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. Author manuscript; available in PMC: 2021 Mar 27.
Published in final edited form as: J Am Chem Soc. 2020 Dec 29;143(4):1836–1845. doi: 10.1021/jacs.0c09970

Conformational Plasticity in Human Heme-Based Dioxygenases

Khoa N Pham 1, Ariel Lewis-Ballester 2, Syun-Ru Yeh 3
PMCID: PMC8000349  NIHMSID: NIHMS1682643  PMID: 33373218

Abstract

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) are two important heme proteins that degrade the essential amino acid, L-tryptophan (Trp), along the kynurenine pathway. The two enzymes share a similar active site structure and an analogous catalytic mechanism, but they exhibit a variety of distinct functional properties. Here we used carbon monoxide (CO) as a structural probe to interrogate how the functionalities of the two enzymes are encoded in their structures. With X-ray crystallography, we detected an unexpected photochemical intermediate trapped in a crystal of the hIDO1-CO-Trp complex, where CO is photolyzed from the heme iron by X-rays at cryogenic temperatures (100 K). The CO photolysis triggers a large-scale migration of the substrate Trp, as well as the photolyzed CO, from the active site to a temporary binding site, Sa*. It is accompanied by a large conformational change to an active site loop, JK-LoopC, despite the severely restricted protein motion under the frozen conditions, which highlights the remarkable conformational plasticity of the hIDO1 protein. Comparative studies of a crystal of the hTDO-CO-Trp complex show that CO and Trp remain bound in the active site under comparable X-ray illumination, indicating a much more rigid protein architecture. The data offer important new insights into the structure and function relationships of the heme-based dioxygenases and provide new guidelines for structure-based design of inhibitors targeting them.

Graphical abstract

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INTRODUCTION

Human indoleamine 2,3 dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) catalyze the first and rate-limiting step of the kynurenine pathway: the conversion of l-tryptophan (Trp) to N-formylkynurenine (NFK).1-4 Over-activation of the two enzymes upregulates the kynurenine pathway, thereby leading to a variety of diseases, such as Alzheimer’s disease, Huntington’s disease, amyotrophic lateral sclerosis, dementia, depression, schizophrenia, and cancer.5-13 Accordingly, they have been recognized as two important therapeutic drug targets.

The expression of hIDO1 is inducible by proinflammatory stimuli, such as IFN-γ, in almost all human tissues as a key part of the intricate immunomodulatory mechanism, while hTDO is mostly expressed constitutively in the liver to regulate Trp flux along the kynurenine pathway.3 There is no direct sequence homology between the two enzymes; in addition, hIDO1 is a monomer,14 while hTDO is a homotetramer.15 As such, they were originally considered as two unrelated enzymes.

Recent crystallographic studies surprisingly revealed that hIDO1 and hTDO share a similar active site structure, albeit possessing distinct overall protein folds.14-16 Resonance Raman and computational studies suggest that the two enzymes follow an analogous two-step dioxygenase mechanism (Figure 1).17-19 The reaction is initiated by radical addition of the ferric heme iron bound superoxide to the C2 of the Trp to generate an alkylperoxo transition state. The O–O bond is then homolytically cleaved to generate a compound II type of ferryl and Trp-epoxide intermediate. Subsequently, the epoxide ring in the Trp epoxide is protonated by the ammonium group of the Trp, which opens up the ring, thereby promoting the addition of the ferryl oxygen to C2 of the Trp to make NFK. This mechanism is supported by additional computational20 and experimental21-24 studies and is now widely accepted in the field.

Figure 1.

Figure 1.

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Trp dioxygenation reaction catalyzed by hIDO1 and hTDO. The active ternary complex and ferryl intermediate are shown to illustrate the hypothesized ferryl-based radical addition mechanism.

Despite their similar active site structure and catalytic mechanism, hIDO1 and hTDO exhibit a variety of functional differences. The hIDO1 protein is active toward various indoleamine derivatives, while hTDO is specific for Trp.3 The hIDO1 protein is easily autooxidizable during enzyme turnover,25 while hTDO can turn over continuously without losing any electrons.26 The inactive ferric derivative of hIDO1, but not hTDO, can be activated by binding superoxide to generate the active ternary complex, thereby sustaining the enzyme turnover.27-29 Substrate binding in hIDO1 retards ligand binding to the heme iron,30 while that in hTDO facilitates it.31 The activity of hIDO1 is directly regulated by substrate inhibition through binding a second Trp in an inhibitory binding site on the proximal side of the heme;16 in contrast, the activity of hTDO is regulated at the protein level by controlling the lifetime of the protein through binding a second Trp in an exosite that is ~40 Å away from the active site15.

The static crystal structures of hIDO1 and hTDO have been instrumental for shaping our current understanding of the dioxygenase chemistry and guiding structure-based design of enzyme-selective inhibitors. However, it remains unclear how the distinct functionalities of the two enzymes are encoded in their structures. Here we sought to use CO as a structural probe and employ X-ray crystallography, combined with optical absorption spectroscopy, to interrogate the structure and function relationship of the two heme-based dioxygenases.

■ RESULTS AND DISCUSSION

Crystal Structure of a Photochemical Intermediate of the hIDO1-CO-Trp Complex.

We first crystallized the hIDO1-CO-Trp complex and solved its structure (Figure 2). The overall structure of the complex is similar to that of the hIDO1-CN-Trp complex reported previously.16 However, there is no electron density associated with the ligand or substrate in the active site. As the integrity of the crystal was confirmed by its absorption spectrum prior to the diffraction measurements (inset i), the data suggest that (i) CO is photolyzed from the heme iron by the X-rays used to obtain the diffraction data, as observed in one other heme protein system, cytochrome c oxidase,32,33 and (ii) CO photolysis leads to the dissociation of the Trp from the active site. This photochemical intermediate is denoted as hIDO1-CO-Trp* hereinafter to distinguish it from the intact hIDO1-CO-Trp complex. It is important to note that we were unable to obtain the structure of the intact complex by reducing the X-ray power due to the high photolability of the complex.

Figure 2.

Figure 2.

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Crystal structure of a photochemical intermediate of the hIDO1-CO-Trp complex (PDB code: 6UBP). The DE-Hairpin is colored in green. The A-, D-, and E-Helix, as well as the DE-Loop (linking the DE-Hairpin to the E-Helix), are colored in magenta. The disordered JK-Loop (linking the J-Helix to the K-Helix) is depicted by the dotted line. The substrate Trp and ligand CO are shown as green sticks, while the heme and its proximal histidine ligand are shown as gray sticks. (i) Absorption spectrum of the crystal of the hIDO1-CO-Trp complex obtained prior to the diffraction measurements, showing two characteristic bands at 540 and 570 nm which confirm the integrity of the complex. (ii) The 2mFo-DFc map of the bound Trp and CO in the Sa* site contoured at 1.0 σ. (iii) Surface view of the solvent-exposed Sa* site with Trp and CO (green sticks) bound to it.

Where do the CO and Trp go following the photolysis? Visual inspection of the diffraction data reveals extra electron density in a surface-exposed pocket near the opening of the active site, which can be clearly modeled with Trp and CO (inset ii and Figure S1). This new surface exposed binding site (inset iii), termed Sa* hereinafter, coincides well with the previously identified inhibitor binding site C1, where a hIDO1-selective inhibitor, BMS-986205, transiently docks (Figure S2).34 In the Sa* site, the Trp binds between the D-Helix and the DE-Loop, with its indole ring lying perpendicular to the heme, while the photolyzed CO docks between the indole ring of the Trp and the side chain of R231 in the D-Helix. The CO was modeled with the polar O atom pointing toward the polar side chain of R231, although the two atoms of the CO are indistinguishable.

Structural comparison of the hIDO1-CO-Trp* complex with the hIDO1-CN-Trp complex (used as a model for the intact hIDO1-CO-Trp complex) reveals that CO photolysis induces a >6 Å translocation of the substrate Trp (Figure 3A) which is accompanied by an ~90° rotation of the indole ring down toward the heme plane, such that the carboxylate and ammonium groups point up away from it. The large-scale translocation of the Trp is associated with the outward movement of the R231 side chain and a large conformational change to the JK-LoopC (vide infra) as well as small structural adjustments in the DE-Loop and D-Helix.

Figure 3.

Figure 3.

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Photoinduced ligand and substrate migration in hIDO1. (A) Active site structure of hIDO1-CO-Trp* (gray) superimposed with that of the hIDO1-CN-Trp complex (PDB: 5WMU) (magenta), used as a model for the intact hIDO1-CO-Trp complex, to illustrate the CO photolysis induced relocation of Trp and CO, as indicated by the green arrows. The bottom panel shows sequence alignment of the JK-Loops of hIDO1 and hTDO. The conserved “GTGG” motif is highlighted in magenta. (B) Schematic illustration of the ligand and substrate migration and the accompanied opening of the JK-LoopC induced by X-rays or visible light (steps 1 and 2) and their subsequent rebinding and the closure of the JK-LoopC upon the termination of the photolysis light (steps 3–5).

The JK-Loop in hIDO1 is 22 residues long—11 residues longer than that in hTDO (see the bottom panel in Figure 3A). The N-terminal fragment of the JK-Loop (JK-LoopN) is present in hIDO1, not in hTDO. It contains residues with a high propensity for disorder35 and is structurally unresolved in all the wild-type hIDO1 structures solved to date. The C-terminal fragment of the JK-Loop (JK-LoopC), on the other hand, shows high sequence homology with the JK-Loop in hTDO. It contains a “GTGG” motif that is fully conserved in the IDO1 and TDO family of enzymes. It is totally disordered in the absence of substrate,14 while Trp binding induces its folding into a reverse β-turn structure that sequesters the Trp in the active site.16 The “GTGG” motif is not only critical for stabilizing the substrate in the active site by forming an intricated H-bonding network with it but also essential for locking the JK-LoopC in the closed conformation,36,37 thereby shielding the substrate from the bulk solvent. In the hIDO1-CO-Trp* structure, the [LEAKG] fragment of the JK-LoopC is totally disordered, while the [TGGTDL] fragment converts to a one-helical turn structure extending from the K-Helix. Together they open up the active site and create a temporary docking site for the substrate and ligand.

It is notable that recently Mirgaux et al. reported a substrate-free structure of a hIDO1 mutant (PDB: 7A62),38 where the whole JK-Loop was refined in one of the four molecules in the asymmetric unit. However, the specific JK-Loop conformer is stabilized by a unique crystal packing resulting from the mutation; in addition, the B-factors of the loop residues are extremely high (up to 180) with respect to that of the overall structure (40). All in all, the data support the view that the JK-Loop is highly dynamic in the absence of the substrate.

The CO photolysis induced substrate and ligand migration in the hIDO1-CO-Trp complex is consistent with previous spectroscopic data showing that ligand dissociation in hIDO1 significantly weakens substrate binding.30 It is also in good agreement with flash photolysis studies suggesting that CO dissociation from the heme iron (induced by visible light) triggers the relocation of the substrate from the active site to an unknown binding site, which retards the unimolecular rebinding of the CO to the heme iron.39 The structure of the hIDO1-CO-Trp* complex reported here provides the first glimpse of this previously unidentified binding site, Sa*, where the bound Trp partially blocks the return passage of the CO, accounting for the retarded CO rebinding kinetics. The flash photolysis studies revealed that upon the termination of the photolysis light CO rebinds to the heme iron prior to Trp rebinding to the active site.39

Taken together the data suggest a photoinduced ligand and substrate migration mechanism illustrated in Figure 3B. CO photolysis induced by either X-rays or visible light triggers the migration of the Trp and CO out of the active site into the Sa* site (step 1). It is accompanied by the opening of the JK-LoopC (step 2). Upon the termination of the light, CO first rebinds to the heme iron (step 3), followed by the rebinding of the Trp to the active site (step 4). It is finished with the closure of the JK-LoopC (step 5), which completes the reaction cycle. This substrate and ligand binding trajectory supports the previously proposed scenario that during enzyme turnover O2 binds to the heme iron prior to substrate binding.30,40 It suggests that during turnover the Sa* site functions as a transient docking site to guide the sequential entry of the ligand and substrate, while the JK-LoopC operates as a gate to dynamically control the entry processes.

Flash photolysis induced CO migration in heme proteins, in particular myoglobin (Mb), has been extensively studied as a mean to understand reversible ligand binding processes.41-45 Visible light illumination of Mb-CO crystals at cryogenic temperatures was shown to trap photochemical intermediates, where CO docks in a variety of well-defined docking sites.42-45 Temperature-dependent studies revealed that CO migration following photolysis is limited to the distal heme pocket because of restricted protein motions under the frozen conditions, while at a relatively higher temperature (>180 K) the photolyzed CO can further migrate to the proximal side of the heme because of the opening of additional ligand migration tunnels resulting from increased protein conformational freedom.45

The CO photolysis reaction reported here represents a unique case where CO photolysis triggers large-scale translocation of not only the photolyzed CO but also the bulky substrate Trp, which is accompanied by a large conformational change to an active site loop, the JK-LoopC. The ability of the protein to undergo such a significant structural rearrangement at the cryogenic temperature (100 K), where most molecular motions are frozen, highlights the remarkable conformational plasticity of hIDO1.

Crystal Structure of the hTDO–CO-Trp Complex.

We next crystallized the hTDO-CO-Trp complex and solved its structure (Figure 4) with a similar protocol. The integrity of the crystal is confirmed by its absorption spectrum prior to the diffraction measurements (Figure S3). Unlike hIDO1, clear electron density associated with Trp and CO is evident in the active site (inset i). The hTDO protein is a homotetramer, made by a dimer of dimers as reported previously.15 Each dimer is stabilized by domain swapping of the N-terminal A-Helix that forms the roof of the active site.

Figure 4.

Figure 4.

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Crystal structure of the hTDO-CO-Trp complex (PDB code: 6UD5). The A-, D-, and E-Helix, as well as the DE-Loop (linking the D-Helix to the E-Helix) and the JK-Loop (linking the J-Helix to the K-Helix), are shown in magenta. The A-Helix is taken from the neighboring subunit. The Trp bound in the active site and exosite (Sexo) and the heme iron bound CO are shown as green sticks. (i) The electron density map associated with Trp and CO, as well as a water molecule (red sphere) intervening between them, is contoured at 1.0 σ. The residues in the A-Helix forming the roof of the substrate binding site are labeled in magenta. (ii) The active site structure of hTDO-CO-Trp (gray) is superimposed with that of hTDO-O2-Trp (green) (PDB code: 5TI9) to highlight their structural similarities. (iii) The active site structure of hTDO-CO-Trp (gray) is superimposed with that of hIDO1-CN-Trp (green) (PDB code: 5WMU), used as a model for the hIDO1-CO-Trp complex, to illustrate the structural differences between the CO complexes of the two enzymes.

The substrate Trp sits in a hydrophobic pocket on top of the heme, with its indole ring lying perpendicular to it and the indoleamine group H-bonding with H76 (equivalent to S167 in hIDO1). Its ammonium group forms H-bonds with T342 in the JK-Loop (equivalent to JK-LoopC in hIDO1) and the propionate group of the heme, while its carboxylate group forms bidentate iron pairing with R144. Trp is shielded from the bulk solvent by the JK-Loop with a reverse β-turn structure.

The ligand CO is coordinated to the heme iron with its terminal O atom ~2.9 and 3.4 Å away from the indoleamine and C2 of the Trp, respectively. It forms H-bonds with the ammonium group of the Trp via an intervening water, which leads to a bent Fe–C–O conformation, with ∠Fe–C–O ~ 161°, even though the Fe–C–O moiety typically prefers a linear geometry.46,47 The overall structure of the active site is similar to that of the hTDO-O2-Trp complex15 (inset ii), although the electron density of the intervening water in the latter is too weak to be identified.15

Conformational Plasticity in hIDO1 vs hTDO.

Why is the heme iron-bound CO photolabile in crystals of the hIDO1-CO-Trp complex but not in those of the hTDO-CO-Trp complex? Previous flash photolysis studies showed that CO in the hTDO-CO-Trp complex, like that in the hIDO1-CO-Trp complex, is readily photolyzable by visible light.31 It suggests that CO in the hTDO complex crystals can be photo-dissociated by X-rays, but it presumably rebinds proficiently due to the steric hindrance imposed by the bound Trp, which unlike that in hIDO1 does not migrate out of the active site upon the photolysis.

Why does CO photolysis trigger Trp migration in hIDO1 but not in hTDO? To address this question, we employed optical absorption spectroscopy to interrogate the conformational plasticity of the active site in hIDO1 vs hTDO using three structurally diverse inhibitors—PF-06840003 (IPD),48 epacadostat,16,49 and NLG91950—as structural probes (Figure 5A).

Figure 5.

Figure 5.

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CO complexes of hIDO1 and hTDO perturbed by inhibitor binding. (A) Molecular structures of the three inhibitors tested in this work and a list of the peak maxima of the Soret and visible bands of the CO complexes obtained in the presence of Trp or the inhibitors. The right panel shows superimposed structures of the hIDO1–IPD complex (PDB code: 6PZ1) and the hTDO–IPD complex (PDB code: 6PYZ) to highlight their preferential binding of the R- and S-enantiomer, respectively. The functional groups of each inhibitor occupying the “A” and “B” pockets of the active site are indicated in the structures. The chiral carbon center in IPD is indicated by asterisks. (B) Absorption spectra of the CO complexes of hIDO1 and hTDO in the presence of various inhibitors.

The substrate-free CO complexes of hIDO1 and hTDO display similar spectral features, with the Soret maximum at ~420 and two visible bands at ~540/570 nm, characteristic for CO–heme complexes, while Trp binding introduces only small changes to the spectra, similar to those reported previously.26,51 The replacement of Trp with the inhibitors in either hIDO1 or hTDO introduces minor perturbations to the spectra, except that IPD binding in hIDO1 induces a large shift of the Soret maximum to 430 nm and the merging of the two visible bands into a single band at 560 nm, indicating the dissociation of CO from the heme iron (Figure 5B). What is unique about IPD? IPD is the only indole derivative among the three inhibitors tested; in addition, it is a structural analogue of Trp, with its S enantiomer mimicking the l-enantiomer of tryptophan.52

Crystallographic studies showed that epacadostat16 and NLG91953 bind to the active site of hIDO1 with their main aromatic rings residing in the “A” pocket and their tails occupying the “B” pocket, where they extrude out into the solvent, causing the JK-LoopC to adopt the open conformation. On the other hand, comparative studies of IPD in complex with hIDO1 vs hTDO revealed that the two enzymes selectively bind the R- and S-enantiomer of the inhibitor, respectively.48,52 In both enzymes the inhibitor binds to the active site with the indole and succinimide ring occupying the “A” and “B” pocket, respectively, with the JK-LoopC locked in the closed conformation, as that observed in the Trp complexes.52 The protein structure of the hTDO–IPDS complex is almost identical with that of the Trp complex, where IPDS binds in a pose comparable to that of the substrate Trp. In contrast, the protein structure of the hIDO1–IPDR complex, as well as the binding pose of the inhibitor, significantly deviates from those of the Trp complex.52

Comparison of the structure of the hIDO1–IPDR complex with that of the hTDO–IPDS complex (see right panel in Figure 5A) reveals that the indole ring of IPD is rotated by ~30°, such that its indoleamine group forms a H-bond with S167, while the succinimide ring moves down toward the heme plane to π-stack with it. This unique binding pose of IPDR in hIDO1 is associated with significant protein conformational rearrangements that presumably optimize the protein–inhibitor interactions, thereby accounting for the 400-fold higher efficacy of the inhibitor in hIDO1 with respect to that in hTDO.52 The crystal structure data suggest that the close proximity of the succinimide ring to the ligand binding site in hIDO1, made possible by its unique conformational plasticity, accounts for the IPD-induced CO exclusion (Figure 5).

The incredible conformational plasticity of the active site in hIDO1 with respect to that in hTDO is in good agreement with previous MD simulations,36,54 showing that the Trp complex of hIDO1 is in a dynamic equilibrium between two conformers, cf1 and cf2, while the comparable complex of a bacterial analogue of hTDO is locked in a fixed cf1 conformer. Here cf1 represents the active structure, where the substrate is bound in the active site and the JK-LoopC is fixed in the closed conformation, while cf2 is associated with an inactive structure, where Trp is bound in a remote binding site and the JK-LoopC is fluctuating between the open and closed conformations. Reinspection of this remote Trp binding site in the cf2 conformer of hIDO1 reveals that it coincides well with the Sa* site identified in this work. It accentuates the functional significance of the Sa* site and supports the view that the Sa* site functions as a transient docking site for the substrate during enzyme turnover. Taken together the data suggest that the hIDO1 protein has a rugged conformational energy landscape, with an energy minimum characterized by substates separated by low energy barriers, while hTDO protein has a smooth conformational energy landscape with an energy minimum characterized by a single conformational state.

Unique CO Binding Site Created by the Bound Trp in hIDO1 vs hTDO.

CO has long been recognized as a powerful probe for active site structures of heme proteins.55-57 As illustrated in Figure 6A, when CO is coordinated to a heme iron, the Fe–CO moiety exists in two resonance forms, forms i and ii, due to π back-bonding. A ligand binding pocket with a positive polarity disfavors form i, with a partial positive charge on the O atom of the CO, due to electrostatic repulsion. A reduction in the electrostatic potential of the ligand environment, for example by site-directed mutations, shifts the equilibrium toward form i. As such, the Fe–CO and C–O stretching frequencies, νFe–CO and νC–O, are typically inversely correlated, and the inverse correlation line can be used as a convenient ruler for gauging the electrostatic potential of the ligand environment in heme proteins.

Figure 6.

Figure 6.

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Inverse correlation plot of νFe–CO vs νC–O (A) and schematic illustration of the distinct ligand–substrate–protein interactions in the hIDO1-CO-Trp complex vs the hTDO-CO-Trp complex (B). The location of each data point in (A) depends on the polarity of the ligand environment based on π back-bonding as depicted by the blue arrows in the equation on the top of the plot. The black hollow arrows in the plot indicate the shifts of the data points induced by Trp binding. The interactions in hIDO1-CO-Trp shown in (B) are proposed based on the structure of the hIDO1-CN-Trp complex (PDB code: 5WMU), while those in hTDO-CO-Trp are taken from this work. The intervening water molecules are indicated by “W”.

Previous resonance Raman studies showed that the hIDO1 data point sits in the middle of the inverse correlation line, while Trp binding caused it to shift to the upper left corner, indicating that the bound Trp creates a ligand environment with a positive electrostatic potential.51 In contrast, the hTDO data point resides at a lower position, while Trp binding leads to a downshift of the data point to the lower right corner,26 indicating that the bound substrate produces a distinct ligand environment with a much lower electrostatic potential.

How does the bound Trp exert such a diverse impact on the ligand environment in the two structurally related enzymes? Structural comparison of the hTDO-CO-Trp complex with the hIDO1-CN-Trp complex (used as a model for the hIDO1-CO-Trp complex) (see inset iii in Figure 4) suggests that in hIDO1 the ammonium group of the Trp is responsible for the positive polar environment of the heme iron bound CO, while in hTDO, the presence of the water molecule intervening between the ammonium and the ligand might shield the positive charge on the ammonium, thereby reducing the polarity of the ligand environment.

The unique substrate–ligand interaction in hIDO1 is associated with distinct protein-substrate interactions (Figure 6B), where the protein forms water mediated H-bonds with the indoleamine group of the substrate via S167, in contrast to the direct H-bond between H76 and the indoleamine in hTDO. Mutagenesis studies showed that the permutation of the two residues in hIDO1 and hTDO dramatically reduces their enzyme activities,25,58,59 suggesting that the substrate–ligand interactions in the two enzymes are strongly coupled to their protein conformational plasticity.

CONCLUSION

The dioxygenase reaction catalyzed by hIDO1 and hTDO represents a unique case in heme protein oxygen chemistry, where the dioxygen is activated by the substrate Trp, instead of electrons and protons.3 To initiate the reaction, it is essential for the enzymes to position the substrate Trp in a precise regio-orientaion with respect to the heme iron-bound dioxygen. As such, the critical structural elements important for substrate recognition and binding are mostly conserved in the two enzymes. Nonetheless, our current studies reveal that hIDO1 exhibits remarkable conformational plasticity which distinguishes it from hTDO. This distinctive structural fingerprint of hIDO1 is presumably important for it to execute multitasks under the harsh and complicated (pro)inflammatory conditions.

How is the unique protein conformational plasticity of hIDO1 encoded in its structure? Visual inspection of the structures of hIDO1 and hTDO suggests that, in addition to the replacement of H76 in hTDO with S167 in hIDO1, the insertion of two additional structural domains, (i) a 25 residue long DE-hairpin (between the DE-Loop and the D-Helix) and (ii) a 11 residue long disordered JK-LoopN fragment (between the J-Helix and the JK-LoopC), in the active site of hIDO1 might at least partially account for its unusual protein conformational plasticity. This unique structural feature of hIDO1 does not seem to impact its dioxygenase chemistry, as it exhibits an enzyme efficiency similar to that of hTDO. However, it casts a unique substrate binding site with a much broader selectivity3 and a characteristic ligand environment encouraging autoxidation and superoxide binding.25,27-29

In summary, the good agreement of the crystal structure of the photochemical intermediate, hIDO1-CO-Trp*, reported here with that inferred from the flash photolysis studies in free solution39 underscores its biochemical relevance. It highlights the unusual conformational flexibility of hIDO1 and offers the first glimpse of the previously unidentified transient substrate and ligand binding site, Sa*, which offers new guidelines for structure-based design of hIDO1-selective inhibitors. Furthermore, it provides molecular evidence supporting the previously proposed sequential ligand and substrate binding mechanism.30,40 This work will promote further progress in our understanding of the mechanisms by which the functional properties of hIDO1 and hTDO are encoded in their structures.

MATERIALS AND METHODS

hIDO1-CO-Trp Crystal Preparation.

The hIDO1 protein was expressed and purified as reported previously.60 The crystallization was initiated by mixing the ferric hIDO1-CN-Trp complex protein (40 mg/mL in 50 mM Tris buffer at pH 7.4) with the precipitant solution (100 mM sodium thiosulfate and 20% PEG 8000 in 100 mM CAPS buffer at pH 10) supplemented with 5 mM CN and 10 mM Trp. The crystals were grown at 4 °C by using the under-oil microbatch method as reported previously.16 They were harvested, thoroughly washed with a large amount of CN-free mother solution, and transferred to a sealed tube. The mixture was purged with CO and then reduced with 5 mM dithionite. The reduced crystals were washed with the CO-purged mother solution to remove dithionite and its oxidative products. The crystals were incubated for 18–30 h at 4 °C to ensure CO binding. The integrity of the hIDO1-CO-Trp crystals was confirmed by optical absorption spectroscopy before they were harvested and cryoprotected by supplementing the CO-purged mother solution with 25% (v/v) ethylene glycol. The crystals were then mounted and flash-frozen in liquid nitrogen for data collection.

hTDO-CO-Trp Crystal Preparation.

hTDO protein was expressed and purified as reported previously.26 The crystallization was initiated by mixing the ferrous hTDO-Trp complex protein (40 mg/mL) with N2-purged precipitant solution (2% Tacsimate and 5% PEG 3350 in 50 mM sodium citrate at pH 5.6) in the presence of 10 mM Trp inside a glovebox at room temperature as reported previously.15 The crystals were grown by using the under-oil microbatch method and were harvested and transferred into a sealed tube containing CO-purged mother solution supplemented with 10 mM Trp. The integrity of the hTDO-CO-Trp crystals was confirmed by optical absorption spectroscopy before they were harvested and cryoprotected by supplementing CO-purged mother solution with 25% (v/v) ethylene glycol. The crystals were then mounted and flash-frozen in liquid nitrogen for data collection.

Crystallographic Data Collection and Analysis.

The crystallographic data of the hIDO1-CO-Trp* complex were collected at Beamline 17-ID-2 of the National Synchrotron Light Source II, while those of the hTDO-CO-Trp complex were collected by the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline staff at Sector 31 of the Advanced Photon Source. The diffraction images were indexed, integrated, and scaled with XDS61 and Aimless.62 The Karplus–Diederichs method63 was used to find a proper resolution cutoff for each structure. Molecular replacement was conducted with Phaser64 through the CCP4i graphic interface65 using the hIDO1-CN-Trp complex structure (PDB code: 5WMU) and the hTDO-Trp complex structure (PDB code: 5TIA) as the search model for hIDO1 and hTDO, respectively. Further model building was performed using COOT.66 Structure refinements were performed by using Refmac5.65,67,68 Data processing and refinement statistics are summarized in Table S1. The polder maps were generated by using the software Phenix.69,70 The structural models were displayed with PyMOL (http://www.pymol.org/).

Spectroscopic Measurements.

All the optical absorption spectra were obtained with the UV2100 spectrophotometer with a spectral slit width of 1 nm. The hIDO1 samples (4 μM) and hTDO samples (4 μM) were prepared in 50 mM Tris buffer (pH 7.4). They were purged in a sealed cuvette with CO and then reduced with a minimum amount of dithionite, followed by the addition of each inhibitor (1 or 5 mM) prepurged with CO. IPD, epacadostat, and NLG919 were purchased from Advanced ChemBlocks Inc., Selleck Chemicals LLC, and BioVision Inc., respectively.

Supplementary Material

Supplemental Material

ACKNOWLEDGMENTS

We thank Dr. Denis L. Rousseau for helpful discussions. This research used Beamline 17-ID-2 of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract DE-SC0012704. The structural data of the TDO-CO-Trp complex were collected by the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline staff at Sector 31 of the Advanced Photon Source. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility. This work was supported by National Institutes of Health Grants GM115773 and GM126297 to S.-R.Y.

ABBREVIATIONS

Trp,

l-tryptophan

CO

carbon monoxide

hIDO1

human indoleamine, 2,3-dioxygenase 1

hTDO

human tryptophan dioxygenase

NFK

N-formylkynurenine

IPD

PF-06840003.

Footnotes

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.0c09970.

Table S1; Figures S1 and S2 (PDF)

The authors declare no competing financial interest.

Contributor Information

Khoa N. Pham, Department of Physiology and Biophysics, Albert Einstein College of Medicine, The Bronx, New York 10461, United States.

Ariel Lewis-Ballester, Department of Physiology and Biophysics, Albert Einstein College of Medicine, The Bronx, New York 10461, United States.

Syun-Ru Yeh, Department of Physiology and Biophysics, Albert Einstein College of Medicine, The Bronx, New York 10461, United States.

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