Molecular Biology - The Scripps Research Institute

172 MOLECULAR BIOLOGY 2005
olution structures of our transporters. Through these
united efforts, we will gain a broader understanding of
the structure and function of drug transporters that
cause MDR in cancer and bacterial infection.
Recently, we determined a new structure of the MDR
ATP-binding cassette transporter homolog MsbA in complex
with magnesium, adenosine diphosphate, inorganic
vanadate, and rough-chemotype lipopolysaccharide.
This structure supports a model involving a rigid-body
torque of the 2 transmembrane domains during ATP
hydrolysis and suggests a mechanism by which the
nucleotide-binding domain communicates with the transmembrane
domain. We propose a lipid “flip-flop” mechanism
in which the sugar groups are sequestered in
the chamber while the hydrophobic tails are dragged
through the lipid bilayer (Fig. 1). This posthydrolysis
Fig. 1. Proposed model for sequestering the polar sugar headgroup
of lipopolysaccharide (LPS) in the internal chamber of MsbA (for
clarity, only 1 LPS is shown). A, LPS initially binds to the elbow
helix as modeled onto the closed apo structure. B, Lipid headgroups
modeled to insert into the chamber of the apo closed structure.
C, As the transporter undergoes conformational changes related
to binding and hydrolysis of ATP, the headgroup is “flipped” within
the polar chamber while the LPS hydrocarbon chains are freely exposed
and dragged through the lipid bilayer. Both LPS and MsbA conformations
are modeled. D, LPS is presented to the outer leaflet of the
membrane as observed in this structure. Reprinted with permission
from Reyes, C.L., Chang, G. Science 308:1028, 2005.
structure of MsbA also gives insight into the possible
drug-binding sites for a number of cancer compounds.
We are continuing our x-ray structural studies of the
small MDR transporter EmrE and of other families of
bacterial MDR transporters to better understand the
molecular basis of the drug-proton antiport. The x-ray
structures of MsbA and EmrE are excellent models for
drug efflux systems that confer MDR to cancer cells and
infectious microorganisms.
PUBLICATIONS
Ma, C., Chang, G. Crystallography of the integral membrane protein EmrE from
Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 60:2399, 2004.
Reyes, C.L., Chang, G. Structure of the ABC transporter MsbA in complex with
ADP•vanadate and lipopolysaccharide. Science 308:1028, 2005.
Published by TSRI Press ®. ©Copyright 2005,
The Scripps Research Institute. All rights reserved.
Structure and Function of
Membrane-Bound Enzymes
C.D. Stout, H. Heaslet, M. Yamaguchi, V. Sundaresan,
L. Hunsicker-Wang, J. Chartron
One focus of our research is the structure and
function of transhydrogenase, an essential
enzyme of respiration in mitochondria and bacteria.
Transhydrogenase couples proton translocation
across the membrane with hydride transfer between
cofactors bound to soluble domains. We are determining
the structure of the enzyme in its membrane-bound
conformation and are studying the structures of the
soluble domains. For studies of enzyme function, we
are using biochemical methods and mutagenesis. Structural
studies entail x-ray crystallography, electron microscopy
studies done in collaboration with M. Yeager and
B. Carragher, Department of Cell Biology, and nuclear
magnetic resonance experiments done in collaboration
with J. Dyson, Department of Molecular Biology.
In collaboration with E.F. Johnson, Department of
Molecular Biology, and J.R. Halpert, University of Texas
Medical Branch, Galveston, Texas, we are studying highresolution
crystal structures of mammalian cytochrome
P450s. The P450s are monooxygenases involved in
the biosynthesis and oxidation of lipophilic molecules,
and they specifically metabolize a wide range of exogenous
compounds and drugs. More than 60 genes for
P450s occur in the human genome. We are studying
high-resolution structures and drug-bound complexes
of the human P450s 2C8, 2C9, 2A6, 3A4, and 1A2
and the rabbit P450s 2B4 and 2C5.
In collaboration with J.A. Fee, Department of Molecular
Biology, we are studying the structure and mechanism
of cytochrome ba 3 oxidase, the terminal enzyme
of respiration responsible for the reduction of molecular
oxygen to water. The high-resolution crystal structure
of the enzyme from a thermophilic bacterium has
been determined (Fig. 1). Crystallographic experiments,
in combination with mutagenesis and spectroscopy, are
being used to capture intermediates in the reaction
cycle and to define the pathways of proton translocation
to and from the active site within the membrane.
In parallel with these studies, we are developing
the application of nanodiscs for biophysical studies of
integral membrane proteins. These experiments are
being done in collaboration with S.G. Sligar, University
of Illinois, Urbana, Illinois, and M. Yeager, Department