期刊
STRUCTURE
卷 19, 期 2, 页码 265-276出版社
CELL PRESS
DOI: 10.1016/j.str.2010.12.005
关键词
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资金
- Howard Hughes Medical Institute Funding Source: Medline
- NIGMS NIH HHS [R01 GM019301, R01 GM034921, R01 GM019301-40] Funding Source: Medline
PKA holoenzymes containing two catalytic (C) subunits and a regulatory (R) subunit dimer are activated cooperatively by cAMP. While cooperativity involves the two tandem cAMP binding domains in each R-subunit, additional cooperativity is associated with the tetramer. Of critical importance is the flexible linker in R that contains an inhibitor site (IS). While the IS becomes ordered in the R:C heterodimer, the overall conformation of the tetramer is mediated largely by the N-Linker that connects the D/D domain to the IS. To understand how the N-Linker contributes to assembly of tetrameric holoenzymes, we engineered a monomeric RI alpha that contains most of the N-Linker, RI alpha(73-244), and crystallized a holoenzyme complex. Part of the N-linker is now ordered by interactions with a symmetry-related dimer. This complex of two symmetry-related dimers forms a tetramer that reveals novel mechanisms for allosteric regulation and has many features associated with full-length holoenzyme. A model of the tetrameric holoenzyme, based on this structure, is consistent with previous small angle X-ray and neutron scattering data, and is validated with new SAXS data and with an RI alpha mutation localized to a novel interface unique to the tetramer.
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