Large Is Fast, Small Is Tight: Determinants of Speed and Affinity in Subunit Capture by a Periplasmic Chaperone

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• 作者：Xiao Di Yu¹ ; ^&dagger ; ; Laura J. Fooks² ; ^&dagger ; ; Elham Moslehi-Mohebi² ; Vladimir M. Tischenko³ ; Gelareh Askarieh¹ ; Stefan D. Knight¹ ; Sheila MacIntyre² ; ^{s.macintyre@reading.ac.uk} ; Anton V. Zavialov¹ ; ⁴ ; ^{anton.zavialov@molbio.slu.se}
• 关键词：DSC ; donor strand complementation ; DSE ; donor strand exchange ; IEF ; isoelectrofocusing ; SPR ; surface plasmon resonance
• 刊名：Journal of Molecular Biology
• 出版年：2012
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：6 April, 2012
• 卷：417
• 期：4
• 页码：294-308
• 文件大小：1417 K

摘要

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 鈥榙onor strand complementation鈥? each subunit completes the immunoglobulin (Ig)-like fold of the neighboring subunit by donating the seventh 尾-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 m>Yersinia pestism> 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.