plays a critical role in Mec1 activation (16, 17).
The helix bundles and upper loops are stabilized by the Leu-mediated hydrophobic interactions (Fig. 2D). The bottom loops in the Ddc2
dimer interface are stabilized by the intermolecular
Asn196-Arg197 salt bridges (Fig. 2D). In addition, Ddc2 also contributes to the Ddc2-Mec1
dimer interface and interacts with the a-solenoid
of another Mec1 by putative Arg260-Asp317, Lys263-
Asp322, and Lys263-Gln323 salt bridges (Fig. 2E).
The Mec1 kinase domain has the signatures of
an active conformation, indicating that it is an
intrinsically active enzyme. The 23-residue activation loop (2241HVDFDCLFEKGKRLPVPEIVPFR2263)
adopts a fully extended conformation that stabilizes the active site. The conserved residues of
both the Asp-Arg-His (DRH) and Asp-Phe-Gly
(DFG) motifs of Mec1 are positioned toward
the active site (Fig. 3A). The 23-residue PRD
(2315SIQKALKVLRNKIRGIDPQDGLV2337) is targeted by Dpb11 as a direct activator of Mec1 (16, 18).
The ka9b helix of Mec1 packs against the
activation loop and caps the catalytic site. Furthermore, the Met2312 residue in the hinge between ka9 and ka9b directly contacts with the
exposed Phe2244 of the DFG motif on the activation loop by hydrophobic interaction (Fig. 3B).
Thus, the Mec1 PRD could clamp the activation
loop through the Phe2244-Met2312 interaction
and block substrate entry into the active site. In
addition, five basic residues (Lys2318, Lys2321,
Lys2326, Arg2324, and Arg2328) condense in the
short ka9b helix of the PRD of Mec1. In human
ATR, the Lys2589 → Glu (K2589E) mutation in
the corresponding basic patch specifically
affects TopBP1 activation (16), highlighting a
critical role of the PRD in both ATR and Mec1
activation (Fig. 3B).
The structure clearly resolves the side-chain
densities in the majority of the N-terminal
a-solenoid of Mec1 (figs. S6 and S7). The N-
terminal Spiral region (residues 236 to 1121) is
followed by a linker (residues 1122 to 1148) that
runs along the surface of the FAT and kinase
domains, thereby connecting the Spiral region
to a region that we refer to as the “Bridge” (resi-
dues 1149 to 1409). Strikingly, two extended
helices of the N-terminal solenoid (HEAT 32R)
that are in close proximity to the active site
may generate steric hindrance for substrate
entry to the catalytic cavity of Mec1 (Fig. 3C).
A point mutation of Ser1333 in ATR creates a
hyperactive kinase (19), which is in proximity
to the linker region stabilizing the Bridge region
(Fig. 3, D and E). The critical regulatory sites
separating the S-phase and G2-phase functions
of both human ATR (Thr1566, Thr1578, Thr1589)
and yeast Mec1 (Phe1179) are coincidentally lo-
cated in the Bridge domain (20, 21). Interestingly,
the structural features of Bridge are shared by
ATM (8), DNA-PKcs (22), and m TOR (9) (figs. S13
and S14). In both ATM and DNA-PKcs, the Bridge
domain contains critical regulatory autophos-
phorylation sites (20). The m TOR Bridge domain
is responsible for Raptor binding (9). These ob-
servations all indicate that the conserved Bridge
domain critically regulates the kinase activity
of PIKKs and constitutes an important regu-
The kinase activation loop generally under-
goes substantial conformational changes during
catalysis that are intimately tied to activation
(23). The structure discloses that the PRD, LBE
domain, and FATC domain of Mec1 inhibit the
substrate binding and enzyme activation by en-
closing the catalytic and activation loops (Fig. 3,
A and B). The relief of such inhibition critically
relies on several specific activators, such as Dpb11.
The ATR/Mec1 activation domain (AAD) of these
activators is generally unstructured and contains
two critical aromatic residues, which may initiate
AAD binding to Mec1 (or, in humans, to ATR)
Although the known AADs share little sequence homology, the region around the conserved aromatic residues generally contains
several consecutive pairs of acid residues (Fig. 4A).
The Mec1 activation mechanism may be mediated
by both the conserved aromatic residues and
acidic patch in the specific AADs (Fig. 4B). In
agreement with this deduction, the sequence
surrounding a conserved aromatic residue in
AADs is essential for its ability to activate ATR
(26). ATR activation by AAD is salt-sensitive and
the activation effect is lost at higher salt concentration (27). The effects of the ATR K2589E
mutation [i.e., decreasing the TopBP1 association and abolishing ATR activation (16)] probably result from altering the PRD’s basic patch
for AAD binding.
When AAD binds to the PRD, the conserved
aromatic residues could first target the exposed
Phe2244-Met2312 interface and destabilize the hydrophobic interaction, which clamps the activation loop. Then, the acid patch surrounding the
aromatic residues could attract the basic patch
on the ka9b, increasing the overall binding affinity and the stability of the AAD-Mec1 complex (Fig. 4C). These multiple interactions could
further trigger substantial conformational changes
in the PRD and in the activation and catalytic
loops. The synergetic movements may expose
the substrate binding site and culminate in full
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The 3D cryo-EM density map reported in this paper has been
deposited in the EM Databank under accession number EMD-6708
and the corresponding model in the Protein Data Bank as PDB ID
5X6O. EM data were collected at the Center for Bio-imaging,
Institute of Biophysics, Chinese Academy of Sciences. We thank
G. Ji and X. Huang for technical help and support with electron
microscopy, X. Shen (University of Texas M. D. Anderson Cancer
Center) for providing the N-terminal Mec1 tagged yeast strain
MEC1-FLAG (MATa MEC1-FLAG his3 D1 leu2 D0 met15 D0 ura3 D0),
and S. Ma and H. Wu (Shanghai Chenglan Technology Co.) for help
with GPU computation. Supported by National Basic Research
Program grants 2014CB910700 and 2013CB910200 and National
Natural Science Foundation of China grants 31222017 and
31770808. Author contributions: X. W. ran the kinase assay, froze
the grids, performed the 3D reconstructions, analyzed the cryo-EM
reconstruction of the Mec1-Ddc2 complex, and built the models
with W. W. and T.R.; X.Z. and T. W. purified the Mec1-Ddc2; J.X.
prepared the Dpb11; Z.Z. and X. W. collected the cryo-EM data; and
G.C. designed experiments, analyzed data, and wrote the
manuscript with X. W.
Materials and Methods
Figs. S1 to S18
26 May 2017; accepted 1 November 2017