Structural Basis for the Specificity and Catalysis of Human Atg4B Responsible for Mammalian Autophagy
2005; Elsevier BV; Volume: 280; Issue: 48 Linguagem: Inglês
10.1074/jbc.m509158200
ISSN1083-351X
AutoresKenji Sugawara, Nobuo Suzuki, Yūko Fujioka, Noboru Mizushima, Yoshinori Ohsumi, Fuyuhiko Inagaki,
Tópico(s)Cellular transport and secretion
ResumoReversible modification of Atg8 with phosphatidylethanolamine is crucial for autophagy, the bulk degradation system conserved in eukaryotic cells. Atg4 is a novel cysteine protease that processes and deconjugates Atg8. Herein, we report the crystal structure of human Atg4B (HsAtg4B) at 1.9-Å resolution. Despite no obvious sequence homology with known proteases, the structure of HsAtg4B shows a classical papain-like fold. In addition to the papain fold region, HsAtg4B has a small α/β-fold domain. This domain is thought to be the binding site for Atg8 homologs. The active site cleft of HsAtg4B is masked by a loop (residues 259–262), implying a conformational change upon substrate binding. The structure and in vitro mutational analyses provide the basis for the specificity and catalysis of HsAtg4B. This will enable the design of Atg4-specific inhibitors that block autophagy. Reversible modification of Atg8 with phosphatidylethanolamine is crucial for autophagy, the bulk degradation system conserved in eukaryotic cells. Atg4 is a novel cysteine protease that processes and deconjugates Atg8. Herein, we report the crystal structure of human Atg4B (HsAtg4B) at 1.9-Å resolution. Despite no obvious sequence homology with known proteases, the structure of HsAtg4B shows a classical papain-like fold. In addition to the papain fold region, HsAtg4B has a small α/β-fold domain. This domain is thought to be the binding site for Atg8 homologs. The active site cleft of HsAtg4B is masked by a loop (residues 259–262), implying a conformational change upon substrate binding. The structure and in vitro mutational analyses provide the basis for the specificity and catalysis of HsAtg4B. This will enable the design of Atg4-specific inhibitors that block autophagy. Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system (1Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (404) Google Scholar, 2Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar) and plays a critical role in numerous biological processes. Such processes include neurodegeneration, pathogen infection, muscular disorders, cancer, and programmed cell death (3Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2196) Google Scholar). In the process of autophagy, a double-membrane structure called an autophagosome sequesters a portion of the cytoplasm and fuses with the lysosome/vacuole to deliver its contents into the organelle lumen. Genetic approaches in Saccharomyces cerevisiae (4Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar) and subsequent biochemical analyses have identified a novel ubiquitin-like conjugation system called the Atg8 system that is essential for autophagosome formation (5Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 489-493Crossref Scopus (1542) Google Scholar). In the Atg8 system, nascent Atg8 is cleaved at its C-terminal arginine residue by Atg4, a novel cysteine protease (6Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-275Crossref PubMed Scopus (742) Google Scholar), and the exposed C-terminal glycine is conjugated to phosphatidylethanolamine (PE) 2The abbreviations used are: PEphosphatidylethanolamineLC3microtubule-associated protein light chain 3GSTglutathione S-transferaseMADmultiwavelength anomalous diffractionDUBdeubiquitinating enzymeHAUSPherpesvirus-associated ubiquitin-specific proteaseUBPubiquitin-specific processing proteaseUCHubiquitin C-terminal hydrolaseULPubiquitin-like proteaseE1ubiquitin-activating enzymeE2ubiquitin carrier proteinGATE-16Golgi-associated ATPase enhancer 16 kDaGABARAPGABAA receptor-associated proteinPDBProtein Data Bank. by Atg7, an E1-like enzyme, and Atg3, an E2-like enzyme (5Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 489-493Crossref Scopus (1542) Google Scholar). The Atg8-PE is then further deconjugated by Atg4. The reversible modification of Atg8 is crucial for the normal progression of autophagy (6Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-275Crossref PubMed Scopus (742) Google Scholar). Although the conjugation mechanism adopted by the Atg8 system is similar to those of other ubiquitin-like conjugation systems, the Atg8 system has several unique features. The most outstanding feature is that the target for Atg8 is a lipid, not a protein. Therefore, in contrast to other deconjugating enzymes, which function to deconjugate soluble proteins, Atg4 has to deconjugate Atg8-PE, which is localized in the membranes. Another unique feature is that in addition to the ubiquitin-fold region, Atg8 has two α-helices at its N terminus. These helices have been elucidated by structural studies of mammalian homologs (7Sugawara K. Suzuki N.N. Fujioka Y. Mizushima N. Ohsumi Y. Inagaki F. Genes Cells. 2004; 9: 611-618Crossref PubMed Scopus (140) Google Scholar, 8Kouno T. Mizuguchi M. Tanida I Ueno T. Kanematsu T. Mori Y. Shinoda H. Hirata M. Kominami E. Kawano K. J. Biol. Chem. 2005; 280: 24610-24617Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 9Knight D. Harris R. McAlister M.S. Phelan J.P. Geddes S. Moss S.J. Driscoll P.C. Keep N.H. J. Biol. Chem. 2002; 277: 5556-5561Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 10Paz Y. Elazar Z. Fass D. J. Biol. Chem. 2000; 275: 25445-25450Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). This feature of Atg8 may also distinguish Atg4 from other deconjugating enzymes. phosphatidylethanolamine microtubule-associated protein light chain 3 glutathione S-transferase multiwavelength anomalous diffraction deubiquitinating enzyme herpesvirus-associated ubiquitin-specific protease ubiquitin-specific processing protease ubiquitin C-terminal hydrolase ubiquitin-like protease ubiquitin-activating enzyme ubiquitin carrier protein Golgi-associated ATPase enhancer 16 kDa GABAA receptor-associated protein Protein Data Bank. Recent investigations have suggested that the molecular machinery of autophagosome formation is evolutionarily conserved from yeast to higher eukaryotes (4Klionsky D.J. Cregg J.M. Dunn Jr., W.A. Emr S.D. Sakai Y. Sandoval I.V. Sibirny A. Subramani S. Thumm M. Veenhuis M. Ohsumi Y. Dev. Cell. 2003; 5: 539-545Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). In mammals, an Atg8-like conjugation system, called the LC3 (microtubule-associated protein light chain 3) system, has been shown to exist (11Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5510) Google Scholar, 12Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 13Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). LC3, the first identified mammalian homolog of Atg8, was originally found as a light chain of microtubule-associated proteins 1A and 1B in the rat brain (14Mann S.S. Hammarback J.A. J. Biol. Chem. 1994; 269: 11492-11497Abstract Full Text PDF PubMed Google Scholar). Like the Atg8 system in yeast, the C-terminal region of LC3 is cleaved by mammalian Atg4 (mAtg4) homologs (11Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5510) Google Scholar, 15Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1132) Google Scholar, 16Hemelaar J. Lelyveld V.S. Kessler B.M. Ploegh H.L. J. Biol. Chem. 2003; 278: 51841-51850Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). The processed form, called LC3-I, has a glycine residue at its C terminus (11Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5510) Google Scholar) and resides in the cytosol. After activation by mammalian Atg7 (mAtg7) and Atg3 (mAtg3) homologs (12Tanida I. Tanida-Miyake E. Ueno T. Kominami E. J. Biol. Chem. 2001; 276: 1701-1706Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 13Tanida I. Tanida-Miyake E. Komatsu M. Ueno T. Kominami E. J. Biol. Chem. 2002; 277: 13739-13744Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar), LC3-I is further modified to another form, called LC3-II, which is most likely the PE-conjugated form (15Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1132) Google Scholar), as in the Atg8 system. Because LC3-II is localized in the autophagosomal membrane, it is now widely used as a probe to monitor autophagosomes and autophagy activity in mammalian systems (11Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5510) Google Scholar). In addition to LC3, three mammalian Atg8 homologs are identified: Golgi-associated ATPase enhancer of 16 kDa (GATE-16) (18Legesse-Miller A. Sagiv Y. Porat A. Elazar Z. J. Biol. Chem. 1998; 273: 3105-3109Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), GABAA receptor-associated protein (GABARAP) (19Wang H. Bedford F.K. Brandon N.J. Moss S.J. Olsen R.W. Nature. 1999; 397: 69-72Crossref PubMed Scopus (491) Google Scholar), and most recently, Atg8L (16Hemelaar J. Lelyveld V.S. Kessler B.M. Ploegh H.L. J. Biol. Chem. 2003; 278: 51841-51850Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). GATE-16 and GABARAP have also been shown to be the substrates for mAtg7, mAtg3 (20Tanida I. Komatsu M. Ueno T. Kominami E. Biochem. Biophys. Res. Commun. 2003; 300: 637-644Crossref PubMed Scopus (85) Google Scholar), and mAtg4 (15Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1132) Google Scholar, 16Hemelaar J. Lelyveld V.S. Kessler B.M. Ploegh H.L. J. Biol. Chem. 2003; 278: 51841-51850Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 17Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 21Scherz-Shouval R. Sagiv Y. Shorer H. Elazar Z. J. Biol. Chem. 2003; 278: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) in a similar manner to LC3 and can be conjugated to PE. However, among these homologs LC3 is most abundant in autophagosomal membranes, indicating the crucial role of LC3 in mammalian autophagy. Thus far, four human homologs of yeast Atg4 have been reported: HsAtg4A/autophagin-2, HsAtg4B/autophagin-1, autophagin-3, and autophagin-4 (6Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-275Crossref PubMed Scopus (742) Google Scholar, 22Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). HsAtg4A cleaves GATE-16 most efficiently (15Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1132) Google Scholar, 21Scherz-Shouval R. Sagiv Y. Shorer H. Elazar Z. J. Biol. Chem. 2003; 278: 14053-14058Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), whereas HsAtg4B has a broad specificity for mammalian Atg8 homologs (GATE-16, GABARAP, and LC3) (15Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1132) Google Scholar, 17Tanida I. Sou Y.S. Ezaki J. Minematsu-Ikeguchi N. Ueno T. Kominami E. J. Biol. Chem. 2004; 279: 36268-36276Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Among human Atg4 homologs, HsAtg4B is the sole enzyme reported to efficiently cleave LC3 (15Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1132) Google Scholar). Thus HsAtg4B is considered to play an important role in mammalian autophagy by processing and deconjugating LC3. Herein, we report the crystal structure of HsAtg4B at 1.9-Å resolution. The structure and structure-based mutagenesis studies provide the molecular basis for the specificity and catalysis of HsAtg4B. Expression, Purification, and Crystallization—The region coding the full length of HsAtg4B was inserted into the pGEX6p vector (Amersham Biosciences), and HsAtg4B was expressed in Escherichia coli BL21 DE3 cells. The GST-fused protein was first purified using a glutathione-Sepharose 4B column (Amersham Biosciences) followed by excision of GST from the protein with PreScission protease (Amersham Biosciences). Further purification was performed using Resource Q followed by Superdex 75 columns (Amersham Biosciences). The purified protein was concentrated to 7 mg ml-1 in 0.15 m NaCl with 20 mm Tris-HCl at pH 7.4 and 2 mm dithiothreitol. Crystallization trials were performed with the sitting-drop vapor diffusion method at 293 K. HsAtg4B was crystallized with a reservoir solution consisting of 0.2 m NaH2PO4, 0.8 m K2HPO4, 0.1 m NaCl, and 0.1 m appropriate buffer at a broad range of pH 5.6 to 10.0. The crystals belong to the orthorhombic space group C2221, with unit cell parameters of a = 51.9, b = 88.3, c = 160.1 Å. The selenomethionine derivative was expressed in E. coli, B834 (DE3) using an amino acid medium (23LeMaster D.M. Richards F.M. Biochemistry. 1985; 24: 7263-7268Crossref PubMed Scopus (212) Google Scholar) containing selenomethionine instead of methionine and was crystallized under the same conditions as those for native crystals. Data Collection—Crystals were immersed in a reservoir solution supplemented with 16% glycerol for several seconds and then flash-cooled and maintained under nitrogen gas at 90 K during data collection. All diffraction data were collected on the ADSC Quantum 315 charge-coupled device detector using the SPring-8 beamline BL41XU. Multiwavelength anomalous diffraction (MAD) data were collected from a single crystal of selenomethionine-substituted HsAtg4B at three wavelengths. Each data set was independently integrated, scaled, and processed using the HKL2000 program suite (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). Statistics for x-ray diffraction data are summarized in TABLE ONE.TABLE ONEData collection, phasing, and refinement statistics For all data, the x-ray source is SPring-8 beamline BL41XU.Native HsAtg4BSeMet-labeled HsAtg4BData collection statistics Wavelength (Å)1.00000.97920.97950.9700 Temperature (K)90909090 Resolution range (Å)50–1.9050–2.3150–2.3150–2.31 Outer shell (Å)1.97–1.902.39–2.312.39–2.312.39–2.31 Observed reflections124,64959,25458,89060,470 Unique reflections26,76925,42425,32525,990 Completeness (%)90.8 (60.3)82.9 (32.7)81.8 (28.7)81.9 (30.3) Rmerge(I)aRmerge (I) = (ΣΣ|Ii – 〈I〉 |)/ΣΣIi, where Ii is the intensity of the ith observation and 〈I〉 is the mean intensity. Values in parentheses refer to the outer shell0.054 (0.248)0.078 (0.345)0.076 (0.310)0.061 (0.319) I/σ(I)21.6 (5.73)9.5 (1.59)9.6 (1.66)9.5 (1.43)Phasing statistics Resolution range (Å)50–3.00 No. of sites9 Mean figure of merit0.61Refinement statistics Resolution range (Å)30–1.90 No. of protein atoms2,616 No. of water molecules202 R/Rfree0.215/0.247r.m.s.d.br.m.s.d., root mean square deviation from ideality bond length (Å)0.006 angles (°)1.25a Rmerge (I) = (ΣΣ|Ii – 〈I〉 |)/ΣΣIi, where Ii is the intensity of the ith observation and 〈I〉 is the mean intensity. Values in parentheses refer to the outer shellb r.m.s.d., root mean square deviation Open table in a new tab Structure Determination and Refinement—The structure of HsAtg4B was determined by the MAD phasing method. All selenium sites with the exception of the N-terminal Met were found and refined, and the initial phases were calculated using the programs SOLVE and RESOLVE (25Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). About 60% of the residues were automatically modeled as a polyalanine chain by RESOLVE. Further model construction was performed manually using the molecular modeling program O (26Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar), and the refinement was performed using the crystallography and NMR system program (27Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar). The refinement statistics are summarized in TABLE ONE. The coordinates and structure factors of HsAtg4B have been deposited in the Protein Data Bank (code 2CY7). In Vitro Cleavage Assay—For the in vitro cleavage assay, two kinds of rat LC3 (homolog of human LC3B) were constructed with different tags fused to the C-terminal Gly-120. One consisted of a 15-amino acid sequence, GSPEFPGRLHHHHHH, which originated from the pGEX vector and a hexahistidine tag (this construct is called LC3-His); the other was the GST tag (this construct is called LC3-GST). Single mutant LC3s (Q116A and F119A) were prepared by PCR-mediated site-directed mutagenesis and sequenced to confirm their identities. LC3-His and LC3-(1–115) were inserted into the pGEX6p vector, whereas LC3-GST was inserted into the pET11a vector (Novagen). They were expressed in E. coli and purified on a glutathione-Sepharose 4B column. Each construct was concentrated to 1 mg/ml (LC3-His and LC3-GST) or 3 mg/ml (LC3-(1–115)) in the reaction buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1 mm dithiothreitol). The 9-mer peptide QETFGTALA, which corresponds to amino acids 116–124 of unprocessed LC3, was purchased from Sigma Genosys. Constructs of various mutant HsAtg4Bs were also prepared by PCR-mediated site-directed mutagenesis and sequenced to confirm their identities. The mutant HsAtg4Bs were expressed and purified according to the same procedure described for wild-type HsAtg4B above. LC3-His, LC3-GST, and mutant LC3s (1 mg/ml) fused to GST were mixed with 0.3, 0.03, or 0.003 mg/ml HsAtg4B at a volume ratio of 1:1 and incubated at 293 K for 10 min. The reaction was stopped by the addition of SDS sample buffer and analyzed by SDS-PAGE using Coomassie Brilliant Blue staining. The 9-mer peptide (1 mg/ml) was mixed with 3 mg/ml HsAtg4B or its deletion mutant Δ(259–262) with or without 3 mg/ml LC3 (1–115) and incubated at 293 K for 6 h. The reaction solutions were applied to a Resource RPC column (Amersham Biosciences) equilibrated with 0.1% trifluoroacetic acid solution and eluted using a gradient of 0–80% CH3CN with 0.1% trifluoroacetic acid solution. For mutational analyses using mutant HsAtg4Bs, wild-type and mutant HsAtg4Bs (0.1 mg/ml) in reaction buffer were mixed with LC3-GST (1 mg/ml) at a volume ratio of 1:1. After 10 min at 293 K, the reaction was stopped by the addition of SDS sample buffer and analyzed by SDS-PAGE using Coomassie Brilliant Blue staining. Overall Structure of HsAtg4B—The crystal structure of HsAtg4B was refined against 1.9-Å data to an R-factor of 0.215 and a free R-factor of 0.247. The region corresponding to amino acids 5–377 was modeled along with 202 water molecules. All non-glycine residues are within the most favored or additional allowed regions of the Ramachandran plot (28Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The four N-terminal residues, 16 C-terminal residues, and four loop regions corresponding to residues 190–216, 288–290, 343–346, and 356–361 lacked defined electron density and were omitted from the model (Fig. 1). The overall structure of HsAtg4B consists of an α/β-fold with nine α-helices and 13 β-strands. It is organized around a central antiparallel β-sheet composed of strands β6, β7, β8, β9, β11, and β12. In addition to the six strands, strand β4 forms a parallel sheet with β6, and strands β1 and β2 form an antiparallel β-sheet with β8. Four α-helices (F–I) and two β-strands (β5 and β13) are located on one side of the central β-sheet, whereas five α-helices (A–E) and two β-strands (β3 and β10) are located on the other side. HsAtg4B Has a Papain-like Fold—Comparison of the HsAtg4B structure with the Protein Data Bank data base using the DALI search engine (29Holm L. Sander C. Science. 1996; 273: 595-602Crossref PubMed Scopus (1289) Google Scholar) revealed that HsAtg4B has high structural similarity to papain family cysteine proteases as well as deubiquitinating enzymes (DUBs) (Fig. 2A). Fig. 2B shows the topology of the proteins shown in Fig. 2A. α-Helices and β-strands of HsAtg4B observed in common with those of papain are colored red and cyan, respectively. These conserved secondary elements are designated after the nomenclature used for those of HsAtg4B. Among these proteins, HsAtg4B shows the highest structural similarity to papain and its homologous proteases. The root mean square deviation between HsAtg4B and papain (Protein Data Bank code 1PE6) is 3.4 Å for 156 Cα atoms. Both HsAtg4B and papain have six antiparallel central strands (β6, β12, β7, β8, β9, and β11 of HsAtg4B) and four α-helices (B, C, E, G of HsAtg4B) in the same order. Among the deconjugating enzymes, HsAtg4B shows the most structural similarity to herpesvirus-associated ubiquitin-specific protease (HAUSP) (Protein Data Bank code 1NBF), a UBP (ubiquitin-specific processing protease) family protease with a root mean square deviation of 4.0 Å for 148 Cα atoms. HAUSP also has six antiparallel central strands and α-helices B and E in the same order. However, the other helices are not superimposed. HsAtg4B lacks a detectable sequence homology with either papain or HAUSP (10 and 7% identity for 156 and 148 superimposed residues, respectively). Nevertheless, the topologies are very similar. The topology of other deconjugating enzymes differs more significantly. For ubiquitin C-terminal hydrolases (UCHs), helix B (containing the catalytic cysteine) is inserted between β7 and β8 (30Johnston S.C. Larsen C.N. Cook W.J. Wilkinson K.D. Hill C.P. EMBO J. 1997; 16: 3787-3796Crossref PubMed Scopus (214) Google Scholar) (Fig. 2B). For ubiquitin-like proteases (ULPs), the topology, including the central β-sheet, differs significantly (31Mossessova E. Lima C.D. Mol. Cell. 2000; 5: 865-876Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar) (Fig. 2B). Deconjugating enzymes have functional differences. UBP family proteases deconjugate ubiquitin from proteins, whereas UCH family proteases deconjugate ubiquitin from small adducts, and ULP family proteases deconjugate ubiquitin-like proteins, such as SUMO or NEDD8, from proteins (32Amerik A.Y. Hochstrasser M. Biochim. Biophys. Acta. 2004; 1695: 189-207Crossref PubMed Scopus (757) Google Scholar). It should be noted that the Atg4 family proteases deconjugate Atg8 family proteins from a small adduct, PE. Based on these facts and no sequence homology with other deconjugating enzymes, Atg4 family proteases have been considered the most distinct member among deconjugating enzymes. However, the structure of HsAtg4B is significantly similar to that of the UBP family protease, and surprisingly, the structural similarity between HsAtg4B and the UBP family protease is higher than that between the two DUB families, UBPs and UCHs, or UBPs and ULPs. A Small Domain Unique to HsAtg4B—Compared with papain, HsAtg4B has an inserted region composed of the α-helices F, H, and I and the β-strands 5 and 13 (enclosed by broken lines in Fig. 2A). HAUSP also has an inserted region, positionally similar to that of HsAtg4B, although its shape and size are different (Fig. 2). The inserted region of HAUSP, designated as the fingers domain (33Hu M. Li P. Li M. Li W. Yao T. Wu J.W. Gu W. Cohen R.E. Shi Y. Cell. 2002; 11: 1041-1054Abstract Full Text Full Text PDF Scopus (493) Google Scholar), consists of a large β-sheet and protrudes from the papain fold region as far as 30 Å. The fingers domain is unique to UBP family proteases and is responsible for the recognition of ubiquitin (33Hu M. Li P. Li M. Li W. Yao T. Wu J.W. Gu W. Cohen R.E. Shi Y. Cell. 2002; 11: 1041-1054Abstract Full Text Full Text PDF Scopus (493) Google Scholar). Compared with the fingers domain of HAUSP, the inserted region of HsAtg4B is appreciably shorter; therefore, it is called the short fingers domain. UCHs and ULPs do not have an inserted domain (Fig. 2). In addition to the domain insertion, HsAtg4B and HAUSP have an insertion of strand β4 in common, near the inserted domain that is parallel to β6 of the central sheet (Fig. 2B). Catalytic Residues—Fig. 3A shows the active site structure of HsAtg4B. The Nϵ2 atom of His-280 is 3.4 Å from the Sγ atom of Cys-74, and the Nδ1 atom of His-280 is 2.6 Å from the Oδ1 atom of Asp-278. This is the geometry observed in the canonical catalytic triad of cysteine proteases. These three residues are strictly conserved among Atg4 homologs (22Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar), suggesting that these are the catalytic triad of HsAtg4B. Fig. 3B shows the superimposition of the catalytic residues of HAUSP (PDB code 1NBF), papain (PDB code 1PE6), UCH-L3 (PDB code 1UCH), and Ulp1 (PDB code 1EUV) on those of HsAtg4B. Here, the structure of the HAUSP-ubiquitin aldehyde complex was used for superimposition because the catalytic triad of HAUSP is properly aligned only when it forms a complex with its substrate (33Hu M. Li P. Li M. Li W. Yao T. Wu J.W. Gu W. Cohen R.E. Shi Y. Cell. 2002; 11: 1041-1054Abstract Full Text Full Text PDF Scopus (493) Google Scholar). Cys-74 and Asp-278 of HsAtg4B are superimposed on the nucleophilic cysteine and the Asp/Asn residue comprising the catalytic triad of cysteine proteases, respectively (Fig. 3B). Cys-74 is located at the N terminus of helix B, and Asp-278 is located at the C terminus of β9. Both of these positions are conserved among papain and DUBs (Fig. 2B). In contrast, although the side chain of His-280 of HsAtg4B can be superimposed on that of the general base histidine of papain and DUBs, its main-chain location is different. His-280 of HsAtg4B is located at a loop between β9 and β11, whereas the general base histidine of papain and DUBs is located at the N terminus of the strand equivalent to β8 of HsAtg4B (Figs. 2B and 3B). Although ULP family proteases have a different topology, their catalytic triad residues are located in the same geometry as those of both papain and DUBs (Figs. 2B and 3B). Thus the different location of the histidine residue is a unique feature of Atg4 family proteases. In addition to the catalytic triad, cysteine proteases have a conserved Asn/Gln residue that participates in the formation of the oxianion hole crucial for catalysis: Gln-19 for papain, Asn-218 for HAUSP, Gln-89 for UCH-L3, and Gln-574 for Ulp1. Atg4 family proteases also have a conserved Gln residue near the nucleophilic cysteine sequence (Gln-80 for HsAtg4B) that was suggested to be a component of the oxianion hole (22Marino G. Uria J.A. Puente X.S. Quesada V. Bordallo J. Lopez-Otin C. J. Biol. Chem. 2003; 278: 3671-3678Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). However, a conserved Tyr residue (Tyr-54), not Gln-80, is superimposed on these Asn/Gln residues (Figs. 2B and 3B), and Gln-80 is located distally. This is another unique feature of Atg4 family proteases. Conformational Changes Prerequisite for Catalysis—Fig. 3C shows the surface representation of HsAtg4B. Residues conserved among four human Atg4 homologs and yeast Atg4 are colored green, whereas catalytic triad residues are colored red. The green region is localized near the catalytic triad (Fig. 3C), suggesting the importance of the region for substrate recognition. There is a concave surface at the left side of the green region. This surface consists of the short fingers domain, strand β4, and helix E and its N-terminal loop (Fig. 3D). HAUSP also has a similar concave surface consisting of the fingers domain, strand β4, helix E, and other regions (Fig. 2), where the core region of ubiquitin aldehyde binds (33Hu M. Li P. Li M. Li W. Yao T. Wu J.W. Gu W. Cohen R.E. Shi Y. Cell. 2002; 11: 1041-1054Abstract Full Text Full Text PDF Scopus (493) Google Scholar). Therefore, the concave surface of HsAtg4B is the putative binding site for the LC3 core. In contrast,
Referência(s)