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Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease

2021; Elsevier BV; Volume: 297; Issue: 4 Linguagem: Inglês

10.1016/j.jbc.2021.101239

ISSN

1083-351X

Autores

Shanshan Li, Kan‐Yen Hsieh, Shih-Chieh Su, Grigore Pintilie, Kaiming Zhang, Chung‐I Chang,

Tópico(s)

Enzyme Structure and Function

Resumo

The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding. The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 Å resolution. Our data indicate that substrate interactions are mediated by the dual pore loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding. The Lon AAA+ protease (LonA) is an ATP-dependent protease conserved in prokaryotes and eukaryotic organelles. LonA assembles as a homohexamer with each protomer containing an N-terminal domain, a middle ATPase domain, and a C-terminal protease domain (1Rotanova T.V. Botos I. Melnikov E.E. Rasulova F. Gustchina A. Maurizi M.R. Wlodawer A. Slicing a protease: Structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains.Protein Sci. 2006; 15: 1815-1828Crossref PubMed Scopus (66) Google Scholar). In addition to LonA, other Lon-like proteases (LonB and LonC) with distinct ATPase domains have been characterized in thermophilic archaeal and bacterial species (2Liao J.-H. Kuo C.-I. Huang Y.-Y. Lin Y.-C. Lin Y.-C. Yang C.-Y. Wu W.-L. Chang W.-H. Liaw Y.-C. Lin L.-H. Chang C.-I. Wu S.-H. A Lon-like protease with no ATP-powered unfolding activity.PLoS One. 2012; 7e40226Crossref PubMed Scopus (10) Google Scholar, 3Rotanova T.V. Melnikov E.E. Khalatova A.G. Makhovskaya O.V. Botos I. Wlodawer A. Gustchina A. Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains.Eur. J. Biochem. 2004; 271: 4865-4871Crossref PubMed Scopus (76) Google Scholar). LonA plays a major role in cellular protein homeostasis by degrading damaged or misfolded abnormal proteins, which prevents these unwanted protein species from forming toxic aggregates. LonA is also involved in the regulation of many biological processes by degrading specific regulatory proteins (4Gur E. The Lon AAA+ protease.Subcell. Biochem. 2013; 66: 35-51Crossref PubMed Scopus (45) Google Scholar). Sharing the fused AAA+ and protease domain organization of Lon, the membrane-anchored AAA+ proteases FtsH and related mitochondrial intermembrane-space (i) and matrix (m) AAA (i/m-AAA) proteases are involved in quality control of membrane proteins; however, the ATPase domains belong to different clades of the superfamily and the protease domains are nonhomologous (5Puchades C. Sandate C.R. Lander G.C. The molecular principles governing the activity and functional diversity of AAA+ proteins.Nat. Rev. Mol. Cell Biol. 2020; 21: 43-58Crossref PubMed Scopus (72) Google Scholar, 6Sauer R.T. Baker T.A. AAA+ proteases: ATP-fueled machines of protein destruction.Annu. Rev. Biochem. 2011; 80: 587-612Crossref PubMed Scopus (502) Google Scholar). Previous results have revealed many functional and structural features of LonA. The protease activity of LonA is dependent on the presence of Mg2+, which binds to the protease domain and induces the formation of open active-site structure (7Su S.-C. Lin C.-C. Tai H.-C. Chang M.-Y. Ho M.-R. Babu C.S. Liao J.-H. Wu S.-H. Chang Y.-C. Lim C. Chang C.-I. Structural basis for the magnesium-dependent activation and hexamerization of the Lon AAA+ protease.Structure. 2016; 24: 676-686Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The Lon protease domain with an open active-site conformation adopts a sixfold symmetric hexameric ring (7Su S.-C. Lin C.-C. Tai H.-C. Chang M.-Y. Ho M.-R. Babu C.S. Liao J.-H. Wu S.-H. Chang Y.-C. Lim C. Chang C.-I. Structural basis for the magnesium-dependent activation and hexamerization of the Lon AAA+ protease.Structure. 2016; 24: 676-686Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8Botos I. Melnikov E.E. Cherry S. Tropea J.E. Khalatova A.G. Rasulova F. Dauter Z. Maurizi M.R. Rotanova T.V. Wlodawer A. Gustchina A. The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site.J. Biol. Chem. 2004; 279: 8140-8148Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 9Cha S.-S. An Y.J. Lee C.R. Lee H.S. Kim Y.-G. Kim S.J. Kwon K.K. De Donatis G.M. Lee J.-H. Maurizi M.R. Kang S.G. Crystal structure of Lon protease: Molecular architecture of gated entry to a sequestered degradation chamber.EMBO J. 2010; 29: 3520-3530Crossref PubMed Scopus (72) Google Scholar, 10Liao J.-H. Ihara K. Kuo C.-I. Huang K.-F. Wakatsuki S. Wu S.-H. Chang C.-I. Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1395-1402Crossref PubMed Scopus (18) Google Scholar). By contrast, LonA with an inactive proteolytic active-site conformation exhibits an open-spiral hexameric structure (11Botos I. Lountos G.T. Wu W. Cherry S. Ghirlando R. Kudzhaev A.M. Rotanova T.V. de Val N. Tropea J.E. Gustchina A. Wlodawer A. Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery.Curr. Res. Struct. Biol. 2019; 1: 13-20Crossref PubMed Scopus (8) Google Scholar, 12Duman R.E. Löwe J. Crystal structures of Bacillus subtilis Lon protease.J. Mol. Biol. 2010; 401: 653-670Crossref PubMed Scopus (54) Google Scholar). Therefore, the protease domain plays an important role in the closed-ring assembly of LonA. While ATP is required for LonA to degrade protein substrates efficiently, ADP is known to inhibit the protease activity of Lon. The crystal structure of the ADP-bound LonA hexamer has revealed how ADP may inhibit LonA activity by inducing the formation of a closed degradation chamber (13Lin C.-C. Su S.-C. Su M.-Y. Liang P.-H. Feng C.-C. Wu S.-H. Chang C.-I. Structural insights into the allosteric operation of the Lon AAA+ protease.Structure. 2016; 24: 667-675Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A substrate-translocation model was posited based on the ADP-bound structure by assuming a structural transition between the nucleotide-free and ADP-bound states from two pairs of three nonneighboring protomers (13Lin C.-C. Su S.-C. Su M.-Y. Liang P.-H. Feng C.-C. Wu S.-H. Chang C.-I. Structural insights into the allosteric operation of the Lon AAA+ protease.Structure. 2016; 24: 667-675Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, the simple one-step transition model is not supported by recent cryo-EM structures of substrate-bound AAA+ proteins with the ATPase domains organized in spiral staircase arrangement around a centrally positioned substrate polypeptide chain (14Monroe N. Han H. Shen P.S. Sundquist W.I. Hill C.P. Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase.Elife. 2017; 6e24487Crossref PubMed Scopus (9) Google Scholar, 15Yu H. Lupoli T.J. Kovach A. Meng X. Zhao G. Nathan C.F. Li H. ATP hydrolysis-coupled peptide translocation mechanism of Mycobacterium tuberculosis ClpB.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E9560-E9569Crossref PubMed Scopus (44) Google Scholar, 16de la Peña A.H. Goodall E.A. Gates S.N. Lander G.C. Martin A. Substrate-engaged 26 proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation.Science. 2018; 362eaav0725Crossref PubMed Scopus (153) Google Scholar, 17Ripstein Z.A. Huang R. Augustyniak R. Kay L.E. Rubinstein J.L. Structure of a AAA+ unfoldase in the process of unfolding substrate.Elife. 2017; 6e25754Crossref PubMed Scopus (87) Google Scholar, 18Ho C.-M. Beck J.R. Lai M. Cui Y. Goldberg D.E. Egea P.F. Zhou Z.H. Malaria parasite translocon structure and mechanism of effector export.Nature. 2018; 561: 70-75Crossref PubMed Scopus (95) Google Scholar, 19Gates S.N. Yokom A.L. Lin J. Jackrel M.E. Rizo A.N. Kendsersky N.M. Buell C.E. Sweeny E.A. Mack K.L. Chuang E. Torrente M.P. Su M. Shorter J. Southworth D.R. Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104.Science. 2017; 357: 273-279Crossref PubMed Scopus (166) Google Scholar, 20White K.I. Zhao M. Choi U.B. Pfuetzner R.A. Brunger A.T. Structural principles of SNARE complex recognition by the AAA+ protein NSF.Elife. 2018; 7e38888Crossref PubMed Scopus (42) Google Scholar, 21Han H. Monroe N. Sundquist W.I. Shen P.S. Hill C.P. The AAA ATPase Vps4 binds ESCRT-III substrates through a repeating array of dipeptide-binding pockets.Elife. 2017; 6e31324Crossref PubMed Scopus (56) Google Scholar, 22Ripstein Z.A. Vahidi S. Houry W.A. Rubinstein J.L. Kay L.E. A processive rotary mechanism couples substrate unfolding and proteolysis in the ClpXP degradation machinery.Elife. 2020; 9e52158Crossref PubMed Scopus (46) Google Scholar). These structures support a processive rotary mechanism, which was also suggested by recent cryo-EM structures of human and Yersinia pestis Lon proteases (23Shin M. Watson E.R. Song A.S. Mindrebo J.T. Novick S.J. Griffin P.R. Wiseman R.L. Lander G.C. Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.Nat. Commun. 2021; 12: 3239Crossref PubMed Scopus (8) Google Scholar, 24Shin M. Puchades C. Asmita A. Puri N. Adjei E. Wiseman R.L. Karzai A.W. Lander G.C. Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.Sci. Adv. 2020; 6eaba8404Crossref PubMed Scopus (21) Google Scholar). However, in these works the bacterial Lon used for reconstruction was a Walker-B mutant; the structures of human and bacterial Lon show different engagement with the bound substrate from the six protomers, with different sets of nucleotide-binding states (23Shin M. Watson E.R. Song A.S. Mindrebo J.T. Novick S.J. Griffin P.R. Wiseman R.L. Lander G.C. Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.Nat. Commun. 2021; 12: 3239Crossref PubMed Scopus (8) Google Scholar, 24Shin M. Puchades C. Asmita A. Puri N. Adjei E. Wiseman R.L. Karzai A.W. Lander G.C. Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.Sci. Adv. 2020; 6eaba8404Crossref PubMed Scopus (21) Google Scholar). Here, we report cryo-EM structures of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state, determined at 3.6 Å resolution. Structural analysis suggests that both the substrate and ATP play an important role to induce a spiral staircase arrangement of the ATPase domains. Moreover, our structure of wild-type bacterial LonA reveals how binding of ATP and hydrolysis of ATP to ADP induce distinct nucleotide-induced conformational states in the six ATPase domains to enable a processive rotary translocation mechanism by LonA-specific allosteric coordination. To capture MtaLonA in the substrate-engaged state, we assessed the degradation of Ig2 (25 kDa), a model substrate used previously (7Su S.-C. Lin C.-C. Tai H.-C. Chang M.-Y. Ho M.-R. Babu C.S. Liao J.-H. Wu S.-H. Chang Y.-C. Lim C. Chang C.-I. Structural basis for the magnesium-dependent activation and hexamerization of the Lon AAA+ protease.Structure. 2016; 24: 676-686Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 25Tzeng S.-R. Tseng Y.-C. Lin C.-C. Hsu C.-Y. Huang S.-J. Kuo Y.-T. Chang C.-I. Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease.Elife. 2021; 10e64056Crossref PubMed Google Scholar), by wild-type MtaLonA using the slowly hydrolyzable ATP analog, ATP-γ-S. At the optimal reaction temperature (55 °C), MtaLonA is able to degrade Ig2 with ATP-γ-S, albeit much slower compared with ATP (Fig. S1). The result suggests that Ig2 is productively translocated by MtaLonA utilizing ATP-γ-S. Therefore, for cryo-EM imaging, we purified MtaLonA in complex with Ig2, ATP-γ-S, and a peptidyl boronate, MMH8709 (13Lin C.-C. Su S.-C. Su M.-Y. Liang P.-H. Feng C.-C. Wu S.-H. Chang C.-I. Structural insights into the allosteric operation of the Lon AAA+ protease.Structure. 2016; 24: 667-675Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), which inhibits Lon protease activity by covalently binding to the catalytic serine in the protease domain, in order to trap MtaLonA in the process of translocating Ig2 without undergoing subsequent substrate proteolysis (7Su S.-C. Lin C.-C. Tai H.-C. Chang M.-Y. Ho M.-R. Babu C.S. Liao J.-H. Wu S.-H. Chang Y.-C. Lim C. Chang C.-I. Structural basis for the magnesium-dependent activation and hexamerization of the Lon AAA+ protease.Structure. 2016; 24: 676-686Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10Liao J.-H. Ihara K. Kuo C.-I. Huang K.-F. Wakatsuki S. Wu S.-H. Chang C.-I. Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1395-1402Crossref PubMed Scopus (18) Google Scholar) (See Experimental procedures for details). The structure of the MtaLonA-Ig2-MMH8709-ATP-γ-S complex was determined to 3.6-Å resolution by cryo-EM (Fig. 1A, Figs. S1 and S2 and Table S1). The six protease domains associate into a closed-ring structure with C6 symmetry (Fig. 1B). The six ATPase domains also form a closed ring (Fig. 1B). However, those from four consecutive protomers P1 to P4 (designated in the PDB coordinate file as chains A–D) organize into a spiral staircase arrangement, with protomers P1 and P4 occupying the highest and the lowest positions, respectively (Fig. 1C). These four protomers spiral around an extended 11-residue polypeptide chain, likely representing a segment of unfolded substrate Ig2, of which the backbone is well resolved in the map. By contrast, the two relatively mobile promoters P5 and P6 (corresponding to chains E and F, respectively, in the PDB file) make no substrate contacts (Fig. 1D). These nonengaged protomers are defined as the “seam” protomers as they detach from the staircase and make a loose association with each other (Fig. 1, A–C). Although the full-length construct of wild-type MtaLonA includes the N-terminal regions (residues 1–243), which is critical for substrate interaction and is required to degrade misfolded or native protein substrates (25Tzeng S.-R. Tseng Y.-C. Lin C.-C. Hsu C.-Y. Huang S.-J. Kuo Y.-T. Chang C.-I. Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease.Elife. 2021; 10e64056Crossref PubMed Google Scholar), no density was found for these regions in our structures, likely due to their flexible nature. The sequence of Ig2 and the polarity of the bound polypeptide chain could not be determined from the density map. A strand of 11 alanine residues was modeled, with the C-terminus facing inside the chamber because Lon is known to recognize exposed C-terminal degron from model substrates and kinetics study indicates that the order of substrate scissile-site delivery occurs from the C- to N-terminal direction (26Mikita N. Cheng I. Fishovitz J. Huang J. Lee I. Processive degradation of unstructured protein by Escherichia coli Lon occurs via the slow, sequential delivery of multiple scissile sites followed by rapid and synchronized peptide bond cleavage events.Biochemistry. 2013; 52: 5629-5644Crossref PubMed Scopus (10) Google Scholar, 27Gur E. Sauer R.T. Recognition of misfolded proteins by Lon, a AAA protease.Genes Dev. 2008; 22: 2267-2277Crossref PubMed Scopus (163) Google Scholar). Contacts to the substrate polypeptide chain are made by protomers P1 to P4, mediated by residue Tyr397 from pore-loop 1 and residue Trp431 from pore-loop 2; both have been shown to be required for degrading Ig2 (13Lin C.-C. Su S.-C. Su M.-Y. Liang P.-H. Feng C.-C. Wu S.-H. Chang C.-I. Structural insights into the allosteric operation of the Lon AAA+ protease.Structure. 2016; 24: 667-675Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The four pore-loop 1 Tyr397 residues contact Ig2 in a right-handed spiral arrangement, with amino acid residues i, i + 2, i + 4, and i + 6 of the substrate (Fig. 1D and Video S1). Contrary to the well-resolved pore loops of substrate-engaged protomers P1 to P4, those of the seam protomers P5 and P6 do not make contact with the substrate and are less well resolved in the map (Fig. 1, A and D). To understand how the staircase arrangement of the pore-loops 1 and 2 engaging the substrate correlates with nucleotide-binding and hydrolysis states in the protomers, we analyzed the cryo-EM map of the ATPase site located in between the large AAA-α/β and the small AAA-α domains. The resolution of the map is sufficient to identify the bound nucleotides in the ATPase sites in protomers P1 to P4 (Figs. S3 and S4 and Video S1). In protomer P1, the ATPase pocket is well ordered, with a bound ATP-γ-S, whose γ-phosphate interacts with the arginine finger (Arg finger) Arg484 from the clockwise neighboring protomer P2; the α- and β-phosphates are contacted by the sensor II, Arg541, in the cis protomer (Fig. 2A). Similarly, in protomers P2 and P3, ATP-γ-S is found in the ATPase pocket with well-resolved density, with the γ-phosphate also contacted by the Arg finger. However, the sensor II Arg541 engages the β- and γ-phosphates of the bound ATP-γ-S in protomer P2 but solely with the γ-phosphate in protomer P3 (Fig. 2, B and C). As a result, the Arg finger and sensor II residues appear to present the scissile Pγ-Oβ bond of ATP-γ-S in the most suitable conformation for hydrolysis only in protomer P3. In contrast, the ATPase pocket of protomer P4, which occupies the lowest step of the substrate-bound staircase, is ADP-bound and more open than those of protomers P1 to P3; the Arg finger from the protomer P5 is 15 Å away from the β-phosphate, and the adjacent sensor II residue contacting α-phosphate is flexible (Fig. 2D). Interestingly, the AAA-α/β domain of the disengaged seam protomer P5 makes the least interprotomer interaction and is highly mobile as indicated in the resolution map (Fig. S2E) and Q-scores (Fig. S4), which measures the local resolution (28Kucukelbir A. Sigworth F.J. Tagare H.D. Quantifying the local resolution of cryo-EM density maps.Nat. Methods. 2014; 11: 63-65Crossref PubMed Scopus (1064) Google Scholar) and structure resolvability (29Pintilie G. Zhang K. Su Z. Li S. Schmid M.F. Chiu W. Measurement of atom resolvability in cryo-EM maps with Q-scores.Nat. Methods. 2020; 17: 328-334Crossref PubMed Scopus (61) Google Scholar), respectively. The ATPase site has only broken density for the bound nucleotide and the Walker-A/B motifs (Fig. S3); the Arg finger from protomer P6 and the sensor II are also unresolved. As mentioned above, the Arg finger R484 from protomer P5 is far from the bound ADP in protomer P4; therefore, the protomer P5 ATPase site is likely bound to ADP also, but not ATP-γ-S. The seam protomer P6 also has a mobile AAA-α/β domain. The resolution of the map at the ATPase pocket is nevertheless sufficient to reveal a bound ADP (Fig. S3), probably due to its ordered neighboring protomer P1. The side chains of the Arg finger from protomer P1 and the sensor II residues, though located nearby, are not well resolved. In the substrate-engaged state, the spiral staircase arrangement of the AAA-α/β domains in the closed AAA+ ring, which is covalently fused to the closed protease ring, is made possible by several previously found LonA-specific structural features (13Lin C.-C. Su S.-C. Su M.-Y. Liang P.-H. Feng C.-C. Wu S.-H. Chang C.-I. Structural insights into the allosteric operation of the Lon AAA+ protease.Structure. 2016; 24: 667-675Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). They include nucleotide-dependent rigid-body rotation of the AAA-α/β domain, which presents the dual pore loops, to adopt different positions with respect to the AAA-α domain. In addition, the flexible protease-domain (PD) linker bridging the AAA-α and the protease domains accommodates further rotational movement of the AAA-α domain (Fig. 3). Notably, the cis Arg finger Arg484 in the ATP-γ-S-bound protomers is positioned within the hydrogen-bonding distance near the bound nucleotide in the ATPase pocket of the counterclockwise neighboring protomer, making contact only with bound ATP-γ-S but not ADP (Fig. 2). Interestingly, Arg484 is contacted by Asp444 and Pro445 in the N-terminal base loop of the pre-sensor-1 β-hairpin (PS1βH), a motif conserved in LonA, HslU, and Clp AAA+ proteases (30Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. Evolutionary history and higher order classification of AAA+ ATPases.J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (615) Google Scholar, 31Erzberger J.P. Berger J.M. Evolutionary relationships and structural mechanisms of AAA+ proteins.Annu. Rev. Biophys. Biomol. Struct. 2006; 35: 93-114Crossref PubMed Scopus (567) Google Scholar). In the substrate-engaged, spirally arranged protomers P1 to P3, the base-loop residues Asp444 and Glu446 form a trans-ATP-interacting group together with Arg484 to bind ATP-γ-S (Fig. S5). Taken together, based on the structural results, it may be assumed that interaction with substrate polypeptide chain appears to induce the formation of a spiral staircase arrangement of ATP-bound protomers, enabling the interaction of the PS1βH base loop and the Arg finger with the ribose and the phosphate groups of ATP, respectively, and to facilitate ATP hydrolysis in the ATPase site. Our structure may thus offer a straightforward explanation of how the presence of protein or peptide substrates stimulates the ATPase activity of LonA, a hallmark feature also shared by other AAA+ proteases (32Cheng I. Mikita N. Fishovitz J. Frase H. Wintrode P. Lee I. Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity.J. Mol. 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Chem. 2003; 278: 50182-50187Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 37Zhang X. Wigley D.B. The “glutamate switch” provides a link between ATPase activity and ligand binding in AAA+ proteins.Nat. Struct. Mol. Biol. 2008; 15: 1223-1227Crossref PubMed Scopus (90) Google Scholar). In this work, we have determined the structure of MtaLonA in the act of translocating the model substrate Ig2, which is partially folded protein (25Tzeng S.-R. Tseng Y.-C. Lin C.-C. Hsu C.-Y. Huang S.-J. Kuo Y.-T. Chang C.-I. Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease.Elife. 2021; 10e64056Crossref PubMed Google Scholar). The substrate-bound structure forms an asymmetric close ring. Interestingly, recent cryo-EM structures of substrate-free Lon show an open spiral structure, where the side opening potentially allows access of the pore loops to substrates from outside (11Botos I. Lountos G.T. Wu W. Cherry S. Ghirlando R. Kudzhaev A.M. Rotanova T.V. de Val N. Tropea J.E. Gustchina A. Wlodawer A. Cryo-EM structure of substrate-free E. coli Lon protease provides insights into the dynamics of Lon machinery.Curr. Res. Struct. Biol. 2019; 1: 13-20Crossref PubMed Scopus (8) Google Scholar, 23Shin M. Watson E.R. Song A.S. Mindrebo J.T. Novick S.J. Griffin P.R. Wiseman R.L. Lander G.C. Structures of the human LONP1 protease reveal regulatory steps involved in protease activation.Nat. Commun. 2021; 12: 3239Crossref PubMed Scopus (8) Google Scholar, 24Shin M. Puchades C. Asmita A. Puri N. Adjei E. Wiseman R.L. Karzai A.W. Lander G.C. Structural basis for distinct operational modes and protease activation in AAA+ protease Lon.Sci. Adv. 2020; 6eaba8404Crossref PubMed Scopus (21) Google Scholar). Nevertheless, the substrate Ig2 is more likely to be translocated from the top of the AAA+ ring, where the substrate-binding N-terminal domain, essential for recognition and proteolysis of Ig2 (25Tzeng S.-R. Tseng Y.-C. Lin C.-C. Hsu C.-Y. Huang S.-J. Kuo Y.-T. Chang C.-I. Molecular insights into substrate recognition and discrimination by the N-terminal domain of Lon AAA+ protease.Elife. 2021; 10e64056Crossref PubMed Google Scholar), is located. The main goal of the present work is to address the molecular basis for substrate translocation by LonA. Our structure of Ig2-bound wild-type MtaLonA suggests a LonA-specific processive rotary mechanism to drive translocation of substrate polypeptide chains (Fig. 4). As shown in the schematic diagrams, during the translocation process, a polypeptide chain is gripped by four protomers, each of these protomers traverses in clockwise direction through successive cycles of six conformational states, of which four are in substrate-engaged modes and two are in nonengaged detached modes. Of the four engaged modes, two are coordinated by binding of ATP, one presumably triggers ATP hydrolysis (indicated by the lightning symbol), and one is bound to ADP. Protomers of LonA in the engaged states communicate with one another by the Arg finger and the PS1βH base loop. In the two detached modes, the protomers adopt mobile conformations permitting exchange of ADP by ATP, after which they are switched sequentially back into the substrate-engaged mode. In the next cycle, ATP hydrolysis proceeds counterclockwise to the next ATPase site. The mechanism featuring “hand-over-hand” substrate translocation coupled to counterclockwise sequential hydrolysis of ATP is conserved in a variety of different AAA+ proteins (14Monroe N. Han H. Shen P.S. Sundquist W.I. Hill C.P. Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase.Elife. 2017; 6e24487Crossref PubMed Scopus (9) Google Scholar, 15Yu H. Lupoli T.J. Kovach A. Meng X. Zhao G. Nathan C.F. Li H. ATP hydrolysis-coupled peptide translocation mechanism of Mycobacterium tuberculosis ClpB.Proc. Natl. Acad. Sci. U. S. 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