Interaction mode between catalytic and regulatory subunits in glucosidase II involved in ER glycoprotein quality control

Abstract

The glycoside hydrolase family 31 (GH31) α‐glucosidases play vital roles in catabolic and regulated degradation, including the α‐subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N‐glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β‐subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα‐binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme.

Short abstract

Interactive Figure 1A; Interactive Figure 1B; Interactive Figure 2 | PDB Code(s): 5JQP

Introduction

The α‐glucosidases are responsible for catabolic and regulated degradation of carbohydrate chains. These enzymes are primarily identified as members of glycoside hydrolase (GH) families 13 and 31.1 The GH13 family contains a variety of glucoside‐processing enzymes such as α‐amylase, and their enzymatic properties have been extensively investigated.2 In contrast, the glycoside hydrolase family 31 (GH31) family includes lysosomal α‐glucosidase, a deficiency of which results in Pompe's disease,3 and other digestive enzymes typified by sucrase–isomaltase and maltase–glucoamylase, the targets of inhibition by anti‐diabetic drugs such as acarbose.4, 5

Endoplasmic reticulum (ER) glucosidase II (GII) contains a catalytic subunit belonging to the GH31 family, which is used for trimming of the terminal glucose residues of N‐glycan, and plays a regulatory role in glycoprotein processing coupled with quality control.6, 7, 8, 9 Among the known GH31 enzymes, only the catalytic subunit of GII (termed α‐subunit; GIIα) works with its specific binding partner (termed regulatory β‐subunit; GIIβ), giving rise to a heterodimeric enzyme structure.10, 11, 12 Regulatory β‐subunit of glucosidase II (GIIβ) contains an N‐terminal GIIα‐binding (G2B) domain (GIIβ^G 2 ^B),13, 14 a putative coiled‐coil segment,13 and a C‐terminal mannose 6‐phosphate receptor homology (MRH) domain15 followed by an ER retention signal [Fig. 1(A)]. The MRH domain acts as a lectin that recognizes substrate high‐mannose‐type glycans, thereby contributing to the efficient enzymatic activity of GIIα.16, 17, 18, 19 Mutations in GANAB and PRKCSH encoding GIIα and GIIβ cause autosomal‐dominant polycystic kidney and liver diseases (ADPKD and ADPLD).20, 21, 22 To date, the 3D structures of GIIα23 and the MRH domain of GIIβ have been determined.24, 25 However, structural information of GIIβ^G 2 ^B as well as its mode of interaction with GIIα has not been elucidated. Therefore, the structural basis for recruitment of the regulatory subunit by the catalytic subunit in GII remains to be addressed. In view of this situation, we performed biophysical experiments including X‐ray crystallographic analysis to characterize the GIIα–GIIβ interaction in this unique GH31 enzyme.

Figure 1.

Overall structure of the GIIα complexed with GIIβ^G 2 ^B. (A) Domain structure of C. thermophilum GIIβ. The G2B domain (residues 21–162) was crystallized in this study. The “SS” and “CC” represent putative signal sequence and coiled‐coil domains, respectively. An interactive view is available in the electronic version of the article. (B) Ribbon model of GIIα (bluish colors) and GIIβ^G 2 ^B (yellow) is represented with positions of the N and C termini. The individual domains of GIIα are colored as follows: N‐terminal domain (slate), unique subdomain B1 (blue), (β/α)₈ barrel domain (marine‐blue), subdomain B2 (purple‐blue), subdomain B3 (magenta), proximal C‐terminal domain (pale‐cyan), and distal C‐terminal domain (cyan). Disulfide bonds of GIIβ^G 2 ^B and active site residues of GIIα are shown as sticks and spheres, respectively. The bound Ca²⁺ ions are shown by pink‐colored spheres. (C) Three‐dimensional structure of GIIβ^G 2 ^B. Residues coordinating Ca²⁺ ions and bonds are represented by sticks and solid lines, respectively. Disulfide bonds of GIIβ^G 2 ^B are shown as sticks together with residue numbers. The insertion loop is colored in orange. An interactive view is available in the electronic version of the article.

Results and Discussion

Overall structure of the glucosidase II heterodimer complex

We selected the thermophilic fungus Chaetomium thermophilum, which can survive at temperatures of up to 60°C,26 as the organism of choice for the structural study. Using this microorganism, we have successfully determined crystal structures of enzymes involved in ER quality control, including GIIα.23, 27 It has been confirmed that closely related species, such as Aspergillus oryzae and Schizosaccharomyces pombe, have enzymatically active GII with the same substrate specificity as that of the mammalian counterparts.17, 18, 24, 25 These results encouraged us to perform a structural study of GIIβ derived from C. thermophilum. We expressed a recombinant GIIβ^G 2 ^B (residues 21–162) [Fig. 1(A)] as an inclusion body in Escherichia coli, which was subjected to oxidative refolding. The crystal structure of the complex formed between GIIα and GIIβ^G 2 ^B was solved by molecular replacement and refined to 2.20‐Å resolution. The final model of the GIIα/GIIβ^G 2 ^B complex has an R _work of 15.3% and R _free of 20.6% (Table 1), visualizing residues 34–213, 235–801, 804–898, and 902–977 of GIIα and 24–154 of GIIβ^G 2 ^B. In the crystal structure, several loop residues and a few N‐ and C‐terminal residues gave no interpretable electron density, possibly due to their flexible nature. The crystal belongs to space group P2₁22₁ with one 1:1 complex molecule per asymmetric unit, which is consistent with an analytical ultracentrifugation result indicating that majority of GIIα/GIIβ^G 2 ^B creates a 1:1 complex in solution (Supporting Information, Fig. S1).

Table 1.

Data collection and refinement statistics for GIIα/GIIβ^G 2 ^B complex

Data collection
Space group	P21221
Unit cell parameters
a, b, c (Å)	82.7, 88.7, 173.1
Beamline	PF BL1A
Wavelength (Å)	1.10000
Resolution range (Å)	50.0–2.20 (2.24–2.20)
Number of total reflections	283,730
Number of unique reflections	64,671
Completeness (%)	99.4 (100.0)
R merge (%)	10.8 (49.9)
I/σ(I)	11.8 (2.2)
Redundancy	4.4 (4.6)
Refinement
Resolution range (Å)	20.0–2.20
R work/ R free (%)	15.3/20.6
R.m.s.d. bond length (Å) and angles (°)	0.015/1.60
Ramachandran plot (%)
Most favored regions	86.4
Additional allowed regions	13.0
Generously allowed	0.6
Number of molecules
Protein atoms (GIIα/GIIβG 2 B)	7419/960
Water molecules	655
Ca2+ ions	2
Tris molecules	1
Average B‐values (Å2)
Protein atoms	25.0/26.2
Water molecules	28.4
Ca2+ ions	20.6
Tris molecules	24.7

As expected, the overall structure of GIIα consisted of four major domains and three subdomains as observed in its uncomplexed form [Fig. 1(B)].23 Among GH31 enzymes,28, 29, 30, 31, 32, 33, 34, 35 this enzyme subunit possesses a unique B1 subdomain in the N‐terminal domain [Fig. 1(B)]. Cellvibrio japonicus α‐xylosidase possesses PA14 domain at the corresponding position (Supporting Information, Fig. S2),34 which is proposed to play a key role as catalytic assistant accommodating large xyloglucan substrates into the active site. Based on the observations, we originally hypothesized that this B1 subdomain is responsible for interaction with the β‐subunit.23 However, unexpectedly, GIIβ^G 2 ^B binds an opposite surface of GIIα mainly through its distal C‐terminal domain [Fig. 1(B)]. GIIβ^G 2 ^B binding caused little or no conformational change in GIIα including the active site, except for the distal C‐terminal domain [Supporting Information, Fig. S3]. This observation is consistent with previous reports that the enzymatic activities of the α‐subunit of GII probed with a p‐nitrophenyl glucose did not depend on GIIβ, whereas hydrolysis of bulky glucosylated high‐mannose‐type oligosaccharides depended on GIIβ through MRH domain.17, 18, 36

Structure of the G2B domain of glucosidase II β‐subunit

Our crystallographic data demonstrated that the 130‐residue segment (24–154) of GIIβ^G 2 ^B is composed of two Ca²⁺‐binding subdomains with five disulfide bonds [Fig. 1(C)]. In several fungal species such as C. thermophilum and Saccharomyces cerevisiae, a 15–25‐residue loop is inserted between the two subdomains (Supporting Information, Fig. S4). The N‐terminal subdomain contains one β‐sheet comprising β1 and β2 strands and two 3₁₀ helices (termed H1 and H2), whereas the C‐terminal subdomain contains one β‐sheet comprising β3 and β4 strands and two 3₁₀ helices (termed H and H5). All 10 cysteine residues in the G2B domain formed disulfide bonds, that is, Cys44–Cys64, Cys62–Cys76, Cys107–Cys132, Cys127–Cys147, and Cys133–Cys151 [Fig. 1(C)]. Although the sequence homology of GIIβ^G 2 ^B between thermophilic fungi and humans was modestly low (26–34% identities) (Supporting Information, Fig. S4), the cysteine and the Ca²⁺‐coordinating residues (vide infra) are highly conserved across species, suggesting their common fold of the G2B domain.

In the first Ca²⁺‐binding site located at the N‐terminal subdomain, the side‐chain oxygen atoms of Asp59, Asp63, Asp69, and Glu70 and the main‐chain carbonyl atoms of Gln56 and Ser61 coordinate with Ca²⁺ ion at distances of 2.2–2.5 Å [Fig. 1(C)]. As for the second Ca²⁺‐binding site located at the C‐terminal subdomain, the side‐chain oxygen atoms of Asp124, Asp128, Asp137, and Glu138 and main‐chain carbonyl atoms of Tyr121 and Val126 coordinate with Ca²⁺ ion at distances of 2.3–2.4 Å [Fig. 1(C)]. Previous mutational data on the G2B domain of GII β‐subunit from budding and fission yeast suggested that Glu70 (Glu73 in S. pombe), Asp128 (Asp125 in S. cerevisiae), and Glu138 (Glu114 in S. pombe and Glu132 in S. cerevisiae) of GIIβ^G 2 ^B are involved in GIIα interaction and the glucose trimming reaction.13, 36 Based on our structural data, we speculated that these mutations impaired Ca²⁺‐binding of this domain and consequently, its structural integrity required for the interaction with GIIα.

Interface between catalytic α‐subunit and its binding G2B domain of the β‐subunit

Our structural data also revealed that GIIβ^G 2 ^B binds to the distal C‐terminal domain of GIIα through potential 16 hydrogen bonds and nine salt bridges, burying a total surface area of 1,212 Ų which were calculated by PISA program37 (Fig. 2). In the complex with GIIα, the C‐terminal subdomain of GIIβ^G 2 ^B is mainly involved in the interaction with GIIα. The electrostatic interface is composed of Asp59, Asp80, Asp124, and Asp128 of GIIβ^G 2 ^B and Arg717, Arg834, Arg835, and Lys963 of GIIα. These interacting residues of GIIα and GIIβ^G 2 ^B are essentially identical across species (Supporting Information, Figs. S4 and S5), indicating that interaction mode between catalytic and regulatory subunits in GII is evolutionally conserved. Among these interacting residues of GIIα, contiguous double Arg834/835 residues are primarily involved in GIIβ^G 2 ^B interaction [Fig. 2(C)]. Our mutational analysis using isothermal titration calorimetry (ITC) assay indicated that alanine substitutions at Arg834 and Arg835 of GIIα impaired interaction with GIIβ^G 2 ^B (Supporting Information, Fig. S6), suggesting that the electrostatic interactions observed in the crystal structure significantly contribute to the stabilization of the GIIβ^G 2 ^B/GIIα complex. From genetic studies, three missense mutations of GANAB have been shown to be cause of ADPKD and ADPLD.22 These mutations include amino acid substitutions in the (β/α)₈ barrel domain (T405R and R422L) and distal C‐terminal domain (R839W). Thr405, Arg422, and Arg839 in human GIIα are highly conserved across species (Thr397, Arg414, and Arg834 in C. thermophilum) (Supporting Information, Fig. S5). Based on our structural data, we speculated that the T405R or R422L mutation perturbs active‐site's structure and thereby affects the enzymatic activity of GIIα. By contrast, The R839W mutation in human GIIα subunit appears to impair its interaction with GIIβ as observed in the R834A/R835A mutation in this study (Supporting Information, Fig. S6), consequently affecting enzymatic efficiency and subcellular localization of GIIα because only GIIβ carries the substrate‐recognizing lectin domain and the ER retention signal.

Figure 2.

Close‐up view of binding mode between GIIα and GIIβ^G 2 ^B. The GIIα/GIIβ^G 2 ^B interface is shown indicating amino acid residues of GIIα (black) and GIIβ^G 2 ^B (red) at the N‐ and C‐terminal subdomains of GIIβ^G 2 ^B in (A) and (B), respectively. Residues mediating the intersubunit interactions are shown as sticks. Ca²⁺‐coordinating bonds are solid lines, and hydrogen bonds and/or ionic interactions are represented by dotted lines. (C) Schematic representation of the binding interface between GIIα and GIIβ^G 2 ^B. Black and grey lines indicate hydrogen bonds and/or ionic interactions and hydrophobic interactions. An interactive view is available in the electronic version of the article

Among the known GH31 enzymes, only GIIα forms a heterodimeric structure through its distal C‐terminal domain [Fig. 1(B)]. Comparison of the amino acid residues of GIIα among GH31 enzymes revealed that, in addition to active site residues located at the center of the (β/α)₈ barrel domain, the GIIβ^G 2 ^B‐binding residues including the contiguous double Arg834/835 residues are highly conserved in GIIs but not in the other GH31 enzymes (Supporting Information, Fig. S7). These structural differences account for the specific recruitment of GIIβ^G 2 ^B by GIIα among GH31 family enzymes.

Previous ultracentrifugation data revealed that a rat liver GII holoenzyme showed a highly extended shape and is susceptible to proteolysis12 presumably at the non‐globular segment connecting the G2B and MRH domains [Fig. 1(A)]. These data implies a flexible nature of β‐subunit which enables this enzyme to cleave the glucosyl linkages at various distances from a tethering point in a multiply glycosylated protein (Supporting Information, Fig. S8) as suggested in previous in vitro and in vivo studies.25, 38, 39 To obtain further insight into the working mechanism of GII, structural information of full‐length GII interacting with substrates would be necessary.

Conclusion

In the present study, we determined the first crystal structure of the heterodimeric complex of a GH31 enzyme. This crystal structure revealed how the catalytic α‐subunit of ER GII recruits the regulatory β‐subunit, which harbors the MRH domain for cooperative substrate recognition. Our findings provide insight into the structural basis underlying the functional extension of this unique GH31 enzyme.

Materials and Methods

Protein expression and purification

Protein expression and purification of GIIα (residues 31–977) from C. thermophilum in E. coli were performed as previously described.23 Recombinant GIIβ^G 2 ^B (residues 21–162) was expressed as an inclusion body in E. coli and subjected to oxidative refolding. Further details are in the Supporting Information.

Crystallization, X‐ray data collection, and structure determination

The GIIα/GIIβ^G 2 ^B protein was concentrated to 8.0 mg/mL and used for crystallization. The crystallization screening was performed by hanging‐drop vapor diffusion and microseeding methods. The seed crystals were grown in a buffer containing 20% PEG3350, 100 mM Tris‐HCl (pH 7.5), and 0.2 M ammonium phosphate dibasic at 20°C for a week. Using the seed crystals, we obtained the final diffraction‐quality crystals in the above crystallization buffer in which the Tris‐HCl buffer was replaced with 100 mM Bis‐Tris‐HCl (pH 6.5). For X‐ray diffraction data collection, the crystals were cryoprotected with a crystallization buffer supplemented with 15% glycerol. The crystal of GIIα/GIIβ^G 2 ^B complex belonged to space group P2₁22₁ with one molecule per asymmetric unit and diffracted up to a resolution of 2.20 Å. Diffraction data were integrated and scaled using HKL2000.40 The crystallographic parameters are shown in Table 1.

The 2.20‐Å crystal structure of GIIα/GIIβ^G 2 ^B was solved by molecular replacement using the program MOLREP41 with GIIα (Protein Data Bank code: 5DKX) as a search model. Model fitting to the electron density maps and refinement procedure were performed with COOT42 and REFMAC5,43 respectively. The stereochemical quality of the final model was validated with PROCHECK.44 The refinement statistics are summarized in Table 1. The molecular graphics were prepared using PyMOL (http://www.pymol.org/).

Accession number

The coordinate and structural factor of the crystal structure of GIIα/GIIβ^G 2 ^Bcomplex has been deposited in the PDB under accession number 5JQP.

Additional information

During peer review processes of our manuscript, crystal structure of the trypsinolyzed murine GII comprising GIIα and the G2B domain of β‐subunit has been reported.45 The murine GII structure was essentially identical with our fugal GII structure, indicating the evolutional conservation of the architecture of GII. In this report, a small‐angle X‐ray scattering structure of full‐length GII in the absence of substrate ligand has also been reported, illustrating its highly extended structure that supports the hypothetical model described in this article.

Conflict of Interest

The authors declare that they have no competing financial interests.

Supporting information

Supporting Information