Sammanfattning

The chaperone/usher pathway assembles surface virulence organelles of Gram-negative bacteria, consisting of fibers of linearly polymerized protein subunits. Fiber subunits are connected through 'donor strand complementation': each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh beta-strand in trans. Whereas the folding of Ig domains is a fast first-order process, folding of Ig modules into the fiber conformation is a slow second-order process. Periplasmic chaperones separate this process in two parts by forming transient complexes with subunits. Interactions between chaperones and subunits are also based on the principle of donor strand complementation. In this study, we have performed mutagenesis of the binding motifs of the Caf1M chaperone and Caf1 capsular subunit from Yersinia pestis and analyzed the effect of the mutations on the structure, stability, and kinetics of Caf1M-Caf1 and Caf1-Caf1 interactions. The results suggest that a large hydrophobic effect combined with extensive main-chain hydrogen bonding enables Caf1M to rapidly bind an early folding intermediate of Caf1 and direct its partial folding. The switch from the Caf1M-Caf1 contact to the less hydrophobic, but considerably tighter and less dynamic Caf1-Caf1 contact occurs via the zip-out-zip-in donor strand exchange pathway with pocket 5 acting as the initiation site. Based on these findings, Caf1M was engineered to bind Caf1 faster, tighter, or both faster and tighter. To our knowledge, this is the first successful attempt to rationally design an assembly chaperone with improved chaperone function. (C) 2012 Elsevier Ltd. All rights reserved.

Nyckelord

assembly; folding; secretion; structure; protein design

Publicerad i

Journal of Molecular Biology
2012, Volym: 417, nummer: 4, sidor: 294-308
Utgivare: ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

SLU författare

• Yu, Xiaodi

  • Institutionen för molekylärbiologi, Sveriges lantbruksuniversitet
    

• Askarieh, Glareh

  • Institutionen för molekylärbiologi, Sveriges lantbruksuniversitet
    

• Knight, Stefan David

  • Institutionen för molekylärbiologi, Sveriges lantbruksuniversitet
    

• Zavialov, Anton

  • Institutionen för molekylärbiologi, Sveriges lantbruksuniversitet
    
  • Turun Yliopisto

UKÄ forskningsämne

Cellbiologi
Mikrobiologi


Publikationens identifierare

DOI: https://doi.org/10.1016/j.jmb.2012.01.020

Permanent länk till denna sida (URI)

https://res.slu.se/id/publ/42985