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• W2028387879 abstract "In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide. In Archaea, an hexameric ATPase complex termed PAN promotes proteins unfolding and translocation into the 20 S proteasome. PAN is highly homologous to the six ATPases of the eukaryotic 19 S proteasome regulatory complex. Thus, insight into the mechanism of PAN function may reveal a general mode of action mutual to the eukaryotic 19 S proteasome regulatory complex. In this study we generated a three-dimensional model of PAN from tomographic reconstruction of negatively stained particles. Surprisingly, this reconstruction indicated that the hexameric complex assumes a two-ring structure enclosing a large cavity. Assessment of distinct three-dimensional functional states of PAN in the presence of adenosine 5′-O-(thiotriphosphate) and ADP and in the absence of nucleotides outlined a possible mechanism linking nucleotide binding and hydrolysis to substrate recognition, unfolding, and translocation. A novel feature of the ATPase complex revealed in this study is a gate controlling the “exit port” of the regulatory complex and, presumably, translocation into the 20 S proteasome. Based on our structural and biochemical findings, we propose a possible model in which substrate binding and unfolding are linked to structural transitions driven by nucleotide binding and hydrolysis, whereas translocation into the proteasome only depends upon the presence of an unfolded substrate and binding but not hydrolysis of nucleotide. In eukaryotic cells most protein breakdown in the cytosol and nucleus is catalyzed by the 26 S proteasome. This ∼2.5-MDa (1Hölzl H. Kapelari B. Kellermann J. Seemüller E. Sümegi M. Udvardy A. Medalia O. Sperling J. Müller S.A. Engel A. Baumeister W. J. Cell Biol. 2000; 150: 119-130Crossref PubMed Scopus (135) Google Scholar) complex degrades ubiquitin-conjugated and certain non-ubiquitinated proteins in an ATP-dependent manner (2Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2234) Google Scholar, 3Voges D. Zwickl P. Baumeister W. Annu. Rev. Biochem. 1999; 68: 1015-1068Crossref PubMed Scopus (1595) Google Scholar). The 26 S complex is composed of one or two 19 S regulatory particles situated at the ends of the cylindrical 20 S proteasome. Within the 26 S complex, proteins are hydrolyzed in the 20 S proteasome. Tagged substrates, however, first bind to the 19 S regulatory particle, which catalyzes their unfolding and translocation into the 20 S subcomplex (4Braun B.C. Glickman M. Kraft R. Dahlmann B. Kloetzel P.M. Finley D. Schmidt M. Nat. Cell Biol. 1999; 1: 221-226Crossref PubMed Scopus (388) Google Scholar, 5Strickland E. Hakala K. Thomas P.J. DeMartino G.N. J. Biol. Chem. 2000; 275: 5565-5572Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The 19 S regulatory particle consists of at least 17 different subunits (1Hölzl H. Kapelari B. Kellermann J. Seemüller E. Sümegi M. Udvardy A. Medalia O. Sperling J. Müller S.A. Engel A. Baumeister W. J. Cell Biol. 2000; 150: 119-130Crossref PubMed Scopus (135) Google Scholar, 6Glickman M.H. Rubin D.M. Fried V.A. Finley D. Mol. Cell. Biol. 1998; 18: 3149-3162Crossref PubMed Google Scholar). Nine of these subunits form a “lid,” whereas the other eight subunits, including six ATPases, comprise the base of the 19 S particle. Electron microscopy (7Walz J. Erdmann A. Kania M. Typke D. Koster A.J. Baumeister W. J. Struct. Biol. 1998; 121: 19-29Crossref PubMed Scopus (160) Google Scholar, 8Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar, 9Nickell S. Mihalache O. Beck F. Hegerl R. Korinek A. Baumeister W. Biochem. Biophys. Res. Commun. 2007; 353: 115-120Crossref PubMed Scopus (31) Google Scholar, 10da Fonseca P.C. Morris E.P. J. Biol. Chem. 2008; 283: 23305-23314Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) as well as cross-linking experiments (11Hendil K.B. Hartmann-Petersen R. Tanaka K. J. Mol. Biol. 2002; 315: 627-636Crossref PubMed Scopus (52) Google Scholar, 12Hartmann-Petersen R. Tanaka K. Hendil K.B. Arch. Biochem. Biophys. 2001; 386: 89-94Crossref PubMed Scopus (61) Google Scholar) have demonstrated that the six homologous ATPases are associated with the α rings of the 20 S particle. Unlike eukaryotes, Archaea and certain eubacteria contain homologous 20 S particles but lack ubiquitin. Their proteasomes degrade proteins in association with a hexameric ATPase ring complex termed PAN (13Zwickl P. Ng D. Woo K.M. Klenk H.P. Goldberg A.L. J. Biol. Chem. 1999; 274: 26008-26014Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). PAN appears to be the evolutionary precursor of the 19 S base, predating the coupling of ubiquitination and proteolysis in eukaryotes (14Zwickl P. Seemüller E. Kapelari B. Baumeister W. Adv. Protein Chem. 2001; 59: 187-222Crossref PubMed Scopus (50) Google Scholar). In addition, PAN recognizes the bacterial targeting sequence ssrA (in analogy to the polyubiquitin conjugates in eukaryotes) and efficiently unfolds and translocates globular substrates, like green fluorescent protein, when tagged with ssrA (15Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In both PAN and the 19 S proteasome regulatory complexes, ATP is essential for substrate unfolding and translocation and for opening of the gated channel in the α ring through which substrates enter the 20 S particle (15Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 16Köhler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 17Benaroudj N. Zwickl P. Seemüller E. Baumeister W. Goldberg A.L. Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Because this portal is quite narrow (18Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1377) Google Scholar, 19Groll M. Ditzel L. Löwe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1941) Google Scholar, 20Groll M. Bajorek M. Köhler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (653) Google Scholar), only extended polypeptides can enter the 20 S proteasome. Consequently, a globular substrate must be unfolded by the associated ATPase complex to be translocated and digested within the 20 S particle. PAN and the six ATPases found at the base of the 19 S particle are members of the AAA+ superfamily of multimeric ATPases which also includes the ATP-dependent proteases Lon and FtsH and the regulatory components of the bacterial ATP-dependent proteases ClpAP, ClpXP, and HslUV (8Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar, 21Larsen C.N. Finley D. Cell. 1997; 91: 431-434Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). For mechanistic studies of the roles of ATP, the simpler archaeal PAN-20 S system offers many technical advantages over the much more complex 26 S proteasome. For example, prior studies of PAN (17Benaroudj N. Zwickl P. Seemüller E. Baumeister W. Goldberg A.L. Mol. Cell. 2003; 11: 69-78Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 22Benaroudj N. Goldberg A.L. Nat. Cell Biol. 2000; 2: 833-839Crossref PubMed Scopus (122) Google Scholar) demonstrated that unfolding of globular substrates (e.g. green fluorescent protein-ssrA) requires ATP hydrolysis. The same was also shown for the Escherichia coli ATP-dependent proteases ClpXP (23Kenniston J.A. Baker T.A. Fernandez J.M. Sauer R.T. Cell. 2003; 114: 511-520Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) and ClpAP (24Weber-Ban E.U. Reid B.G. Miranker A.D. Horwich A.L. Nature. 1999; 401: 90-93Crossref PubMed Scopus (362) Google Scholar). We have also shown that unfolding by PAN can take place on the surface of the ATPase ring in the absence of translocation (15Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Thus, unfolding seems to proceed independently from protein translocation into the 20 S proteolytic particle. It is noteworthy that other studies suggest that proteins are unfolded by energy-dependent translocation through the ATPase ring (25Lee C. Schwartz M.P. Prakash S. Iwakura M. Matouschek A. Mol. Cell. 2001; 7: 627-637Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 26Matouschek A. Curr. Opin. Struct. Biol. 2003; 13: 98-109Crossref PubMed Scopus (131) Google Scholar). These studies have suggested that the translocation of an unfolded polypeptide from the ATPase into the 20 S core is an active process that is coupled to ATP hydrolysis. A key to underline a detailed molecular mechanism for substrate binding, unfolding, and translocation by the proteasome regulatory ATPase complex is improved understanding of its architecture and the nucleotide-dependent structural transitions that afford these functions. To date we and others have failed to generate micrographs suitable for three-dimensional reconstruction of PAN using single-particle EM analysis. Likewise, structural information regarding the three-dimensional architecture and subunit organization within the 19 S particle is very limited. In fact, high resolution three-dimensional information on the 19 S complex is not yet available. Most knowledge available is based on cross-linking experiments (11Hendil K.B. Hartmann-Petersen R. Tanaka K. J. Mol. Biol. 2002; 315: 627-636Crossref PubMed Scopus (52) Google Scholar, 12Hartmann-Petersen R. Tanaka K. Hendil K.B. Arch. Biochem. Biophys. 2001; 386: 89-94Crossref PubMed Scopus (61) Google Scholar) as well as EM structural analysis (7Walz J. Erdmann A. Kania M. Typke D. Koster A.J. Baumeister W. J. Struct. Biol. 1998; 121: 19-29Crossref PubMed Scopus (160) Google Scholar, 8Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1304) Google Scholar, 9Nickell S. Mihalache O. Beck F. Hegerl R. Korinek A. Baumeister W. Biochem. Biophys. Res. Commun. 2007; 353: 115-120Crossref PubMed Scopus (31) Google Scholar, 10da Fonseca P.C. Morris E.P. J. Biol. Chem. 2008; 283: 23305-23314Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), which provided a three-dimensional model outline of the general architecture of the 26 S complex. Unlike the 19 S complex, the structure of the 20 S subcomplex was determined by x-ray crystallography (18Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1377) Google Scholar, 19Groll M. Ditzel L. Löwe J. Stock D. Bochtler M. Bartunik H.D. Huber R. Nature. 1997; 386: 463-471Crossref PubMed Scopus (1941) Google Scholar). In contrast to the highly homogenous structure of the 20 S complex, the structural heterogeneity and flexibility of the 19 S subcomplex is presumably reflected in multiple conformations, which in turn also contribute to the difficulty in generating a high resolution three-dimensional structural model of the 26 S proteasome. Accordingly, the initial goal of this study was to generate a three-dimensional model of PAN that will allow us to determine its general architecture and to correlate unique conformational transitions within this ATPase with the nucleotide state of the complex (i.e. in the presence of ATPγS, ADP, or in the absence of nucleotides). Smith et al. (27Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) suggested a general architecture for the PAN-20 S complex based on two-dimensional averaging of a Thermoplasma acidophilum (TA) 3The abbreviations used are: TAT. acidophilumATPγSadenosine 5′-O-(thiotriphosphate)EMelectron microscopyMJM. jannaschiiANS1-anilinonaphthalene-8-sulfonic acidYFPyellow fluorescent protein. 3The abbreviations used are: TAT. acidophilumATPγSadenosine 5′-O-(thiotriphosphate)EMelectron microscopyMJM. jannaschiiANS1-anilinonaphthalene-8-sulfonic acidYFPyellow fluorescent protein. 20 S proteasome and Methanococcus jannaschii (MJ) PAN hybrid complex in the presence of ATPγS. Based on side-view projections of that complex, these authors proposed that PAN assumes an overall structure similar to E. coli HslU (28Rohrwild M. Pfeifer G. Santarius U. Müller S.A. Huang H.C. Engel A. Baumeister W. Goldberg A.L. Nat. Struct. Biol. 1997; 4: 133-139Crossref PubMed Scopus (175) Google Scholar, 29Bochtler M. Hartmann C. Song H.K. Bourenkov G.P. Bartunik H.D. Huber R. Nature. 2000; 403: 800-805Crossref PubMed Scopus (378) Google Scholar, 30Song H.K. Hartmann C. Ramachandran R. Bochtler M. Behrendt R. Moroder L. Huber R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14103-14108Crossref PubMed Scopus (135) Google Scholar). T. acidophilum adenosine 5′-O-(thiotriphosphate) electron microscopy M. jannaschii 1-anilinonaphthalene-8-sulfonic acid yellow fluorescent protein. T. acidophilum adenosine 5′-O-(thiotriphosphate) electron microscopy M. jannaschii 1-anilinonaphthalene-8-sulfonic acid yellow fluorescent protein. We realized that although PAN appears heterogeneous in electron micrographs, it does not occupy all possible orientations when adsorbed to carbon-coated electron microscopy (EM) grids, a prerequisite for single particle analysis. This problem was overcome by applying electron tomography in conjunction with a three-dimensional averaging procedure that accounts for the missing wedge in the Fourier space of electron tomograms (31Beck M. Förster F. Ecke M. Plitzko J.M. Melchior F. Gerisch G. Baumeister W. Medalia O. Science. 2004; 306: 1387-1390Crossref PubMed Scopus (401) Google Scholar, 32Förster F. Medalia O. Zauberman N. Baumeister W. Fass D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4729-4734Crossref PubMed Scopus (239) Google Scholar). The three-dimensional model generated revealed an unexpected architecture leading to a possible molecular mechanism describing the function of PAN and presumably the 19 S ATPases. PAN was purified according to Navon and Goldberg (15Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The α and β subunits of the MJ 20 S proteasome were cloned into the pET22 plasmid downstream of the T7 promoter. The MJ 20 S complex was expressed in E. coli BL21 and purified by heating the resuspended bacterial pellet (50 mm Hepes, pH 7.5) and removing the aggregated E. coli proteins by centrifugation. The cleared lysate was loaded onto an anion exchange column (Mono Q), and PAN-containing fractions were pooled, concentrated, and dialyzed against 50 mm Hepes, pH 7.5. For electron tomography, an aliquot of PAN was diluted into buffer A (50 mm Tris-HCl, pH 7.5, and 1 mm MgCl2) to a final protein concentration of 25 μg/ml. When stated, the mixture was also complemented with 50 μm ATPγS, ADP, or no nucleotide. A 5-μl drop was applied onto 200 mesh carbon-coated copper grids. The specimens were transferred into a Phillips CM 200 FEG microscope equipped with a 2k TVIPS CCD camera. Data were collected in a fully automated manner using TVIPS tomography software. Tilt series were collected, typically covering an angular range from −60° to 60°, and sampled at 2° tilt increments with a 2-μm underfocus. The pixel size was 0.41 nm at the specimen level, which corresponds to a magnification of ×27,500. All image processing operations were carried out with the EM package (33Harauz G. Cicicopol C. Hegerl R. Cejka Z. Goldie K. Santarius U. Engel A. Baumeister W. J. Struct. Biol. 1996; 116: 290-301Crossref PubMed Scopus (12) Google Scholar) and the TOM- and av3-toolbox (32Förster F. Medalia O. Zauberman N. Baumeister W. Fass D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4729-4734Crossref PubMed Scopus (239) Google Scholar, 34Nickell S. Förster F. Linaroudis A. Net W.D. Beck F. Hegerl R. Baumeister W. Plitzko J.M. J. Struct. Biol. 2005; 149: 227-234Crossref PubMed Scopus (333) Google Scholar) for Matlab (MathWorks). A total number of 500 single particle-containing sub-tomograms of PAN-ATPγS volumes, 450 of PAN-ADP, 500 of PAN without nucleotide, and 250 of PAN Δ1–73 volumes were selected. The sub-tomograms containing particle volumes were extracted in silico and subjected to a three-dimensional averaging procedure (32Förster F. Medalia O. Zauberman N. Baumeister W. Fass D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4729-4734Crossref PubMed Scopus (239) Google Scholar). The resolutions of the different PAN structures were determined by Fourier shell correlation with 0.5 criteria. After a few iterations, the 6-fold symmetry of the structure became apparent. Therefore, imposed 6-fold symmetry was used in the refinement iterations. The PAN-ATPγS structure was resolved to 2.0-nm resolution, whereas the structures of PAN-ADP and PAN without nucleotide were resolved to 2.3 nm each. The PAN Δ1–73 structure was resolved to 2.5 nm. Three-dimensional surface-rendered visualizations were created with Amira 3.1 (TGS). Electron micrographs were recorded digitally using a FEI Polara microscope equipped with a GIF energy filter (Gatan), a CCD camera at 1.5 μm underfocus, and a pixel size of 0.41 nm. A set of 1157 images of negatively stained proteasomes were aligned and averaged using the single particle procedures of the EM software package (33Harauz G. Cicicopol C. Hegerl R. Cejka Z. Goldie K. Santarius U. Engel A. Baumeister W. J. Struct. Biol. 1996; 116: 290-301Crossref PubMed Scopus (12) Google Scholar). Electron micrographs were recorded as above. A set of 7000 images of negatively stained complexes were analyzed with the EMAN package (35Tang G. Peng L. Baldwin P.R. Mann D.S. Jiang W. Rees I. Ludtke S.J. J. Struct. Biol. 2007; 157: 38-46Crossref PubMed Scopus (2087) Google Scholar). The images were low pass-filtered at 1.7 nm (below the first zero of the contrast transfer function). The structure was resolved to 2 nm of resolution, determined by Fourier shell correlation with 0.5 criteria. The unfolding and degradation of YFP-ssrA was monitored by recording fluorescence over time. The reactions were performed in buffer B (50 mm Hepes, pH 7.5, 5 mm MgCl2, 1 mm ATP, and 30 mm KCl) at 45 °C. YFP-ssrA was excited at 485 nm, and fluorescence emission was recorded at 529 nm. Unless stated differently, the final concentration of YFP-ssrA in a typical reaction was 10 μm, and PAN-20 S complexes were present at a final concentration of 16 nm. To gain insight into how substrates interact with PAN and how the conformation of PAN is affected by ATP binding and hydrolysis, we undertook an integrative approach combining biochemistry and structural analysis. Three-dimensional reconstruction of PAN (Fig. 1 and supplemental Fig. S1 and movie (PAN ATPγS.mpg)) indicated that the hexameric complex assumes a two-ring structure with a large cavity in its center. Fig. 1A depicts successive 0.4-nm-thick slices through the reconstructed volume of the PAN complex in the presence of ATPγS. The two rings have similar thicknesses of about 3 nm each, with connecting stems that converge in the mid-plane of the complex to form six symmetrical protruding “spikes” (Fig. 1Bc). Overall, the complex has a diameter of 14.5 nm and a height of 9.5 nm. It is noteworthy that two-dimensional projection of our three-dimensional model (see Fig. 7B) resembles the view seen by Smith et al. (27Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar).FIGURE 7Three-dimensional model of the MJ PAN-20 S complex. A, a three-dimensional model of the PAN-20 S proteasome complex generated in silico based on the three-dimensional model of PAN and a reconstructed three-dimensional model of the MJ proteasome using negatively stained complexes. B, projection view through the PAN-20 S complex demonstrating spatial interactions between the MJ 20 S proteasome and PAN. The dimensions of the pseudo-ring and the cavity in the C-terminal ring of PAN allow direct interaction between the N-terminal region of the α-ring and the C-terminal residues of PAN.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm that the structure depicted in Fig. 1B represents the architecture of a single hexameric PAN complex rather than that of a dodecamer (a dimer of hexamers), we determined the molecular weight of the complex by mass spectrometry. The detected mass of 294,570 ± 23 Da agrees with the predicted molecular mass of a single hexameric complex (36Medalia N. Sharon M. Martinez-Arias R. Mihalache O. Robinson C.V. Medalia O. Zwickl P. J. Struct. Biol. 2006; 156: 84-92Crossref PubMed Scopus (13) Google Scholar). In addition, to verify this result and localize the N and the C domains of the subunits in the complex, we engineered a mutant of PAN in which each of the subunits lacks the first 73 residues (PAN Δ1–73). We reconstructed the three-dimensional structure of this deletion mutant under similar conditions as employed with wild type PAN (supplemental movie PAN Δ1–73.mpg). As depicted in Fig. 1C, the bottom ring did not change, maintaining the same dimensions as in wild type PAN (compare Fig. 1, Bb to Cb). By contrast, the top ring was altered in response to the N-terminal deletions, with the “shoulders” supporting the connecting stems between the two rings being contracted and the protruding spikes becoming less pronounced. Thus, the sequence of PAN encodes for a protein, which upon oligomerization forms a structure comprising two rings. One ring, reminiscent in structure of the classical AAA domain (37Dreveny I. Kondo H. Uchiyama K. Shaw A. Zhang X. Freemont P.S. EMBO J. 2004; 23: 1030-1039Crossref PubMed Scopus (156) Google Scholar), depicted in Fig. 1Bb, is presumably formed by the C-terminal domains of the six PAN monomers (residues 160–430, based on homology models). An additional ring (Fig. 1Ba) is assembled by the N-terminal domains of the monomers (residues 1–160, based on the predicated N-terminal coiled-coil sequence and N-terminal deletion mutants). In addition, the data also demonstrate that the coiled-coil region (or at least part of it) contributes to the formation of the spikes seen in the side view of the PAN complex (Fig. 1Bc) and appear to be trimmed in the ΔN mutant (Fig. 1Cc). Despite the critical roles and conserved structural motifs shared by members of the AAA superfamily of ATPases, their mode of action is not well understood. In particular, the link between the ATPase catalytic cycle and the conformational changes responsible for the biological activity of these proteins has yet to be elucidated. Likewise, it is still unclear how nucleotide binding, hydrolysis, and release by PAN promote substrate binding, unfolding, and translocation into the 20 S proteasome. To address these outstanding questions, we generated a three-dimensional model of PAN in the presence of ADP and in the absence of nucleotides. In the presence of ATPγS, PAN is presumably locked in a conformation similar to that of the ATP-bound state. The ADP-bound form simulates the conformation that PAN assumes upon nucleotide hydrolysis. The structure realized in the absence of nucleotide represents the conformation of the complex upon nucleotide release, before reloading with ATP. As shown in Fig. 2, although the overall structural character of PAN did not change, the size of the internal cavity of the complex contracted upon nucleotide hydrolysis (as reflected in the structure of PAN preincubated with ADP) and became even smaller in the absence of nucleotides (Fig. 2B). In addition, the size and orientation of the spikes were also affected (Fig. 2C). As presented in Fig. 2C, the shoulders connecting the spikes to the top ring change their orientation and move inward upon ATP hydrolysis (i.e. the ADP state) and release (i.e. the no nucleotide state). We measured the dimensions of the upper ring by calculating the projection of these elements from averaged structures. The measured difference in radius between the ATPγS state and the “no nucleotide” state is about 2 nm (Fig. 2C), whereas in the presence of ADP the complex adopts an intermediate state. Together these data suggest that upon nucleotide binding (i.e. the ATPγS-bound state), PAN assumes a conformation in which the internal cavity is more accessible. Upon nucleotide hydrolysis, which probably occurs after or concomitantly with substrate entry into the internal chamber of the complex, the spikes change their conformation, presumably to exclude binding of additional substrate molecules. The expansion and contraction that are associated with nucleotide binding, hydrolysis, and release may promote the unfolding of the globular domain trapped (or partially trapped) within the internal chamber of PAN. In the chaperonin GroEL, structural transitions are linked with direct nucleotide-dependent modulation of the hydrophobic character of the complex. To directly assess the hydrophobicity of the PAN complex in the various nucleotide-related states, we made use of 1-anilinonaphthalene-8-sulfonic acid (ANS). ANS is essentially non-fluorescent in water, only becoming appreciably fluorescent when bound to membranes (quantum yield ∼0.25) or proteins (quantum yield ∼0.7). These properties make ANS a sensitive indicator of conformational changes that modify the exposure of the probe to water (38Arighi C.N. Rossi J.P. Delfino J.M. Biochemistry. 1998; 37: 16802-16814Crossref PubMed Scopus (29) Google Scholar, 39Raha T. Chattopadhyay D. Chattopadhyay D. Roy S. Biochemistry. 1999; 38: 2110-2116Crossref PubMed Scopus (17) Google Scholar). Indeed, this probe was previously used to correlate the hydrophobicity of the GroEL complex with specific nucleotide-associated conformations (40Smoot A.L. Panda M. Brazil B.T. Buckle A.M. Fersht A.R. Horowitz P.M. Biochemistry. 2001; 40: 4484-4492Crossref PubMed Scopus (66) Google Scholar, 41Kusmierczyk A.R. Martin J. J. Biol. Chem. 2000; 275: 33504-33511Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 42Golbik R. Zahn R. Harding S.E. Fersht A.R. J. Mol. Biol. 1998; 276: 505-515Crossref PubMed Scopus (41) Google Scholar). We, thus, incubated PAN (1 μm) in the presence of ATPγS or ADP or in the absence of nucleotides in standard reaction buffer complemented with ANS (5 μm). Fluorescence emission spectra (400–600 nm) were then recorded. Interestingly, the fluorescence intensity of a reaction mixture containing PAN and ATPγS was about 50% (±8%) higher than that of PAN incubated in the presence of ADP (Fig. 3, left panel). Although the no nucleotide conformation appeared reproducibly to be about 5–10% more fluorescent than was the ADP-associated conformation, these differences were not statistically significant. Our findings, thus, predict that incubating PAN with ATP and ANS should initially confer a conformation similar to that of PAN in the presence of ATPγS, reflected by high ANS fluorescence. Over time, as ATP is being hydrolyzed, the conformation would shift into the ADP-bound state, and ANS fluorescence intensity should drop accordingly. Indeed, as observed in Fig. 3 (right panel), the initial fluorescent intensity of ANS incubated with PAN and ATP was reduced over time, whereas the intensity of fluorescence of a similar sample incubated with ADP (or ATPγS) remained unchanged. Thus, the hydrophobic character of PAN increases as the internal cavity of PAN expands in response to nucleotide binding (i.e. the ATPγS state) and decreases upon nucleotide hydrolysis, with the associated reduction of PAN internal chamber volume (i.e. the ADP state). Our structural analysis reveals that the opening in the C-terminal portion of the PAN complex is gated, as density is detected at the center of the bottom ring (Fig. 1Bb). However, we cannot exclude the existence of a low density region at the center of the bottom ring. Such an opening would have been revealed only through higher resolution structural det" @default.
• W2028387879 created "2016-06-24" @default.
• W2028387879 creator A5000049607 @default.
• W2028387879 creator A5006382114 @default.
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• W2028387879 date "2009-08-01" @default.
• W2028387879 modified "2023-10-02" @default.
• W2028387879 title "Architecture and Molecular Mechanism of PAN, the Archaeal Proteasome Regulatory ATPase" @default.
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