action of the Ile¹⁹⁹ side chain in 2-BFI binding affinity and in the potentiation by tranylcypromine inhibition.

Reports in the literature suggesting that the I₂ site on MAO B is distinct from the active site are based on observations that irreversible inhibitors such as pargyline, deprenyl, or phenelzine fail to prevent I₂ ligand binding (25). Our previous structural data on MAO B have shown that these inhibitors are covalently bound to the flavin cofactor and are situated in the substrate cavity and do not extend into the entrance cavity (24). Fluorescence quenching titration experiments of 2-BFI to test the binding of these inhibited forms of MAO B give no evidence for any binding of the imidazoline ligand in the nanomolar concentration range. The structure of rasagiline-inhibited MAO B after co-crystallization with 2-BFI shows binding of the imidazoline in the entrance cavity (Fig. 4c), as found in all other 2-BFI bound structures. These data demonstrate the feasibility of weak (micromolar) 2-BFI binding to enzyme that has been inhibited by irreversible inhibitors and that the site for binding is independent of the nature of the inhibitor used. However, in the rasagiline complex, the structural determinants identified previously in tranylcypromine-inhibited MAO B including the side chains of Gln²⁰⁶ and Ile¹⁹⁹ are not in conformations to support high affinity binding of 2-BFI (Fig. 4, a and c).

The reversible MAO inhibitor, isatin, has been shown to bind in the substrate cavity of MAO B with a closed conformation of the Ile¹⁹⁹ side chain (18). Inhibition studies indicate that there is no observable cooperativity in binding by the two ligands (data not shown). Structural studies of MAO B show that both ligands are bound to MAO B with isatin in the substrate cavity and 2-BFI in the entrance cavity (Fig. 4d). The Ile¹⁹⁹ side chain is in a closed conformation, but the side chain conformation of Gln²⁰⁶ is unaltered from the native conformer and, therefore, not able to participate in H-bond formation with Tyr³²⁶ as observed with tranylcypromine-inhibited enzyme (compare Fig. 4, a and d). Taken together these data show that neither the conformational change of Ile¹⁹⁹ nor the conformational change of Gln²⁰⁶ alone is able to generate the high affinity binding site, but both are required at the same time to have the potentiation effect. These properties appear to be observed only with tranylcypromine-inhibited MAO B, although the possibility exists that similar conformational forms can be induced by other inhibitor ligands in the MAO B substrate cavity.

DISCUSSION AND CONCLUSIONS

The structural data presented show that the imidazoline-binding site on human MAO B is located in the entrance cavity. Thus, the early idazoxan photoaffinity labeling data of Raddatz et al. (2) suggesting that the domain labeled is localized to residues Lys¹⁴⁹–Met²²² is fully consistent with the structure. The combination of structural, mutagenesis, and binding data contained in this study provides strong evidence for the roles of the side chains of Ile¹⁹⁹ and Gln²⁰⁶ as major factors in mediating the enhanced affinity of MAO B for 2-BFI. The alterations in side chain conformations and natures of side chains found in MAO B for the residues Ile¹⁹⁹ and Ile³¹⁶ required for potentiation of the binding affinity of 2-BFI are expected to increase the hydrophobicity of the binding site, and therefore, enhance the H-bond strength of the 2-BFI-protein H-bonds identified in the structures. An increase in hydrophobicity on H-bond energetics has been demonstrated to provide a positive cooperativity in binding affinities with thrombin inhibitors (26), and a similar situation appears to be operative in MAO B. Consistently, conversion of Ile¹⁹⁹ to Ala results in the loss of the hydrophobic sandwich for the benzofuran ring with reduced binding affinity and no tranylcypromine potentiation (Fig. 4a).

It is of interest that rat MAO B exhibits a lower binding affinity for 2-BFI (Table 2) and does not exhibit the increased affinity for 2-BFI after inhibition by tranylcypromine (13) although the two sources of MAO B exhibit ∼90% sequence identity. Examination of the rat MAO B sequence shows a Val substitution for Ile at position 316 (Fig. 1c). This rather conservative amino acid change appears to result in large changes in the hydrophobic interactions with 2-BFI accounting for the reduced affinity in the rat protein. Altered hydrophobic contributions to function in comparisons with Ile and Val have been observed in tRNA synthetase (27, 28) and in the PXP domain of the K⁺ channel (29) involved in gate loop opening and closing in ion translocation. The rat MAO B comparison with human MAO B provides an additional example. These results may also explain why human MAO A exhibits such differential behavior from human MAO B in binding the imidazolines. MAO A has a monopartite cavity (19, 20), and therefore, imidazoline analog binding is suggested to occur at or near the substrate site (7) and to exhibit quite different protein-ligand interactions than those observed in MAO B. This is demonstrated by a lower binding affinity for 2-BFI (Table 2) and no observable enhancement of binding by tranylcypromine inhibition.

Three main conclusions highly relevant for the field of MAO pharmacology can be made as a result of insights gained from this study. 1) The binding affinity of imidazolines to MAO B is dependent on the mammalian source, and therefore, discrepancies in the literature appear to arise from comparisons of human with rat, rabbit, or mouse MAO B. 2) Our data suggest that the subpopulation of MAO B able to interact with nanomolar affinity with 2-BFI (9–11) represents an endogenous ligand-bound form structurally similar to the tranylcypromine-inhibited enzyme. Validation of this hypothesis will require further studies. 3) The potentiation of ligand binding to the entrance cavity of MAO B (not found in MAO A) by ligand occupancy in the substrate cavity opens up possibilities for the design of highly specific MAO B inhibitors that specifically target the entrance cavity space. Molecular dynamics calculations on these interactions may point out additional approaches for the exploitation of conformational equilibria and conformational sampling for the creation of cooperative binding and ligand potentiation.