of the resolved LAMTOR1 portions are in proximity to the LAMTOR2 and LAMTOR3 regions
that were previously thought to be potential effector interaction sites (Fig. 1A) (12). Therefore, we
tested whether the Rags might directly bind
LAMTOR through their CTDs. We performed mass
spectrometry after cross-linking to analyze the
heptameric LAMTOR-RagA T21N-RagC Q120L
complex (supplementary text and figs. S3 and S10).
We detected intra- and intermolecular contacts
among the LAMTOR subunits coherent with the
crystal structure. Consistent with a previous model
(23), we detected cross-linked peptides between
the RagA CTD and LAMTOR2 and between the
RagC CTD and LAMTOR3 but not between the G
domains of the Rags and LAMTOR components.
As deduced from the crystal structure of het-erodimeric Gtr1 and Gtr2, the CTDs of the Rags
are predicted to form a stable roadblock heterodimer (23, 24) and coimmunoprecipitate LAMTOR1
in vivo (23). Purified Rag CTDs and LAMTOR eluted
as a stable heptameric complex, as confirmed by
size exclusion chromatography and mass spectrometry (fig. S11). Thus, in vitro, the Rags CTDs were
sufficient to interact with the LAM TOR complex.
The crystal structure of LAMTOR with Rag
CTDs revealed CTDs binding to the predicted
region of LAMTOR2 and LAMTOR3 with additional contact surfaces provided by LAMTOR1
(Fig. 4, fig. S12, and table S1) (23). In contrast to
the pentameric LAMTOR structure, LAMTOR1
residues 47 to 64 form a helix in the heptameric
complex (Fig. 4 and figs. S13 and S14). We have
reconstituted the LAMTOR1HM cell line with a
version of LAMTOR1 in which N64, I66, and V68
(hereafter NIV) were mutated to alanines (fig. S1).
Immunoprecipitated LAMTOR1_ NIV.HA failed
to recruit the Rags or SLC38A9 despite restoring
the assembly of the LAMTOR complex (fig. S9B),
and colocalization of endogenous RagC with
LAMTOR1_NIV.HA (fig. S9C) was abolished.
The additional density observed at the C terminus of LAMTOR1 (residues 150 to 158) revealed
that the LVV motif contacts the N terminus of
LAMTOR2 (unstructured in the pentamer crystal)
that mediates most of the interactions between
LAMTOR and the RagA CTD. Residues 157 to 160,
adjacent to the LVV motif of LAMTOR1, come close
to the RagA CTD and may further contribute to this
interaction (figs. S12 to S14). Together with the
functional data and the cross-linking analysis (fig.
S10C), where three different pairs of cross-linked
peptides identified the N terminus of LAMTOR2
linked to the C terminus of RagA (K295 and K299),
a second essential region for Rags recruitment in
the LAMTOR was identified.
We superimposed the structure of the CTDs of
yeast Gtr1 and Gtr2 to the heptameric complex
(fig. S15). With some minor deviations, the CTDs
of Rag and Gtr proteins adopted a similar fold.
This defined the approximate orientation of
the G domains of the Rags for interaction with
mTORC1 (Fig. 4B). Targeting of Gtr1 and Gtr2
to the membrane in the absence of the Ego com-
plex is sufficient to promote TORC1 activity (15),
indicating that in yeast the Ego complex serves
as scaffold for the Gtr proteins. In contrast, mam-
malian LAMTOR exhibits GEF activity toward
RagA and RagB (1). Extrapolating from our hep-
tameric complex, the G domains and associated
nucleotide binding sites would be far away from
the LAMTOR components, raising the possibility
of an unusual or allosteric mechanism of nucleo-
tide exchange, if no other cellular components
are required for GEF activity.
Altering the Rag-LAMTOR interaction might
represent a previously unknown mechanism for
specific inhibition of m TORC1 (25). The identifi-
cation of two small structural motifs (LVV and
NIV) in LAMTOR1, necessary for Rag recruitment,
may be used to design compounds interfering
with this interaction.
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We thank J. Frankel and E. M. Nelsen for depositing 12G10 anti–
a-tubulin to the Developmental Studies Hybridoma Bank. We also
thank M. Gstaiger for providing the p TO-HA-STREPIIc-GW-FR T and
pCDNA5/FRT/TO/SH/GW constructs, I. Berger (European Molecular
Biology Laboratory Grenoble) for support on the design of the
synthetic LAMTOR gene construct and for the acceptor vector,
M. Nanao and D. Sanctis at the European Synchrotron Radiation
Facility for support with data acquisition, C. Herrmann for technical
support, K. Pansi for technical support and maintenance of the
insect cell culture, and W. Kabsch (Max Planck Institute Heidelberg,
Germany) and F. McCormick (University of California, San Francisco)
for valuable comments and discussion. The obtained data sets
from LAM TOR pentamer and LAM TOR with RagA and RagC C TDs
have been deposited under Protein Data Bank (PDB) IDs 6EHP and
6EHR, respectively. The work presented in this manuscript was
supported by grants from the Austrian Science Funds (FWF): P26682
to L. A. H. and P28975 to K.S.
Materials and Methods
Figs. S1 to S16
Tables S1 to S3
6 July 2017; accepted 11 September 2017
Published online 21 September 2017
D4 dopamine receptor high-resolution
structures enable the discovery of
Sheng Wang,1*† Daniel Wacker,1*† Anat Levit,2 Tao Che,1 Robin M. Betz,3,4,5,6
John D. McCorvy,1 A. J. Venkatakrishnan,3,4,5 Xi-Ping Huang,1 Ron O. Dror,3,4,5,6
Brian K. Shoichet,2† Bryan L. Roth1,7,8†
Dopamine receptors are implicated in the pathogenesis and treatment of nearly every
neuropsychiatric disorder. Although thousands of drugs interact with these receptors, our molecular
understanding of dopaminergic drug selectivity and design remains clouded. To illuminate dopamine
receptor structure, function, and ligand recognition, we determined crystal structures of the D4
dopamine receptor in its inactive state bound to the antipsychotic drug nemonapride, with
resolutions up to 1.95 angstroms. These structures suggest a mechanism for the control of
constitutive signaling, and their unusually high resolution enabled a structure-based campaign for
new agonists of the D4 dopamine receptor. The ability to efficiently exploit structure for specific
probe discovery—rapidly moving from elucidating receptor structure to discovering previously
unrecognized, selective agonists—testifies to the power of structure-based approaches.
Dopamine (DA) receptors are G protein– coupled receptors (GPCRs) that are ther- apeutic targets for treating schizophrenia, Parkinson’s disease, and drug abuse (1). DA receptors are divided into two subfamilies:
the Gas/olf-coupled D1-like family (e.g., D1 and D5
dopamine receptors) and the Gai/o-coupled D2-
like family [e.g., D2, D3, and D4 dopamine receptors (DRD2, DRD3, and DRD4)] (2). Given
that DRD4 has been implicated in many disorders, including attention deficit disorder,
metastatic progression, and erectile dysfunction,