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. 2014 Jun 16;289(31):21267–21275. doi: 10.1074/jbc.R114.579607

FIGURE 2.

FIGURE 2.

Evidence in support of a KaiCII binding mode by KaiB. A, six KaiB monomers forming a third layer on top of the KaiCII ring, consistent with cryo-EM density (16-Å resolution) and a more extensive buried surface at the KaiCII end based on molecular dynamics flexible fitting (MDFF) simulations when compared with a KaiCI binding mode (30). B, negative stain EM study of KaiC with a C-terminal His6 tail (identical to the construct used for the crystal structure determination; Fig. 1C), bound to KaiB and in the presence of 1.8-nm nickel-nitrilotriacetic acid Nanogold (left panel), bound to KaiB without gold (central panel), and applying a weighted Fourier difference electron density analysis (right panel) (55). The gold labeling approach supports binding of KaiB to KaiCII because of the co-localization of His tag, KaiB, and the gold cloud on top of the CII ring. C, cryo-EM density of the KaiC6B6 complex (30). The strongest density (blue regions) maps to the α-helical regions of KaiCI and KaiCII (Fig. 1C), which is supportive of KaiBs bound to the β-rich side of the KaiCII ring. D, SAXS investigation of the KaiBC complex with crystallographic models of KaiC hexamer (magenta, with one subunit highlighted in cyan) and KaiB dimers (green) modeled into the three-dimensional envelope (34). The observation that the protrusions produced by the C-terminal KaiCII tails (Fig. 1C) and by KaiB appear on the same side supports a KaiCII binding mode. E, side view of three KaiC subunits rendered in surface mode with blue patches indicating regions between subunits that are protected from HDX (left panel). HADDOCK-based models of the KaiC6B6 complex with KaiB monomers bound to either the CII (central panel) or the CI ring (right panel) (44). The former is favored based on MS-based ion mobility spectrometry-derived collisional cross-section data. F–H, native PAGE assays of the complex formations between full-length KaiC hexamer and KaiB (F), KaiCI hexamer and KaiB (G), and KaiCII and KaiB (H) (KaiCII alone does not form a stable hexamer) (29). KaiB binds to KaiC and to KaiCII but not to KaiCI (complex bands are marked by an asterisk).