FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2009593650> ?p ?o ?g. }

• W2009593650 endingPage "34381" @default.
• W2009593650 startingPage "34376" @default.
• W2009593650 abstract "MecA is an adaptor protein that regulates the assembly and activity of the ATP-dependent ClpCP protease in Bacillus subtilis. MecA contains two domains. Although the amino-terminal domain of MecA recruits substrate proteins such as ComK and ComS, the carboxyl-terminal domain (residues 121–218) has dual roles in the regulation and function of ClpCP protease. MecA-(121–218) facilitates the assembly of ClpCP oligomer, which is required for the protease activity of ClpCP. This domain was identified to be a non-recycling degradation tag that targets heterologous fusion proteins to the ClpCP protease for degradation. To elucidate the mechanism of MecA, we determined the crystal structure of MecA-(121–218) at 2.2 Å resolution, which reveals a previously uncharacterized α/β fold. Structure-guided mutagenesis allows identification of surface residues that are essential for the function of MecA. We also solved the structure of a carboxyl-terminal domain of YpbH, a paralogue of MecA in B. subtilis, at 2.4 Å resolution. Despite low sequence identity, the two structures share essentially the same fold. The presence of MecA homologues in other bacterial species suggests conservation of a large family of unique degradation tags. MecA is an adaptor protein that regulates the assembly and activity of the ATP-dependent ClpCP protease in Bacillus subtilis. MecA contains two domains. Although the amino-terminal domain of MecA recruits substrate proteins such as ComK and ComS, the carboxyl-terminal domain (residues 121–218) has dual roles in the regulation and function of ClpCP protease. MecA-(121–218) facilitates the assembly of ClpCP oligomer, which is required for the protease activity of ClpCP. This domain was identified to be a non-recycling degradation tag that targets heterologous fusion proteins to the ClpCP protease for degradation. To elucidate the mechanism of MecA, we determined the crystal structure of MecA-(121–218) at 2.2 Å resolution, which reveals a previously uncharacterized α/β fold. Structure-guided mutagenesis allows identification of surface residues that are essential for the function of MecA. We also solved the structure of a carboxyl-terminal domain of YpbH, a paralogue of MecA in B. subtilis, at 2.4 Å resolution. Despite low sequence identity, the two structures share essentially the same fold. The presence of MecA homologues in other bacterial species suggests conservation of a large family of unique degradation tags. Proteolysis regulated by the AAA+ superfamily is important to both prokaryotes and eukaryotes. It is an important regulatory mechanism to biological processes such as stress responses or protein quality control under non-stress conditions (1.Prepiak P. Dubnau D. Mol. Cell. 2007; 26: 639-647Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In prokaryotic cells, ClpP and its associated adaptor ATPases form distinct proteolytic complexes with an architecture that is similar to the eukaryotic 26 S proteasome. The closely related ATPases include ClpA, ClpB, ClpC, ClpE, ClpL, ClpV, ClpX, and HslU (ClpY) (2.Zolkiewski M. Mol. Microbiol. 2006; 61: 1094-1100Crossref PubMed Scopus (96) Google Scholar). The active, hexameric ATPases unfold and translocate the substrate proteins to the proteolytic chamber within the double ring of ClpP heptamers for degradation (3.Sauer R.T. Bolon D.N. Burton B.M. Burton R.E. Flynn J.M. Grant R.A. Hersch G.L. Joshi S.A. Kenniston J.A. Levchenko I. Neher S.B. Oakes E.S. Siddiqui S.M. Wah D.A. Baker T.A. Cell. 2004; 119: 9-18Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar). Among these ATPases, ClpC is particularly intriguing because it requires an adaptor protein to stimulate its ATPase activity and to facilitate its oligomerization and activation. In Bacillus subtilis, MecA or its paralogue YpbH is required for assembly of the active protease complex ClpCP and recruitment of substrate proteins such as ComK and ComS (4.Schlothauer T. Mogk A. Dougan D.A. Bukau B. Turgay K. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 2306-2311Crossref PubMed Scopus (115) Google Scholar, 5.Persuh M. Mandic-Mulec I. Dubnau D. J. Bacteriol. 2002; 184: 2310-2313Crossref PubMed Scopus (33) Google Scholar, 6.Nakano S. Zheng G. Nakano M.M. Zuber P. J. Bacteriol. 2002; 184: 3664-3670Crossref PubMed Scopus (86) Google Scholar, 7.Turgay K. Hamoen L.W. Venema G. Dubnau D. Genes Dev. 1997; 11: 119-128Crossref PubMed Scopus (168) Google Scholar, 8.Turgay K. Hahn J. Burghoorn J. Dubnau D. EMBO J. 1998; 17: 6730-6738Crossref PubMed Google Scholar, 9.Kirstein J. Schlothauer T. Dougan D.A. Lilie H. Tischendorf G. Mogk A. Bukau B. Turgay K. EMBO J. 2006; 25: 1481-1491Crossref PubMed Scopus (107) Google Scholar). MecA contains two domains, the N-terminal domain and the C-terminal domain, which are connected by a flexible linker sequence. The N-terminal domain of MecA is essential for substrate recognition, whereas the C-terminal domain was reported to mediate the interaction with ClpC and to stimulate the ATPase activity of ClpC (9.Kirstein J. Schlothauer T. Dougan D.A. Lilie H. Tischendorf G. Mogk A. Bukau B. Turgay K. EMBO J. 2006; 25: 1481-1491Crossref PubMed Scopus (107) Google Scholar, 10.Persuh M. Turgay K. Mandic-Mulec I. Dubnau D. Mol. Microbiol. 1999; 33: 886-894Crossref PubMed Scopus (65) Google Scholar). In the accompanying article (23.Mei Z. Wang F. Qi Y. Zhou Z. Hu Q. Li H. Wu J. Shi Y. J. Biol. Chem. 2009; 284: 34366-34375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), we identified MecA-(121–218) to be a structure entity that plays dual roles. On one hand, MecA-(121–218) is the minimal domain that by itself is sufficient to induce ClpC oligomerization, to stimulate the ATPase activity of ClpC, and to facilitate the assembly and activation of the ClpCP complex. On the other hand, we demonstrated that MecA-(121–218) is a non-recycling degradation tag that targets heterologous fusion proteins for degradation by the ClpCP protease. As indicated by the term non-recycling, MecA-(121–218) is also degraded together with the substrate fusion protein. The association and degradation of MecA-(121–218) result in dynamic cycles of assembly and disassembly of the ClpCP complex. We also showed that the C-terminal domain of YpbH (residues 101–194), which share a sequence identity of 31% and sequence similarity of 63% with MecA-(121–218), functions similarly as MecA-(121–218), both for assembly of the ClpCP protease and as a non-recycling degradation tag. The biochemical observations raised the following questions. First, how does the structure of MecA-(121–218) support its function? Second, given the moderate sequence identity and similarity between MecA-(121–218) and YpbH-(101–194), do they have a similar structure? Third and more importantly, does the prokaryotic degradation tag, MecA-(121–218), exhibit any structural similarity to the eukaryotic degradation tag ubiquitin? To address these questions, we crystallized and determined the structures of MecA-(121–218) and YpbH-(101–194) at 2.2 and 2.4 Å resolution, respectively. The two structures share essentially the same fold, a previously uncharacterized α/β fold. We also performed structure-guided mutagenesis for MecA and identified surface residues that may mediate ClpC binding and ComK degradation. The structural study, together with the biochemical observations reported in the accompanying article (23.Mei Z. Wang F. Qi Y. Zhou Z. Hu Q. Li H. Wu J. Shi Y. J. Biol. Chem. 2009; 284: 34366-34375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), provides important insights into the mechanistic understanding of MecA-regulated assembly and activation of the ClpCP protease. All clones were generated using a standard PCR-based cloning strategy, and the identities of individual clones were verified through double-strand plasmid sequencing. ClpC, MecA, and YpbH variants were subcloned into pGEX-6P-1 vector (GE Healthcare) with an N-terminal glutathione S-transferase tag or PKTG vector with a C-terminal glutathione S-transferase tag and overexpressed in Escherichia coli strain BL21(DE3) at 22 °C. The soluble fraction of the E. coli lysate was purified through a glutathione-Sepharose column and cleaved by the PreScissionTM protease (GE Healthcare) wherever necessary. ClpP was overexpressed in E. coli strain BL21 (DE3) as a C-terminal His6-tagged protein using the pET-21b vector (Novagen). The soluble fraction of ClpP-His6 in the E. coli lysate was purified with nickel-nitrilotriacetic acid resin (Qiagen). ComK was overexpressed in E. coli strain BL21 (DE3) in a PLM303 vector (a derivative of pET-27a). The soluble fraction of maltose-binding protein fusion ComK in the E. coli extracts was purified over amylose resin (New England Biolabs) and then cleaved by the PreScission protease (GE Healthcare). After affinity purification, all proteins were further purified by anion-exchange chromatography (Source 15Q, GE Healthcare) and gel filtration chromatography (Superdex 200, GE Healthcare). Protein concentration was determined by UV spectroscopic measurement at 280 nm. In vitro degradation assays were reconstituted as described previously (7.Turgay K. Hamoen L.W. Venema G. Dubnau D. Genes Dev. 1997; 11: 119-128Crossref PubMed Scopus (168) Google Scholar, 8.Turgay K. Hahn J. Burghoorn J. Dubnau D. EMBO J. 1998; 17: 6730-6738Crossref PubMed Google Scholar). ClpC and ClpP were added at 4 μm final concentration. All MecA variants and ComK were added at 6 μm final concentration. All protein concentrations reported in this study refer to those of the monomers, regardless of the oligomerization states of the proteins. Size exclusion chromatography (Superdex 200, 10/30, GE Healthcare) was employed to examine potential protein-protein interaction. In all cases, proteins were incubated at 4 °C for at least 45 min to allow equilibration. The assay buffer contained 10 mm Tris, pH 8.0, 150 mm NaCl, and 2 mm dithiothreitol. The eluted fractions were subjected to SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Crystals were grown using the hanging-drop vapor diffusion method. Crystals of MecA-(121–218) were grown at 18 °C by mixing an equal volume of the protein (10 mg/ml) with reservoir solution containing 14% (w/v) polyethylene glycol 3350, 300 mm CaCl2, 4% ethylene glycol, and 0.1 m Bis-Tris, 4The abbreviations used are: Bis-Tris2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediolSADsingle-wavelength anomalous dispersionr.m.s.d.root mean square deviation. pH 6.2. Crystals of YpbH-(101–194) were grown at 18 °C by mixing an equal volume of the protein (10 mg/ml) with reservoir solution containing 1.85 m (NH4)2SO4, 2% polyethylene glycol 400, and 100 mm HEPES at pH 7.5. 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol single-wavelength anomalous dispersion root mean square deviation. MecA crystals were soaked into 200 mm NaI for 30 s and flash-frozen in the cold nitrogen stream. Single-wavelength anomalous dispersion (SAD) data were measured on a Rigaku FR-E system. All diffraction data were integrated and scaled using DENZO and SCALEPACK (11.Otwinowski Z. Minor W. Methods in Enzymology: Macromolecular Crystallography part A.in: Carter Jr., C.W. Sweet R.M. Vol. 276. Academic Press, New York1997: 307-326Google Scholar). Further processing was carried out using programs from the CCP4 package (12.Collaborative Computational Project, Number FourActa Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19667) Google Scholar). Full data collection and processing statistics are shown in Table 1. The heavy atom positions in the iodine-soaked mutant crystal were determined using SHELXD (13.Schneider T.R. Sheldrick G.M. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1570) Google Scholar). Heavy atom parameters were then refined, and initial phases were generated in the program PHASER (14.McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1590) Google Scholar) with the SAD experimental phasing module. Density modification, including solvent flattening and histogram matching, was performed using DM (15.Cowtan K. Main P. Acta Crystallogr. D. Biol. Crystallogr. 1998; 547: 487-493Crossref Scopus (308) Google Scholar). The initial model was built with BUCCANEER (16.Cowtan K. Acta Crystallogr. D. 2006; 62: 1002-1011Crossref PubMed Scopus (1415) Google Scholar). Additional missing residues in the auto-built model were added in COOT (17.Emsley P. Cowtan K. Acta Crystallogr. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22527) Google Scholar) manually. The structure was refined with PHENIX (18.Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3548) Google Scholar).TABLE 1Data collection and refinement statisticsProteinaValues in parentheses are for the highest resolution shell.MecA 121–218YpbH 104–194YpbH 101–194Data collectionX-ray sourceRigaku FR-ERigaku MM-007HFBL41XU, SPring-8Wavelength (Å)1.54181.54181.0000Space groupC2221P21I4122Unit cell (Å)a = 69.52, b = 107.27, c = 109.61a = 28.93, b = 71.66, c = 39.33, β = 95.35a = 162.06, c = 142.85Number of molecules in ASU426Resolution (outer shell) (Å)2.17 (2.25-2.17)2.09 (2.21-2.09)2.4 (2.49-2.4)RmergebRmerge = ΣhΣi|Ih,i − Ih|/ΣhΣiIh,i, where Ih is the mean intensity of the i observations of symmetry related reflections of h. (outer shell) (%)6.9 (43.1)7.9 (73.2)8.4 (78.2)I/σ (outer shell)36.75 (4.55)9.09 (2.6)25.3 (3.0)Completeness (outer shell) (%)99.4 (98.7)94.1 (88)99.7 (100)Redundancy (outer shell)8.5 (7.9)2.9 (2.7)8.1 (8.3)Wilson B factor (Å2)50.337.754.0Phasing protocolI-SADI-SADMRRefinementR/RfreecR = Σ|Fobs − Fcalc|/ΣFobs, where Fcalc is the calculated protein structure factor from the atomic model. Rfree was calculated with 5% of the reflections selected randomly. (%)22.2/27.921.8/30.423.0/27.0Number of atoms/B-factor:Protein3063/51.31422/43.14332/50.0Water86/52.960/45.8153/45.8Iodine35/69.114/62.0All3184/51.51496/43.44485/49.8Ramachandran plotMost favored (%)86.691.089.1Additional allowed (%)11.49.010.9Generously allowed (%)0.90.00.0Disallowed (%)1.10.00.0r.m.s.d.Bond distances (Å)0.010.010.007Bond angles (°)1.311.161.161Omega values (°)1.1030.8581.034a Values in parentheses are for the highest resolution shell.b Rmerge = ΣhΣi|Ih,i − Ih|/ΣhΣiIh,i, where Ih is the mean intensity of the i observations of symmetry related reflections of h.c R = Σ|Fobs − Fcalc|/ΣFobs, where Fcalc is the calculated protein structure factor from the atomic model. Rfree was calculated with 5% of the reflections selected randomly. Open table in a new tab YpbH-(104–194) crystals were soaked into 500 mm NaI for 60 s and flash-frozen in the cold nitrogen stream. SAD data were measured at a Rigaku MicroMax-007HF system and processed with the XDS package (14.McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1590) Google Scholar). YpbH-(101–194) native data were collected at the SPring-8 beamline BL41XU at the wavelength of 1.0 Å using MX-225 CCD detector, integrated, and scaled using DENZO and SCALEPACK (11.Otwinowski Z. Minor W. Methods in Enzymology: Macromolecular Crystallography part A.in: Carter Jr., C.W. Sweet R.M. Vol. 276. Academic Press, New York1997: 307-326Google Scholar). Full data collection and processing statistics are shown in Table 1. The iodine sites in the YpbH-(104–194) crystal were determined using SHELXD (13.Schneider T.R. Sheldrick G.M. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1570) Google Scholar). Heavy atom parameters were then refined, and initial phases were generated in the program PHASER (14.McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr. D. Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1590) Google Scholar). The real-space constraints, including solvent flattening, histogram matching, and non-crystallographic symmetry averaging, were applied using DM (15.Cowtan K. Main P. Acta Crystallogr. D. Biol. Crystallogr. 1998; 547: 487-493Crossref Scopus (308) Google Scholar). The initial model was built with BUCCANEER (16.Cowtan K. Acta Crystallogr. D. 2006; 62: 1002-1011Crossref PubMed Scopus (1415) Google Scholar). Additional missing residues in the auto-built model were added in COOT (17.Emsley P. Cowtan K. Acta Crystallogr. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22527) Google Scholar) manually. The YpbH-(104–194) model was used for molecular replacement with the program PHASER (17.Emsley P. Cowtan K. Acta Crystallogr. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22527) Google Scholar) into the YpbH-(101–194) crystal form. The YpbH-(101–194) structure was refined with PHENIX (18.Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. D. Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3548) Google Scholar). To decipher the mechanism by which MecA-(121–218) functions, we crystallized this domain and determined its three-dimensional structure at 2.2 Å resolution using iodine-based SAD (Table 1 and Fig. 1). The final atomic model of MecA-(121–218) contains amino acids 127–218, with the N-terminal 6 residues disordered. The compact structure comprises a central β-sheet of four antiparallel β-strands, which is surrounded by three α-helices (α1, α2, and α4) on one side and a single α-helix (α3) on the other side (Fig. 1A). There are four molecules of MecA-(121–218) in each asymmetric unit; these four molecules exhibit identical structure and can be very well superimposed with one another (Fig. 1B). Exhaustive search of the Protein Data Bank (PDB) using the program DALI (19.Holm L. Sander C. J. Mol. Biol. 1993; 233: 123-138Crossref PubMed Scopus (3545) Google Scholar) did not identify any continuous polypeptide or isolated domain that exhibits a similar fold as MecA-(121–218) (Fig. 1C). However, the search identified a large number of structures that display partial similarity to MecA-(121–218). The entry with the highest Z-score (a measure of structural similarity) of 5.3 is the structure of the protein alkyl-dihydroxyacetone phosphate synthase (20.Razeto A. Mattiroli F. Carpanelli E. Aliverti A. Pandini V. Coda A. Mattevi A. Structure. 2007; 15: 683-692Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), of which a discontinuous portion can be aligned with MecA-(121–218) with a root mean square deviation (r.m.s.d.) of 2.9 Å for 78 aligned Cα atoms (Fig. 2, A and B). The second closest entry, with a Z-score of 5.2, is the structure of a small, hypothetical protein from Thermotoga maritima (PDB code 2NZC) (Fig. 2, A and C). We conclude that MecA-(121–218) represents a previously uncharacterized fold (Fig. 1C). Given the sequence conservation of the C-terminal domain of MecA in other bacterial species (Fig. 3A), we propose that, in analogy to MecA, these proteins use their C-terminal domain as a degradation tag. We further posit that each of these proteins may facilitate assembly of the ClpCP protease in its cognate bacterial species. The advent of structural information on MecA-(121–218) greatly facilitated understanding of its function. Sequence alignment of 11 MecA homologues revealed four patches (I, II, III, and IV) of highly conserved amino acids (Fig. 3). Patch II contains mostly hydrophobic amino acids on strands β2 and β3, which likely contribute to the structural stability of the MecA fold. The other three patches are exposed on the surface and may play an important role in MecA function. To understand the biochemical function of these conserved residues, we generated eight full-length, mutant MecA proteins. For six of the eight mutant proteins, there is only a single point mutation in each protein. The other two each contains more than one point mutation. We purified these eight mutant proteins to homogeneity and evaluated their abilities to interact with ClpC and to mediate ClpCP degradation of themselves and ComK (Fig. 4, A–H). The same assays were performed for the wild-type MecA and are reported in the accompanying article (23.Mei Z. Wang F. Qi Y. Zhou Z. Hu Q. Li H. Wu J. Shi Y. J. Biol. Chem. 2009; 284: 34366-34375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). When compared with the wild-type protein, five mutations, E137R in patch I, E176R in patch III, I203A in patch IV, and T211A and H215A,F216A, had little or no impact on ClpCP degradation of the cognate MecA mutant and ComK (Fig. 4, A, B, F, G, and H). Subsequent characterization demonstrated that these mutant proteins retained the ability to oligomerize ClpC and to interact with the ClpCP protease (Fig. 4I). Two mutations, E184R in patch III and E198A,Y199A,K201D in patch IV, led to complete abrogation of ClpCP degradation of the associated MecA mutant and ComK (Fig. 4, C and E). Although still interacting with ClpC, these two mutations abolished the ability to mediate formation of a stable ClpC hexamer (data not shown), thus disrupting assembly of a functional ClpCP complex. Interestingly, the missense mutation R195D eliminated degradation of MecA itself but only weakened degradation of ComK (Fig. 4D). MecA R195D retained interaction with ClpC and weak interaction with the ClpCP protease. These results identified two regions of the MecA surface that are important for its function: one around Glu-184 in patch III and the other around the sequence EYGK in patch IV. These two regions are located on the opposite sides of the MecA fold (Fig. 3B), yet mutations in either region led to compromised ability of MecA to interact with ClpC, suggesting direct involvement of both regions in binding to ClpC. YpbH is a paralogue of MecA. It targets different substrate proteins such as Spx, but not ComK or ComS, to ClpCP for degradation (6.Nakano S. Zheng G. Nakano M.M. Zuber P. J. Bacteriol. 2002; 184: 3664-3670Crossref PubMed Scopus (86) Google Scholar). Similar to MecA-(121–218), YpbH-(101–194) is able to target the BIR1 domain (residues 31–145) of DIAP1 (21.Yan N. Wu J.W. Chai J. Li W. Shi Y. Nat. Struct. Mol. Biol. 2004; 11: 420-428Crossref PubMed Scopus (85) Google Scholar) for degradation as a heterologous fusion protein (supplemental Fig. 1). YpbH-(101–194) and MecA-(121–218) share sequence identity of 31% and sequence similarity of 63%. To examine whether YpbH-(101–194) is structurally similar to MecA-(121–218), we crystallized the C-terminal fragment of YpbH (residues 101–194) and determined its structure at 2.4 Å resolution. Due to the relatively moderate sequence similarity between MecA and YpbH, molecular replacement using MecA-(121–218) as the starting model did not result in an apparent solution. We had to solve the phase problem using the iodine-SAD data collected on crystals of YpbH residues 104–194. Upon completion of the structural refinement, it became obvious that YpbH-(101–194) shares the same fold as MecA-(121–218). There are six molecules of YpbH-(101–194) in each asymmetric unit. The structures of the six molecules are almost identical (Fig. 5A). Despite the moderate sequence similarity, YpbH-(101–194) can be closely superimposed with MecA-(121–218), with an r.m.s.d. of 1.16 Å over 75 aligned Cα atoms. The only apparent differences exist in some of the surface loop regions. The positions of the conserved amino acids are very similar in both structures. In particular, all of the crucial residues identified in MecA-(121–218) are located at nearly identical positions in YpbH-(101–194) (Fig. 5B). The structural conservation further suggests the important role of these residues. In this study, we reported the crystal structures of MecA-(121–218) and YpbH-(101–194). Despite the moderate sequence similarity, the two structures share an almost identical, previously uncharacterized α/β fold. The distinct α/β fold of MecA-(121–218) is reminiscent of ubiquitin (22.Vijay-Kumar S. Bugg C.E. Wilkinson K.D. Cook W.J. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 3582-3585Crossref PubMed Scopus (114) Google Scholar), but close examination revealed little structural similarity between these two degradation tags. BLAST search of the non-redundant protein data base resulted in more than 100 entries, mostly from Gram-positive bacteria, that share significant sequence similarity with MecA-(121–218), suggesting the existence of a large family of unique degradation tags with this novel fold. Preliminary biochemical characterization suggests the involvement of two separate surface regions that are located on opposing sides of the MecA C-terminal domain. It is possible that each MecA molecule may interact with two neighboring ClpC molecules through distinct interfaces, hence strengthening the interactions between neighboring ClpC molecules. Another possibility is that MecA binding to ClpC causes a conformational change in ClpC that allows oligomerization. The structural study reported in this study and biochemical characterizations reported in the accompanying article (23.Mei Z. Wang F. Qi Y. Zhou Z. Hu Q. Li H. Wu J. Shi Y. J. Biol. Chem. 2009; 284: 34366-34375Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) allow us to propose a working model of the MecA-ClpCP system, as discussed in the accompanying article. Despite these tantalizing clues, many important questions remain to be answered. At present, it is mechanistically unknown how MecA facilitates the oligomerization of ClpC and the assembly of ClpCP complex or how MecA binds and delivers the substrate protein to the ClpCP protease. The structure of a MecA-ClpC complex should provide critical insights into these processes. We thank N. Shimizu, T. Kumasaka, and S. Baba at the SPring-8 beamline BL41XU for on-site assistance. Download .pdf (.03 MB) Help with pdf files" @default.
• W2009593650 created "2016-06-24" @default.
• W2009593650 creator A5011823621 @default.
• W2009593650 creator A5013621410 @default.
• W2009593650 creator A5028323792 @default.
• W2009593650 creator A5042919729 @default.
• W2009593650 creator A5044785404 @default.
• W2009593650 creator A5046983295 @default.
• W2009593650 creator A5048395907 @default.
• W2009593650 creator A5070812231 @default.
• W2009593650 creator A5091493201 @default.
• W2009593650 date "2009-12-01" @default.
• W2009593650 modified "2023-09-27" @default.
• W2009593650 title "Crystal Structure of the MecA Degradation Tag" @default.
• W2009593650 cites W1967824952 @default.
• W2009593650 cites W1981024715 @default.
• W2009593650 cites W1983732912 @default.
• W2009593650 cites W1994286350 @default.
• W2009593650 cites W2001641653 @default.
• W2009593650 cites W2006395260 @default.
• W2009593650 cites W2014951926 @default.
• W2009593650 cites W2022058405 @default.
• W2009593650 cites W2030798502 @default.
• W2009593650 cites W2038363407 @default.
• W2009593650 cites W2038470214 @default.
• W2009593650 cites W2070342675 @default.
• W2009593650 cites W2077892784 @default.
• W2009593650 cites W2109184569 @default.
• W2009593650 cites W2115703815 @default.
• W2009593650 cites W2119612051 @default.
• W2009593650 cites W2126018533 @default.
• W2009593650 cites W2143815085 @default.
• W2009593650 cites W2144081223 @default.
• W2009593650 cites W2144908841 @default.
• W2009593650 cites W2146293937 @default.
• W2009593650 cites W2165327361 @default.
• W2009593650 doi "https://doi.org/10.1074/jbc.m109.053033" @default.
• W2009593650 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2797205" @default.
• W2009593650 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19801546" @default.
• W2009593650 hasPublicationYear "2009" @default.
• W2009593650 type Work @default.
• W2009593650 sameAs 2009593650 @default.
• W2009593650 citedByCount "13" @default.
• W2009593650 countsByYear W20095936502012 @default.
• W2009593650 countsByYear W20095936502013 @default.
• W2009593650 countsByYear W20095936502014 @default.
• W2009593650 countsByYear W20095936502018 @default.
• W2009593650 crossrefType "journal-article" @default.
• W2009593650 hasAuthorship W2009593650A5011823621 @default.
• W2009593650 hasAuthorship W2009593650A5013621410 @default.
• W2009593650 hasAuthorship W2009593650A5028323792 @default.
• W2009593650 hasAuthorship W2009593650A5042919729 @default.
• W2009593650 hasAuthorship W2009593650A5044785404 @default.
• W2009593650 hasAuthorship W2009593650A5046983295 @default.
• W2009593650 hasAuthorship W2009593650A5048395907 @default.
• W2009593650 hasAuthorship W2009593650A5070812231 @default.
• W2009593650 hasAuthorship W2009593650A5091493201 @default.
• W2009593650 hasBestOaLocation W20095936501 @default.
• W2009593650 hasConcept C115624301 @default.
• W2009593650 hasConcept C185592680 @default.
• W2009593650 hasConcept C199360897 @default.
• W2009593650 hasConcept C2779679103 @default.
• W2009593650 hasConcept C2781285689 @default.
• W2009593650 hasConcept C41008148 @default.
• W2009593650 hasConcept C70721500 @default.
• W2009593650 hasConcept C76155785 @default.
• W2009593650 hasConcept C8010536 @default.
• W2009593650 hasConcept C86803240 @default.
• W2009593650 hasConceptScore W2009593650C115624301 @default.
• W2009593650 hasConceptScore W2009593650C185592680 @default.
• W2009593650 hasConceptScore W2009593650C199360897 @default.
• W2009593650 hasConceptScore W2009593650C2779679103 @default.
• W2009593650 hasConceptScore W2009593650C2781285689 @default.
• W2009593650 hasConceptScore W2009593650C41008148 @default.
• W2009593650 hasConceptScore W2009593650C70721500 @default.
• W2009593650 hasConceptScore W2009593650C76155785 @default.
• W2009593650 hasConceptScore W2009593650C8010536 @default.
• W2009593650 hasConceptScore W2009593650C86803240 @default.
• W2009593650 hasIssue "49" @default.
• W2009593650 hasLocation W20095936501 @default.
• W2009593650 hasLocation W20095936502 @default.
• W2009593650 hasLocation W20095936503 @default.
• W2009593650 hasLocation W20095936504 @default.
• W2009593650 hasOpenAccess W2009593650 @default.
• W2009593650 hasPrimaryLocation W20095936501 @default.
• W2009593650 hasRelatedWork W1977362542 @default.
• W2009593650 hasRelatedWork W2039374736 @default.
• W2009593650 hasRelatedWork W2119429067 @default.
• W2009593650 hasRelatedWork W2159319742 @default.
• W2009593650 hasRelatedWork W2233838515 @default.
• W2009593650 hasRelatedWork W2360896545 @default.
• W2009593650 hasRelatedWork W2385092867 @default.
• W2009593650 hasRelatedWork W2924175997 @default.
• W2009593650 hasRelatedWork W381926892 @default.
• W2009593650 hasRelatedWork W2004703842 @default.
• W2009593650 hasVolume "284" @default.
• W2009593650 isParatext "false" @default.
• W2009593650 isRetracted "false" @default.

• next