CD72 is an inhibitory pattern recognition receptor that recognizes ribosomes and suppresses production of anti-ribosome autoantibody.

B cell responses to nucleic acid-containing self-antigens that involve intracellular nucleic acid sensors play a crucial role in autoantibody production in SLE. CD72 is an inhibitory B cell co-receptor that down-regulates BCR signaling, and prevents the development of SLE. We previously showed that CD72 recognizes the RNA-containing self-antigen Sm/RNP, a target of SLE-specific autoantibodies, and induces B cell tolerance to Sm/RNP by specifically inhibiting B cell response to this self-antigen. Here, we address whether CD72 inhibits B cell response to ribosomes because the ribosome is an RNA-containing self-antigen and is a target of SLE-specific autoantibodies as well as Sm/RNP. We demonstrate that CD72 recognizes ribosomes as a ligand, and specifically inhibits BCR signaling induced by ribosomes. Although conventional protein antigens by themselves do not induce proliferation of specific B cells, ribosomes induce proliferation of B cells reactive to ribosomes in a manner dependent on RNA. This proliferative response is down-regulated by CD72. These results suggest that ribosomes activate B cells by inducing dual signaling through BCR and intracellular RNA sensors and that CD72 inhibits B cell response to ribosomes. Moreover, CD72-/- but not CD72+/+ mice spontaneously produce anti-ribosome autoantibodies. Taken together, CD72 induces B cell self-tolerance to ribosomes by recognizing ribosomes and inhibiting RNA-dependent B cell response to this self-antigen. CD72 appears to prevent development of SLE by inhibiting autoimmune B cell responses to multiple RNA-containing self-antigens. Because these self-antigens but not protein self-antigens induce RNA-dependent B cell activation, self-tolerance to RNA-containing self-antigens may require a distinct tolerance mechanism mediated by CD72.

Affinity-matured antibody with a disulfide bond in H-CDR3 loop.

Affinity maturation increases antigen-binding affinity and specificity of antibodies by somatic hypermutation. Various monoclonal antibodies against (4-hydroxy-3-nitrophenyl)acetyl (NP) were obtained during affinity maturation. Among them, highly matured anti-NP antibodies, such as E11 and E3, possess Cys96H and Cys100H in the complementarity-determining region 3 of the heavy chain, which would form a disulfide bond. In this study, we evaluated the effects of disulfide bonds on antigen binding by generating single-chain Fv (scFv) antibodies of E11 and its mutants, E11_C96KH/C100EH and E11_C96KH/C100QH, and determined their antigen-binding thermodynamics and kinetics. The binding affinities of the Cys mutants were lower than that of E11 scFv, indicating that the disulfide bond contributed to antigen binding, especially for stable complex formation. This was also supported by the decreased affinity of E11 scFv in the presence of a reducing agent. The crystal structures of NP-free and NP-bound E11 scFvs were determined at high resolution, showing the existence of a disulfide bond between Cys96H and Cys100H, and the antigen recognition mechanism, which could be compared with those of other anti-NP antibodies, such as germline-type N1G9 and matured-type C6, as reported previously. These structures could explain the molecular basis of changes in antigen-binding affinity and thermal stability in the absence or presence of antigens. Small-angle X-ray scattering further showed a local conformational change in E11 scFv upon antigen binding in solution.

Structural analysis of the water channel AQP2 by single-particle cryo-EM.

Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful technique routinely used to determine the structures of membrane proteins. Because the single-particle method enables structural analysis of a whole protein with flexible parts that interfere with crystallization, we have focused our efforts on analyzing water channel structures. Here, utilizing this system, we analyzed the structure of full-length aquaporin-2 (AQP2), a primary regulator of vasopressin-dependent reabsorption of water at the renal collecting ducts. The 2.9 Å resolution map revealed a cytoplasmic extension of the cryo-EM density that was presumed to be the highly flexible C-terminus at which the localization of AQP2 is regulated in the renal collecting duct cells. We also observed a continuous density along the common water pathway inside the channel pore and lipid-like molecules at the membrane interface. Observations of these constructions in the AQP2 structure analyzed without any fiducial markers (e.g., a rigidly bound antibody) indicate that single-particle cryo-EM will be useful for investigating water channels in native states as well as in complexes with chemical compounds.

Varying the Directionality of Protein Catalysts for Aldol and Retro-Aldol Reactions.

Natural aldolase enzymes and created retro-aldolase protein catalysts often catalyze both aldol and retro-aldol reactions depending on the concentrations of the reactants and the products. Here, we report that the directionality of protein catalysts can be altered by replacing one amino acid. The protein catalyst derived from a scaffold of a previously reported retro-aldolase catalyst, catalyzed aldol reactions more efficiently than the previously reported retro-aldolase catalyst. The retro-aldolase catalyst efficiently catalyzed the retro-aldol reaction but was less efficient in catalyzing the aldol reaction. The results indicate that protein catalysts with varying levels of directionality in usually reversibly catalyzed aldol and retro-aldol reactions can be generated from the same protein scaffold.

Structural basis of mutants of PET-degrading enzyme from Saccharomonospora viridis AHK190 with high activity and thermal stability.

The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca2+ -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca2+ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca2+ to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS.

A Guillain-Barré syndrome-associated SIGLEC10 rare variant impairs its recognition of gangliosides.

Guillain-Barré syndrome (GBS), including its variant Miller Fisher syndrome (MFS), is an acute peripheral neuropathy that involves autoimmune mechanisms leading to the production of autoantibodies to gangliosides; sialic acid-containing glycosphingolipids. Although association with various genetic polymorphisms in the major histocompatibility complex (MHC) is shown in other autoimmune diseases, GBS is an exception, showing no such link. No significant association was found by genome wide association studies, suggesting that GBS is not associated with common variants. To address the involvement of rare variants in GBS, we analyzed Siglec-10, a sialic acid-recognizing inhibitory receptor expressed on B cells. Here we demonstrate that two rare variants encoding R47Q and A108V substitutions in the ligand-binding domain are significantly accumulated in patients with GBS. Because of strong linkage disequilibrium, there was no patient carrying only one of them. Recombinant Siglec-10 protein containing R47Q but not A108V shows impaired binding to gangliosides. Homology modeling revealed that the R47Q substitution causes marked alteration in the ligand-binding site. Thus, GBS is associated with a rare variant of the SIGLEC10 gene that impairs ligand binding of Siglec-10. Because Siglec-10 regulates antibody production to sialylated antigens, our finding suggests that Siglec-10 regulates development of GBS by suppressing antibody production to gangliosides, with defects in its function predisposing to disease.

Structural Dynamics of the PET-Degrading Cutinase-like Enzyme from Saccharomonospora viridis AHK190 in Substrate-Bound States Elucidates the Ca2+-Driven Catalytic Cycle.

A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca2+ binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca2+ binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca2+ binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca2+ can greatly accelerate.

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

Structural and biophysical analysis of sero-specific immune responses using epitope grafted Dengue ED3 mutants.

Dengue fever is a re-emerging tropical disease and its severe form is caused by cross-reactivity between its four serotypes (DEN1, DEN2, DEN3 and DEN4). The third domain of the viral envelope protein (ED3) contains the two major putative epitopes and is a highly suitable model protein for examining the molecular determinants of a virus' sero-specificity. Here we examine d the sero-specificity and cross-reactivity of the immune response against DEN3 and DEN4 ED3 using six epitope grafted ED3 variants where the surface-exposed epitope residues from DEN3 ED3 were switched to those of DEN4 ED3 and vice versa. We prepared anti-DEN3 and anti-DEN4 ED3 serum by immunizing Swiss albino mice and measured their reactivities against all six grafted mutants. As expected, both sera exhibited strong reactivity against its own serotype's ED3, and little cross-reactivity against their counterpart serotype's ED3s. E2 played a major role in the sero-specificity of anti-DEN3 serum, whereas E1 was important for DEN4 ED3's sero-specificity. Next, the reactivity patterns corroborated our working hypothesis that sero-specificity could be transferred by grafting the surface exposed epitope residues from one serotype to the other. To analyze the above results from a structural viewpoint, we determined the crystal structure of a DEN4 ED3 variant, where E2 was grafted from DEN3 ED3, at 2.78Å resolution and modeled the structures of the five remaining grafted variants by assuming that the overall backbone remained unchanged. The examination of the electrostatic and molecular surfaces of the variants suggested some further rationale for the sero-specificity of the immune responses.

Modeling and experimental assessment of a buried Leu-Ile mutation in dengue envelope domain III.

Envelope protein domain III (ED3) of the dengue virus is important for both antibody binding and host cell interaction. Here, we focused on how a L387I mutation in the protein core could take place in DEN4 ED3, but cannot be accommodated in DEN3 ED3 without destabilizing its structure. To this end, we modeled a DEN4_L387I structure using the Penultimate Rotamer Library and taking the DEN4 ED3 main-chain as a fixed template. We found that three out of seven Ile(387) conformers fit in DEN4 ED3 without introducing the severe atomic clashes that are observed when DEN3 serotype's ED3 is used as a template. A more extensive search using 273 side-chain rotamers of the residues surrounding Ile(387) confirmed this prediction. In order to assess the prediction, we determined the crystal structure of DEN4_L387I at 2 Å resolution. Ile(387) indeed adopted one of the three predicted rotamers. Altogether, this study demonstrates that the effects of single mutations are to a large extent successfully predicted by systematically modeling the side-chain structures of the mutated as well as those of its surrounding residues using fixed main-chain structures and assessing inter-atomic steric clashes. More accurate and reliable predictions require considering sub-angstrom main-chain deformation, which remains a challenging task.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins.

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported. In the present study, crystal structures of V2Hb from the tube worm Lamellibrachia satsuma have been determined in both the fully oxygenated and deoxygenated states. A glycosylation site and novel metal-binding sites for divalent cations were clearly observed with no intersubunit interactions in V2Hb. A comparison of the oxygenated and the deoxygenated forms of V2Hb reveals that the ternary- and quaternary-structural changes occur in a manner that maintains the molecular D3 symmetry. These structures suggest that the mechanisms of quaternary-structural changes between the oxy and deoxy states for the giant Hbs are identical across species.

Recognition Mechanism of a Novel Gabapentinoid Drug, Mirogabalin, for Recombinant Human α2δ1, a Voltage-Gated Calcium Channel Subunit.

Mirogabalin is a novel gabapentinoid drug with a hydrophobic bicyclo substituent on the γ-aminobutyric acid moiety that targets the voltage-gated calcium channel subunit α2δ1. Here, to reveal the mirogabalin recognition mechanisms of α2δ1, we present structures of recombinant human α2δ1 with and without mirogabalin analyzed by cryo-electron microscopy. These structures show the binding of mirogabalin to the previously reported gabapentinoid binding site, which is the extracellular dCache_1 domain containing a conserved amino acid binding motif. A slight conformational change occurs around the residues positioned close to the hydrophobic group of mirogabalin. Mutagenesis binding assays identified that residues in the hydrophobic interaction region, in addition to several amino acid binding motif residues around the amino and carboxyl groups of mirogabalin, are critical for mirogabalin binding. The A215L mutation introduced to decrease the hydrophobic pocket volume predictably suppressed mirogabalin binding and promoted the binding of another ligand, L-Leu, with a smaller hydrophobic substituent than mirogabalin. Alterations of residues in the hydrophobic interaction region of α2δ1 to those of the α2δ2, α2δ3, and α2δ4 isoforms, of which α2δ3 and α2δ4 are gabapentin-insensitive, suppressed the binding of mirogabalin. These results support the importance of hydrophobic interactions in α2δ1 ligand recognition.

Elucidation of binding mechanism, affinity, and complex structure between mWT1 tumor-associated antigen peptide and HLA-A*24:02.

We have applied our advanced computational and experimental methodologies to investigate the complex structure and binding mechanism of a modified Wilms' Tumor 1 (mWT1) protein epitope to the understudied Asian-dominant allele HLA-A*24:02 (HLA-A24) in aqueous solution. We have applied our developed multicanonical molecular dynamics (McMD)-based dynamic docking method to analyze the binding pathway and mechanism, which we verified by comparing the highest probability structures from simulation with our experimentally solved x-ray crystal structure. Subsequent path sampling MD simulations elucidated the atomic details of the binding process and indicated that first an encounter complex is formed between the N-terminal's positive charge of the 9-residue mWT1 fragment peptide and a cluster of negative residues on the surface of HLA-A24, with the major histocompatibility complex (MHC) molecule preferring a predominantly closed conformation. The peptide first binds to this closed MHC conformation, forming an encounter complex, after which the binding site opens due to increased entropy of the binding site, allowing the peptide to bind to form the native complex structure. Further sequence and structure analyses also suggest that although the peptide loading complex would help with stabilizing the MHC molecule, the binding depends in a large part on the intrinsic affinity between the MHC molecule and the antigen peptide. Finally, our computational tools and analyses can be of great benefit to study the binding mechanism of different MHC types to their antigens, where it could also be useful in the development of higher affinity variant peptides and for personalized medicine.

A P39R mutation at the N-terminal domain of human αB-crystallin regulates its oligomeric state and chaperone-like activity.

Recent structure analyses of αB-crystallin have proposed some models of the N-terminal domain and the manner of oligomerization, whereas the effects of the significantly high content of Pro residues at the N-terminal domain remain unclear. We report the properties of a novel P39R mutant of αB-crystallin. The content of α-helix was increased, and the molecular size of the P39R mutant was larger than that of wild-type αB-crystallin. A slight loss of chaperone-like activity was observed using alcohol dehydrogenase (ADH), while a significant increase was detected by insulin assay. The Pro residue at the N-terminal domain of αB-crystallin is important for oligomerization and function.

Structures of oxygen dissociation intermediates of 400 kDa V2 hemoglobin provide coarse snapshots of the protein allostery.

Ever since the historic discovery of the cooperative oxygenation of its multiple subunits, hemoglobin (Hb) has been among the most exhaustively studied allosteric proteins. However, the lack of structural information on the intermediates between oxygenated and deoxygenated forms prevents our detailed understanding of the molecular mechanism of its allostery. It has been difficult to prepare crystals of intact oxy-deoxy intermediates and to individually identify the oxygen saturation for each subunit. However, our recent crystallographic studies have demonstrated that giant Hbs from annelids are suitable for overcoming these problems and can provide abundant information on oxy-deoxy intermediate structures. Here, we report the crystal structures of oxy-deoxy intermediates of a 400 kDa Hb (V2Hb) from the annelid Lamellibrachia satsuma, following up on a series of previous studies of similar giant Hbs. Four intermediate structures had average oxygen saturations of 78%, 69%, 55%, and 26%, as determined by the occupancy refinement of the bound oxygen based on ambient temperature factors. The structures demonstrate that the cooperative oxygen dissociation is weaker, large ternary and quaternary changes are induced at a later stage of the oxygen dissociation process, and the ternary and quaternary changes are smaller with local perturbations. Nonetheless, the overall structural transition seemed to proceed in the manner of the MWC two-state model. Our crystallographic snapshots of the allosteric transition of V2Hb provide important experimental evidence for a more detailed understanding of the allostery of Hbs by extension of the Monod-Wyman-Changeux (MWC) model.

Lithocholic Acid Amides as Potent Vitamin D Receptor Agonists.

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3, 1] is an active form of vitamin D3 and regulates various biological phenomena, including calcium and phosphate homeostasis, bone metabolism, and immune response via binding to and activation of vitamin D receptor (VDR). Lithocholic acid (LCA, 2) was identified as a second endogenous agonist of VDR, though its potency is very low. However, the lithocholic acid derivative 3 (Dcha-20) is a more potent agonist than 1α,25(OH)2D3, (1), and its carboxyl group has similar interactions to the 1,3-dihydroxyl groups of 1 with amino acid residues in the VDR ligand-binding pocket. Here, we designed and synthesized amide derivatives of 3 in order to clarify the role of the carboxyl group. The synthesized amide derivatives showed HL-60 cell differentiation-inducing activity with potency that depended upon the substituent on the amide nitrogen atom. Among them, the N-cyanoamide 6 is more active than either 1 or 3.

Inter-subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1-ATPase.

V-type ATPases (V-ATPases) are categorized as rotary ATP synthase/ATPase complexes. The V-ATPases are distinct from F-ATPases in terms of their rotation scheme, architecture and subunit composition. However, there is no detailed structural information on V-ATPases despite the abundant biochemical and biophysical research. Here, we report a crystallographic study of V1-ATPase, from Thermus thermophilus, which is a soluble component consisting of A, B, D and F subunits. The structure at 4.5 A resolution reveals inter-subunit interactions and nucleotide binding. In particular, the structure of the central stalk composed of D and F subunits was shown to be characteristic of V1-ATPases. Small conformational changes of respective subunits and significant rearrangement of the quaternary structure observed in the three AB pairs were related to the interaction with the straight central stalk. The rotation mechanism is discussed based on a structural comparison between V1-ATPases and F1-ATPases.

Cutinases from thermophilic bacteria (actinomycetes): From identification to functional and structural characterization.

Thermophilic cutinases are mainly obtained from thermophilic actinomycetes, and are categorized into two groups, i.e., those with higher (>70°C) or lower (<70°C) thermostabilities. The thermostabilities of cutinases are highly relevant to their ability to degrade polyethylene terephthalate (PET). Many crystal structures of thermophilic cutinases have been solved, showing that their overall backbone structures are identical, irrespective of their ability to hydrolyze PET. One of the unique properties of cutinases is that metal ion-binding on the enzyme's surface both elevates their melting temperatures and activates the enzyme. In this chapter, we introduce the methodology for the identification and cloning of thermophilic cutinases from actinomycetes. For detailed characterization of cutinases, we describe the approach to analyze the intricate dynamics of the enzyme, based on its crystal structures complexed with metal ions and model substrates using a combination of experimental and computational techniques.

Coarse snapshots of oxygen-dissociation intermediates of a giant hemoglobin elucidated by determining the oxygen saturation in individual subunits in the crystalline state.

Cooperative oxygen binding of hemoglobin (Hb) has been studied for over half a century as a representative example of the allostericity of proteins. The most important problem remaining to be solved is the lack of structural information on the intermediates between the oxygenated and deoxygenated forms. In order to characterize the intermediate structures, it is necessary to obtain intermediate-state crystals, determine their oxygen saturations and then determine the oxygen saturations of each of their constituent subunits, all of which are challenging issues even now. Here, intermediate forms of the 400 kDa giant Hb from the tubeworm Oligobrachia mashikoi are reported. To overcome the above problems without any artificial modifications to the protein or prosthetic groups, intermediate crystals of the giant Hb were prepared from fully oxygenated crystals by a soaking method. The oxygen saturation of the crystals was measured by in situ observation with a microspectrophotometer using thin plate crystals processed by an ultraviolet laser to avoid saturation of absorption. The oxygen saturation of each subunit was determined by occupancy refinement of the bound oxygen based on ambient temperature factors. The obtained structures reveal the detailed relationship between the structural transition and oxygen dissociation. The dimer subassembly of the giant Hb shows strong correlation with the local structural changes at the heme pockets. Although some local ternary-structural changes occur in the early stages of the structural transition, the associated global ternary-structural and quaternary-structural changes might arise at about 50% oxygen saturation. The models based on coarse snapshots of the allosteric transition support the conventional two-state model of Hbs and provide the missing pieces of the intermediate structures that are required for full understanding of the allosteric nature of Hbs in detail.

Multiple structural states of Ca2+-regulated PET hydrolase, Cut190, and its correlation with activity and stability.

An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190*S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190*, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.