Core Residue Replacements Cause Coiled-Coil Orientation Switching in Vitro and in Vivo: Structure-Function Correlations for Osmosensory Transporter ProP
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- 作者：Yonit Tsatskis ; Stanley C. Kwok ; Elisabeth Becker ; Chad Gill ; Michelle N. Smith ; Robert A. B. Keates ; Robert S. Hodges ; Janet M. Wood
- 刊名：Biochemistry
- 出版年：2008
- 出版时间：January 8, 2008
- 年：2008
- 卷：47
- 期：1
- 页码：60 - 72
- 全文大小：419K
- 年卷期：v.47,no.1(January 8, 2008)
- ISSN：1520-4995
文摘
Protein ProP acts as an osmosensory transporter in diverse bacteria. C-Terminal residues 468-497 of Escherichia coli ProP (ProPEc) form a four-heptad homodimeric -helical coiled coil. Arg 488,at a core heptad a position, causes it to assume an antiparallel orientation. Arg in the hydrophobic coreof coiled coils is destabilizing, but Arg 488 forms stabilizing interstrand salt bridges with Asp 475 andAsp 478. Mutation R488I destabilizes the coiled coil and elevates the osmotic pressure at which ProPEcactivates. It may switch the coiled-coil orientation to parallel by eliminating the salt bridges and increasingthe hydrophobicity of the core. In this study, mutations D475A and D478A, which disrupt the salt bridgeswithout increasing the hydrophobicity of the coiled-coil core, had the expected modest impacts on theosmotic activation of ProPEc. The five-heptad coiled coil of Agrobacterium tumefaciens ProP (ProPAt)has K498 and R505 at a positions. Mutation K498I had little effect on the osmotic activation of ProPAt,and ProPAt-R505I was activated only at high osmotic pressure; on the other hand, the double mutant wasrefractory to osmotic activation. Both a synthetic peptide corresponding to ProPAt residues 478-516 andits K498I variant maintained the antiparallel orientation. The single R505I substitution created an unstablecoiled coil with little orientation preference. Double mutation K498I/R505I switched the alignment, creatinga stable parallel coiled coil. In vivo cross-linking showed that the C-termini of ProPAt and ProPAt-K498I/R505I were antiparallel and parallel, respectively. Thus, the antiparallel orientation of the ProP coiledcoil is contingent on Arg in the hydrophobic core and interchain salt bridges. Two key amino acidreplacements can convert it to a stable parallel structure, in vitro and in vivo. An intermolecular antiparallelcoiled coil, present on only some orthologues, lowers the osmotic pressure required to activate ProP.Formation of a parallel coiled coil renders ProP inactive.