ultimate goal of our studies is to gain a deeper understanding
of the molecular basis for important human diseasessuch
as sudden death, myocardial infarction, rotavirus infection
and HIV infection that cause substantial mortality and suffering.
The structural details revealed by our work may provide
clues for the design of more effective and safer medicines.
basic science level, we are intrigued by biological questions
at the interface between cell biology and structural biology.
How do membrane channels open and close? How are signals
transmitted across a cellular membrane when an extracellular
ligand binds to a membrane receptor? How do viruses attach
and enter host cells, replicate and assemble infectious
laboratory we use high resolution electron cryo-microscopy
(cryo-EM) and image processing to explore the molecular
design of large, multicomponent supramolecular assemblies.
Biological specimens are quick frozen in a physiological
state to preserve their native structure and functional
properties. A special advantage of this rapid-freezing method
is that we can trap and image dynamic states of functioning
macromolecular assemblies, such as open and closed states
of membrane channels and viruses actively transcribing RNA.
Three-dimensional density maps are obtained by digital image
processing of the high-resolution electron micrographs.
The rich detail in the maps reveals the structural organization
of complex biological structures that can be related to
the functional properties of such assemblies.
projects underway include the structure analysis of:
gap junction membrane channels: Cardiac gap junctions
electrically couple adjacent cells, thereby playing a critical
role in the normal coordinated depolarization of heart muscle
as well as cardiac arrhythmias causing sudden death. Our
goal is to determine the structure of these intercellular
channels at a level of detail that will allow us to understand
the molecular mechanism for channel gating. These channels
are formed by protein molecules called connexins. Gap junction
channels in the heart are formed by a 43 kDa protein called
a1 connexin, also
called Cx43. In our previous work, we used site-specific
peptide antibody labeling, protease cleavage, CD spectroscopy
and electron microscopy of 2D crystals to delineate the
membrane topology and quaternary structure of rat heart
gap junctions. We have now expressed a C-terminal truncation
mutant of a1 connexin
and grown 2D crystals of recombinant gap junctions. A projection
density map at 7-Å resolution revealed for the first
time a ring of a-helices that
line the aqueous pore of the channel and a second ring of
a-helices in close contact with
the membrane lipids. Our 3D structure provided the highest
resolution thus far achieved for a gap junction channel.
In fact, to our knowledge, this is the first example where
a mammalian polytopic membrane protein has been expressed
and examined by crystallographic methods. A novel technical
accomplishment of this work was that the structure analysis
was performed using only microgram amounts of material.
The dodecameric channel is formed by the end-to-end docking
of two hexamers, each of which displays 24 rods of density
in the membrane interior, consistent with an a-helical
conformation for the four transmembrane domains of each
connexin subunit. The transmembrane a-helical
rods merge with a double layer of protein density in the
extracellular vestibule, providing a tight seal to exclude
exchange of substances with the extracellular milieu. We
anticipate that this basic molecular design will be a common
folding motif for gap junction channels. High priority projects
include the comparison of open and closed channels and crystallization
of full-length a1
connexin in order to examine the structure of the C-terminal
Cardiovascular disease is the major cause of mortality in
the United States, primarily due to myocardial infarction
resulting from coronary atherosclerosis. Myocardial infarction
almost always results from formation of a thrombus at the
site of a coronary artery stenosis. A key event that stimulates
thrombus formation is platelet aggregation, which is mediated
by the prototypical integrin aIIbb3.
Integrins are a large family of heterodimeric transmembrane
receptor proteins, which modulate cell adhesion, such as
platelet aggregation, as well as other important biological
processes such as development, angiogenesis, wound healing,
and neoplastic transformation. Integrins accomplish these
diverse functions by mediating dynamic linkages between
extracellular adhesion molecules and the intracellular environment.
Integrin functions are regulated by transmembrane signaling,
which can occur as a consequence of binding extracellular
ligands (so-called “outside-in” signaling),
as well as the binding of molecules to the cytoplasmic domains
(so-called “inside-out” signaling). We used
cryo-EM and single particle image reconstruction to derive
a three-dimensional structure at 20 Å resolution of
the unliganded, low-affinity state of the human platelet
The large ectodomain and small cytoplasmic domains are connected
by a rod of density that we interpret as two parallel transmembrane
a-helices. The docking of the X-ray structure of the aVb3.
ectodomain into the electron cryomicroscopy map of aIIbb3.
requires hinge movements at linker regions between domains
in the crystal structure. Comparison of the putative high-
and low-affinity conformations reveals dramatic conformational
changes associated with integrin activation. The structural
details revealed by these studies will provide insight into
the molecular basis of integrin activation that will be
relevant for the rational design of drugs to modulate integrin
channels: Aquaporins are channels that play a critical
role in water transport across membranes. We previously
used electron cryo-crystallography to derive a 3D map of
AQP1 at 6-Å resolution. This is the first water channel
to be visualized in aqueous buffer within a lipid bilayer.
Each monomer is composed of six membrane-spanning, tilted
that form a barrel which encloses a vestibule leading to
the water-selective channel. The structure has an in-plane,
intramolecular pseudo-twofold axis of symmetry located in
the hydrophobic core of the bilayer, which is consistent
with the sequence-related N- and C-terminal halves of the
protein. This folding pattern represents a new motif for
the topology and design of membrane protein channels, and
is a simple and elegant solution to the problem of bidirectional
water transport across the bilayer. We are now focusing
on a-TIP (tonoplast intrinsic
protein isolated from the membranes of plant vacuoles) because
this aquaporin is gated by phosphorylation. Analysis of
tubular crystals shows that the a-helical
design of aquaporins is conserved between the plant and
animal kingdoms. Our aim is to delineate the conformational
changes associated with phosphorylation-dependent gating.
Rotavirus: Our goal is
to understand the molecular design and assembly pathway
of rotavirus, which is the major cause of human infant mortality
in developing countries. Using cryo-EM and icosahedral image
analysis, we showed for the first time that the structural
proteins in rotavirus are organized into three layers: an
outer capsid shell formed by 780 VP7 molecules and 60 VP4
hemagglutinin spikes; an inner capsid shell formed by 260,
pillar-shaped, VP6 trimers; and a core shell formed primarily
by VP2 as well as VP1 and VP3. The molecular design of rotavirus
is therefore substantially more complicated than simple
icosahedral viruses that have a single capsid shell. Essential
steps in infection are trypsin cleavage of VP4, the surface
hemagglutinin, and attachment to susceptible cells. Using
difference map analysis between native and spikeless particles,
we showed that VP4 extends ~110Å from the surface
of the virus and has a bi-lobed head. The spike penetrates
~90Å beneath the virion surface and interacts with
VP7 and VP6, that form the outer and inner capsid shells.
The structural features of VP4 revealed by our analysis
have implications for interactions of the virus with cell
surface receptors as well as for viral morphogenesis. We
have now decorated rotavirus with VP4-specific antibodies
that neutralize the virus. Difference maps between native
and Fab-decorated rotavirus allow us to map the binding
"footprint" of the antibodies on VP4. The maps
are of sufficient clarity that a canonical Fab molecular
structure can be docked within the EM density in order to
infer how the hypervariable loops of the Fab interact with
the surface of the hemagglutinin domains. By recording and
analyzing images of 1000's of virus particles, we aim to
delineate the subtle conformational changes that accompany
activation of VP4 by trypsin cleavage. Understanding this
process will provide specific insight into the pathogenesis
of rotavirus and may provide clues for vaccine and drug
Reovirus: Reovirus is in
the same family as rotavirus and has served as an important
model system for exploring viral assembly and pathogenesis.
We previously used cryo-EM and image analysis to examine
the 3D structure of intact virions, infectious subvirion
particles and core particles. A comparison of the 3D structures
and inspection of difference maps provided considerable
insight into the conformational changes and structural rearrangements
related to virus-cell interactions and viral pathogenesis.
Double-stranded RNA was discovered in reovirus, and we are
using the core particles as a system for exploring RNA transcription.
Cryo-EM and image analysis revealed that transcriptionally
active reovirus core particles display a rod-like density
centered within the channel formed by the pentameric guanylyltransferase,
which we attribute to exiting nascent (+) strand RNA. Comparison
of active and inactive particles reveal that transcription
is associated with dramatic reorganization of the ten dsRNA
genomic segments and small protein conformational changes.
The approach we have taken should be of general use for
exploring the molecular events associated with dynamic transcription/translation
Our goal is to understand the assembly pathway of retroviruses
and the structural rearrangements that occur with cleavage
of the Gag polyprotein, a step which confers infectivity.
We recently used cryo-EM and image analysis to examine the
native structure of immature, protease-deficient Moloney
murine leukemia virus (MuLV) and infectious, wild-type MuLV.
A common assumption in the literature has been that the
core of retorviruses is assembled with icosahedral symmetry.
In fact, our analysis disproved this concept. Instead, the
Gag subunits in the immature particle are packed in paracrystalline
domains, which become further disordered upon cleavage of
the polyprotein. Structural details about the transformations
that take place in the formation of infectious MuLV will
be important for a complete understanding of mechanisms
of retrovirus pathogenesis. To this end, our current aim
is to grow 2D crystals of expressed constructs of the matrix,
capsid and nucleocapsid proteins.