dimer. Models for TSPO-mediated ligand transport are somewhat constrained by the dimeric
structure seen in Rs TSPO crystals, making unlikely an internal pore-like transport mechanism (28)
through the monomer or transport via the tight
dimer interface (14). Thus, an external surface-transport mechanism appears worthy of further
Based on the structures of Rs TSPO, WT, and
A139T determined in this study and current understanding of TSPO function, we propose that
the phenotype of the A147T mutation in humans
arises from altered cholesterol binding and transport caused by the perturbed environment around
the CRAC site, which modifies the binding surface for cholesterol. Concurrently, the changes in
the tilt of the helices give rise to reduced binding
of other ligands, suggesting that the A147T mutation overall favors a lower-affinity conformation.
A proposed external surface-transport mechanism
that probably requires protein partners is consistent with the complex functional and regulatory
properties of this ancient multifaceted protein
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The atomic coordinates and structure factors have been
deposited in the Protein Data Bank under identification numbers
4UC3 (WT), 4UC1 (A139T_C2), and 4UC2 (A139T_P212121).
We thank S. Kaplan, X. Zeng (University of Texas, Houston),
and A. Yeliseev (NIH) for providing the expression plasmid;
R. M. Stroud, J. Lee, and members of the University of California,
San Francisco, Membrane Protein Expression Center (NIH grant
GM094625 to R. M. Stroud) for initial collaboration on the project
and training on the lipidic cubic phase crystallization method, as
well as continued support and discussion; M. Caffrey and D.-F. Li
for helpful discussion of selenomethionine phasing strategies
and supplying some noncommercial lipids; and A. Kruse and
C. Wang for suggestions on data collection and heavy-metal
soaking strategies. We also thank B. Atshaves, C. Najt, and L. Valls
for assistance in obtaining the fluorescence quenching data;
C. Hiser and N. Bowlby for technical assistance in protein
expression and purification and careful reading of the manuscript;
K. Parent for discussion on analyzing the EM map; and C. Ogata,
R. Sanishvili, N. Venugopalan, M. Becker, and S. Corcoran at
beamline 23ID at GM/CA CAT Advanced Photon Source, as well as
M. Soltis, C. Smith, and A. Cohen at BL12-2 at the Stanford
Synchrotron Radiation Light Source, for assistance and
consultation. Funding was provided by NIH grant GM26916
(to S.F.-M.) and Michigan State University Strategic Partnership
Grant, Mitochondrial Science and Medicine (to S.F.-M.).
GM/CA@APS has been funded in whole or in part with federal
funds from the National Cancer Institute (grant ACB-12002)
and the National Institute of General Medical Sciences (grant
AGM-12006). This research used resources of the Advanced
Photon Source, a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science by
Argonne National Laboratory under contract no. DE-AC02-06CH11357.
Materials and Methods
Figs. S1 to S9
29 August 2014; accepted 16 December 2014
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