title: Slippery substrates impair function of a bacterial protease ATPase by unbalancing translocation versus exit.
abstract: BACKGROUND
  ATP-dependent proteases translocate and unfold their substrates.
  
  
  RESULTS
  A human virus sequence with only Gly and Ala residues causes similar dysfunctions of eukaryotic and prokaryotic protease motors: unfolding failure.
  
  
  CONCLUSIONS
  Sequences with amino acids of simple shape and small size impair unfolding of contiguous stable domains.
  
  
  CONCLUSIONS
  Compartmented ATP-dependent proteases of diverse origin share conserved principles of interaction between translocase/effector and substrate/recipient. ATP-dependent proteases engage, translocate, and unfold substrate proteins. A sequence with only Gly and Ala residues (glycine-alanine repeat; GAr) encoded by the Epstein-Barr virus of humans inhibits eukaryotic proteasome activity. It causes the ATPase translocase to slip on its protein track, stalling unfolding and interrupting degradation. The bacterial protease ClpXP is structurally simpler than the proteasome but has related elements: a regulatory ATPase complex (ClpX) and associated proteolytic chamber (ClpP). In this study, GAr sequences were found to impair ClpXP function much as in proteasomes. Stalling depended on interaction between a GAr and a suitably spaced and positioned folded domain resistant to mechanical unfolding. Persistent unfolding failure results in the interruption of degradation and the production of partial degradation products that include the resistant domain. The capacity of various sequences to cause unfolding failure was investigated. Among those tested, a GAr was most effective, implying that viral selection had optimized processivity failure. More generally, amino acids of simple shape and small size promoted unfolding failure. The ClpX ATPase is a homohexamer. Partial degradation products could exit the complex through transient gaps between the ClpX monomers or, alternatively, by backing out. Production of intermediates by diverse topological forms of the hexamer was shown to be similar, excluding lateral escape. In principle, a GAr could interrupt degradation because 1) the translocase thrusts forward less effectively or because 2) the translocase retains substrate less well when resetting between forward strokes. Kinetic analysis showed that the predominant effect was through the second of these mechanisms.
authors.affiliation: Department of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.
authors.forename: Priscilla Hiu-Mei
authors.initials: PH
authors.lastname: Too
authors.forename: Jenny
authors.initials: J
authors.lastname: Erales
authors.forename: Joana Danica
authors.initials: JD
authors.lastname: Simen
authors.forename: Antonija
authors.initials: A
authors.lastname: Marjanovic
authors.forename: Philip
authors.initials: P
authors.lastname: Coffino
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D064211:Connectin; D049071:Endopeptidase Clp / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D058767:Protein Unfolding; D059748:Proteolysis; D020449:Repetitive Sequences, Amino Acid; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry
chemical_list: D064211:Connectin; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D009124:Muscle Proteins; D010446:Peptide Fragments; C577285:TTN protein, human; D013762:Tetrahydrofolate Dehydrogenase; D011494:Protein Kinases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities
keywords: ATP; ATP-dependent Protease; ATPases; Protease; Proteasome; Protein Degradation; Protein Turnover; Translocation; Unfolding