in the evening (39) (Fig. 1). In addition, formation
of the KaiBfs-KaiC complex requires ATPase activity
in the CI domain (44). However, the structural
basis for KaiBfs binding and the requirement of CI
domain ATPase activity in regulating these interactions has been unclear.
The phosphorylation status of the hexameric
CII ring is communicated to the CI ring via phosphoryl S431-dependent ring-stacking interactions
(27, 29). When stacked with CII, the CI ring captures KaiBfs to displace SasA from KaiC via competition for overlapping binding sites on CI (28, 39),
thereby turning off further stimulation of RpaA-mediated expression of class 1 genes (Fig. 1). While
bound by CI, KaiBfs recruits and inhibits KaiA (39).
The KaiBfs-KaiC complex also binds CikA to stimulate its phosphatase activity toward RpaA (21, 39),
repressing class 1 genes and activating class 2
genes (peak expression at dawn) (Fig. 1). Therefore, the KaiBfs-KaiC complex forms a hub that
regulates multiple interactions to disengage SasA,
inhibit KaiA, and activate CikA.
Thermophilic clock proteins from Thermosyn-
echococcus elongatus (fig. S1) are structurally and
functionally similar to those from S. elongatus
and more amenable for structural studies (45).
Using mutations that stabilize the rare fold of
KaiBfs from T. elongatus (39), here we present
high-resolution structures of the KaiBfs-KaiC hub
alone and in complex with domains of its interacting partners, KaiA and CikA. Together, these
structures explain the requirement for CI ATPase
activity and KaiB metamorphosis in the inactivation of KaiA and SasA and the recruitment of CikA.
Basis of SasA inactivation by KaiB
KaiB competes with SasA for an overlapping
binding site on KaiC (46, 47) at the CI domain
(28, 29) to regulate clock output signaling (39).
Previously, we designed a monomeric form of
the isolated CI domain, hereafter referred to as
CImono (fig. S1), that forms a stable complex with
KaiB (29). Here, we designed slightly truncated
forms to favor crystallization, one on the N
terminus of CImono, hereafter referred to as
CIcryst (fig. S1), and another at the C terminus
of a fold-switched KaiB mutant stabilized by
G89A and D91R mutations (KaiBfs), hereafter
referred to as KaiBfs-cryst (fig. S1). Y8A and Y94A
substitutions were also incorporated to enhance
the stability of KaiBfs-cryst. Using these optimized
constructs, we obtained a 1.8-Å resolution crystal
structure of the KaiBfs-cryst-CIcryst complex (Fig. 2A).
The interface predominantly comprises residues from the fold-switched C-terminal half of
KaiB (Fig. 2, B and C), explaining the requirement
of fold-switching for KaiB-KaiC complex formation (39). The interface centers on the B loop
of CIcryst, consistent with findings that deletion
of the B loop and alanyl substitutions of B-loop
residues E117, V118, and F122, as well as R130,
abolished or weakened KaiB-KaiC binding (28, 39).
Because KaiBfs has the same thioredoxin-like fold
as the domain of SasA that binds KaiC (39), their
modes of binding to the CI domain are likely to
be similar. Indeed, SasA binding to CImono was
shown to be weakened by the same set of mutations (28, 39). Thus, our KaiBfs-cryst-CIcryst
structure provides insights into the competition between
KaiB and SasA that explains the down-regulation of
SasA activity at night.
1176 17 MARCH 2017 • VOL 355 ISSUE 6330 sciencemag.org SCIENCE
Fig. 4. The mechanism of KaiA autoinhibition is revealed by the KaiAcryst-
KaiBfs-cryst-CIcryst complex. (A) The ternary KaiAcryst-KaiBfs-cryst-CIcryst com-
plex at 2.6 Å. Orange, KaiBfs-cryst; sky blue, CIcryst; orchid, KaiAcryst. (B) Zoomed-in
view of the boxed region in (A). Dashed lines: electrostatic interactions.
(C) Conformational changes of dimeric KaiA. NTD, N-terminal domain. Prime
symbols denote the other protomer within the dimer. (Left) Crystal structure
(PDB 5C5E) of KaiASe (purple) bound to KaiC CIISe peptides (dark cyan).
(Right) The KaiAcryst-KaiBfs-cryst-CIcryst complex, with same coloring scheme
as in (A). (Middle) Superposition of KaiASe (left) and KaiAcryst in complex
(right); only a5 helices and b6 strands are shown. (D) Fluorescence anisot-
ropy of 6IAF-labeled CII peptides (0.05 mM). Open circles, 0 mM KaiA, titration
with CIcryst and KaiBfs-cryst; triangles, 10 mM KaiA, titration with CIcryst; dia-
monds, 10 mM KaiA, titration with KaiBfs-cryst; squares, 10 mM KaiA, titration with
equal molar of KaiBfs-cryst and CIcryst. Error bars, SD from triplicates. (E) Size-
exclusion chromatography of ternary complex formation. Wild-type KaiA
(green, dashed); wild-type KaiB (red, dashed); CIcryst (orange, dashed); KaiA
+KaiB+CIcryst (green); L155A-KaiA+KaiB+CIcryst (cyan); K158A-KaiA+KaiB+CIcryst
(blue); D212A-KaiA+ KaiB+CIcryst (purple); D266A-KaiA+KaiB+CIcryst (black); and
N212A-D266A-KaiA+ KaiB+CIcryst (red). (F) Bioluminescence rhythms from
strains of S. elongatus: wt-kaiASe (blue), complemented with kaiASe (green),
L156A-kaiASe (purple), and kaiASe knockout (red). L156A of kaiASe is analogous
to L155A in kaiA. See fig. S1 and table S1 for construct details.