De Pinto, Vito

Guarino, Francesca

Impellizzeri, Agata

Magrì, Andrea

Messina, Angela

Reina, Simona

Tomasello, Marianna Flora

Elsevier - Publisher Connector 

Wednesday, February 6, 2013 539aPlatform: Membrane Protein Structure
& Function III
2766-Plat
Transport Dynamics in a Glutamate Transporter Homologue
Nurunisa Akyuz, Roger Altman, Scott Blanchard, Olga Boudker.
Weill Cornell Graduate School, New York, NY, USA.
Glutamate transporters are secondary active transporters, which mediate gluta-
mate uptake from the synaptic cleft into glial cells and neurons allowing mul-
tiple rounds of neural transmission and preventing glutamate-mediated
excitotoxicity. their structural dynamics is key to their function: during trans-
port cycle, they alternate between outward facing and inward facing states,
in which the substrate-binding sites are accessible from the extracellular and
intracellular solutions, respectively. Crystallographic studies of a bacterial
homologue GltPh revealed that this isomerization entails trans-membrane
movements of three discrete transport domains within a trimeric scaffold.
Here, using single-molecule fluorescence resonance energy transfer (smFRET)
imaging, we report real time observations of these movements for the first time.
We labeled GltPh with donor and acceptor fluorescent dye pairs at positions for
which the inter-dye distances differ in the outward and inward states. Our ob-
servations reveal FRET states with efficiencies consistent with those predicted
from the crystal structures. Remarkably, the rates of transitions between these
states are modulated by substrate binding, as well as by lipid surroundings.
2767-Plat
Structural Basis of the Alternate-Access Mechanism in a Bile Acid
Transporter
Xiaoming Zhou1, Elena J. Levin1, Matthias Quick1,2, Yaping Pan1,
Ruchika Sharma1, Ming Zhou1.
1Columbia University, New York, NY, USA, 2New York State Psychiatric
Institute, New York, NY, USA.
The apical sodium-dependent bile acid transporter (ASBT) utilizes the Naþ
concentration gradient to transport bile acids into cells. ASBT is essential in
cholesterol homeostasis and blocking of ASBT reduces cholesterol concentra-
tion in plasma. A recent structure of a bacterial homolog of ASBT in an
inward-open state advanced our understanding of the transporter, but a single
conformation does not resolve fundamental questions about the transport
mechanism. Here we present crystal structures of an ASBT homolog from
Yersinia frederiksenii, ASBTYf in a lipid environment, in an inward-open state
at 1.9 A˚ and an outward-open state at 2.5 A˚. The structures reveal how bile
acids could be transported across the membrane by a large rigid-body rotation
of a six-helix domain, suggest the existence of a substrate binding site critical
for translocation, and show how Naþ could be released into the cytosol. These
results build a structural framework for understanding the mechanism of bile
acid recognition and transport in ASBT.
2768-Plat
Molecular Details of SERCA Regulation by Phospholamban Revealed by
Paramagnetic Relaxation Enhancements and Solid-State NMR
Gianluigi Veglia, Martin Gustavsson, Vitaly V. Vostrikov, Raffaello Verardi.
University of Minnesota, Minneapolis, MN, USA.
Sarcoplasmic reticulum (SR) Ca2þ-ATPase (SERCA) ia a 110 kDa membrane
protein that regulates cardiomyocyte relaxation by transporting Ca2þ into the
SR. SERCA is regulated by PLN, a 52-resdiue membrane protein consisting
of a helical, inhibitory transmembrane domain and a regulatory cytoplasmic
domain. The cytoplasmic domain is in equilibrium between a folded T state
and an unfoled R state. Phosphorylation of PLN at S16 relieves the inhibition
of SERCA and, based on studies in animal models, mimicking the effects of
PLN phosphorylation is a promising path for the development of therapeutics
against heart failure.
Here we co-reconstituted SERCA and PLN in lipid bilayers under fully func-
tional conditions and utilized solid state NMR to probe the molecular mecha-
nisms of SERCA regulation. Asymmetric spin labeling and isotopic labeling
of SERCA, PLN and lipids in combination with magic angle spinning experi-
ments provided paramagnetic relaxation enhancements (PREs) that mapped
the structure of the membrane protein complex. PREs were combined with
oriented restraints from separated local field experiments, chemical shift pertur-
bations and torsion angles from backbone chemical shifts to determine a struc-
tural model of the PLN/SERCA complex. The transmembrane domain of PLN
remains anchored to the ATPase, while the cytoplasmic domain is membrane-
associated in the T state and interacts transiently with the phosphorylation and
nucleotide domains of SERCA in the R state. Phosphorylation increases the R
state population and the affinity of the R state for SERCA. These results explain
the non-inhibitory character of PLN mutants with increased R state populationand provide crucial information for future design of therapeutics to increase
SERCA function. In addition, the methodological advances described here fur-
ther open the landscape to conducting structural studies of membrane protein
complexes of molecular weights beyond 100 kDa.
2769-Plat
Charge Asymmetry in the Proteins of the Outer Membrane
Joanna S.G. Slusky, Roland L. Dunbrack.
Fox Chase Cancer Center, Philadelphia, PA, USA.
Outer membrane b-barrels (OMBBs) are the proteins found in the outer mem-
branes of bacteria, mitochondria and chloroplasts. There are thousands of
b-barrels reported in genomic databases with approximately 2-3% of the genes
in gram-negative bacteria encoding these proteins. These proteins have a wide
variety of biological functions including active and passive transport, cell
adhesion, catalysis, and structural anchoring. Of the non-redundant OMBB
structures in the Protein Data Bank, half have been solved over the past five
years. This influx of information provides new opportunities for understanding
the chemistry of these proteins.
Assessing this class of proteins bioinformatically, we have determined a dra-
matic asymmetry in the charge distribution of these proteins. For the
outward-facing amino acids of the beta barrel within regions of similar amino
acid density for both sides of the membrane, the extracellular side of the mem-
brane contains more than three times the number of charged amino acids as the
periplasmic side of the membrane. Moreover, the overall preference for amino
acid types to be in the outward leaflet of the membrane corresponds roughly
with hydrophobicity. This preference is irrespective the protein’s oligomeric
state and is demonstrably related to the structure of the outer membrane itself.
Finally, the charge asymmetry of proteins in the outer membrane has important
implications for how we understand the mechanism of outer membrane protein
insertion.
2770-Plat
Site-Directed Spin Labeling Reveals Multiple Modes for Regulating
Protein-Protein Interactions in Bacterial Outer Membrane Transport
Jessica Sarver, Audrey Mokdad, David S. Cafiso.
University Virginia, Charlottesville, VA, USA.
Structural transitions in three outer-membrane bacterial transport proteins have
been investigated using site-directed spin labeling and EPR spectroscopy. In
the vitamin B12 transporter BtuB and the ferric citrate transporter FecA,
EPR spectra reveal the existence of a substrate-dependent order-to-disorder
transition in the energy coupling motif (Ton box), which is localized at the
periplasmic surface of the transporter. In BtuB, the Ton box unfolds into the
periplasm, and in FecA, an N-terminal transcriptional domain disengages
from the Ton box. Both these events expose the Ton box and initiate interac-
tions with the inner membrane protein TonB, which drives transport. However,
this disorder transition is not observed in all transporters, and in the ferri-
chrome transporter FhuA the EPR spectra indicate that the Ton box is consti-
tutively disordered. Distance measurements using double electron-electron
resonance (DEER) indicate how the FhuA Ton box is regulated, and each
transporter regulates interactions with the inner membrane protein TonB using
a distinct molecular mechanism. This work indicates that a variety of mecha-
nisms may used to regulate protein-protein interactions in a single transporter
family.
2771-Plat
VDAC1 Topology in the Outer Mitochondrial Membrane: The Final
Answer
Francesca Guarino1, Marianna Flora Tomasello2, Andrea Magrı`1,
Agata Impellizzeri1, Simona Reina1, Angela Messina1, Vito De Pinto3.
1University of Catania and INBB, Catania, Italy, 2University of Catania,
Catania, Italy, 3university of Catania, Catania, Italy.
The first two authors equally contributed.
Voltage Dependent Anion Channel (VDAC) is a pore forming protein located
in outer mitochondrial membrane (1). Its lack is lethal in human (2) and it is
involved in cellular cross-talk, important in the apoptotic cascade (3). Its struc-
ture is a transmembrane b-barrel (4) organized in 19 b-strands and a N-terminal
a-helix probably important for gating (5). However the sidedness of VDAC
inside the membrane still remains unresolved (4). This issue is essential in order
to define the pore structural determinants interacting with soluble proteins. We
expressed, in HeLa cells, a recombinant hVDAC1 carrying C-terminal tag
including the hemagglutinin-tag (HA), the specific caspase 3/7 cleavage site
(DEVD) and 7 histidine residues (7xHis). DEVD amino acidic sequence can
be cleaved upon apoptosis induction by staurosporine. A complementary
Fluorescence Protease Protection assay was performed where cell protein are
exposed to protease digestion after plasma membrane permeabilization. If
540a Wednesday, February 6, 2013the C-terminus of VDAC1 is exposed to the cytosolic side, we expect to lose the
His tag upon DEVD cleavage. As result only the HA can be detected at
mitochondrial level. Conversely if the C-terminus is oriented towards the
IMS, the DEVD is not cleavable and both tags will be detected in mitochondria.
Our results by confocal microscopy and appropriate controls with other
membrane-oriented constructs showed that this strategy is able to define the
sidedness of the VDAC pore.
1) Shoshan-Barmatz, V. et al (2010) Mol Aspects Med 31, 227-285.
2) Huizing, M. et al (1994) 8924, 762-762.
3) Tomasello M.F. et al (2009) Cell Res. 19, 1363-1376.
4) De Pinto V. et al (2008) J. Bioenerg Biomemb 40, 139-147.
5) De Pinto V. et al (2007) Chembiochem 8, 744-756.
Acknowledgements to FIRB_PRIN Grants.
2772-Plat
Deciphering the Interaction of FLAP and 5LO
Ramakrishnan B.Kumar1, Hans Hebert2, Caroline Jegerscho¨ld1.
1Karolinska Institutet, Huddinge, Sweden, 2KTH, Huddinge, Sweden.
Leukotrienes are pro-inflammatory lipid mediators involved in chronic inflam-
matory diseases like asthma and atherosclerosis. Different enzymes and pro-
teins are involved in the leukotriene biosynthesis pathway that stems from
oxygenation of arachidonic acid (AA) by 5LO (5-lipoxygenase). FLAP (5-lip-
oxygenase activating protein) is an integral membrane protein, belonging to the
MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione
Metabolism) family. Till now, there is no prominent evidence for any biochem-
ical or enzymatic activity for FLAP, except involvement as an ‘‘activator’’ in
leukotriene biosynthesis. The hypothesis is that, on increase in intracellular
calcium concentration, 5LO moves from the cytosol to the nuclear membrane
and localizes near FLAP. The AA is then transferred from the nuclear
membrane to 5LO by homo-trimeric FLAP. Then 5LO converts the AA to
leukotriene A4. This interaction between FLAP and 5LO is ambiguous due
to intricate mechanisms which occur at the interface of the nuclear membrane
and also between an enzyme and an integral membrane protein with no
documented function. To understand the function and involvement of FLAP
in leukotriene biosynthesis in vitro, we employ soluble phospholipid bilayers
called ‘‘Nanodiscs’’ which mimic a membrane environment. The nanodisc
will act as a stable platform for structural and functional characterization of the
interaction between 5LO and FLAP. After initial studies of the Ca2þdependent
recruitment of 5LO to empty nanodiscs as well as the reconstitution of FLAP
into nanodiscs, the entire complex 5LO-FLAP-nanodisc can be targeted. We
employ biochemical assays to characterize the interactions and transmission
electron microscopy (single particle analysis) to create a 3D model of the func-
tional complex of 5LO and FLAP. Here, we portray our outcomes from the
above mentioned subprojects encompassed in understanding the interaction
of 5LO and FLAP.
2773-Plat
Membrane Anchoring Amplifies Osmosensing by the Inner Membrane
Receptor EnvZ
Ganesh Anand1,2, Jeremy Wang1, Linda Kenney2,3.
1Department of Biological Sciences, National University of Singapore,
Singapore, Singapore, 2Mechanobiology Institute, National University of
Singapore, Singapore, Singapore, 3Department of Microbiology and
Immunology, University of Illinois-Chicago, Chicago, IL, USA.
A fundamental question in signalling biology is how do receptors transmit the
effects of extracellular stimuli across membranes? Transmembrane segments
that connect a receptor extracellular domain to its intracellular domain are often
considered to be critical elements for transmitting these signals. The dynamics
of receptor signalling is not clearly understood, as there are few biophysical
probes to directly monitor transmembrane regions. Amide Deuterium
Exchange Mass Spectrometry (HDXMS) provides an excellent method to de-
scribe the conformational dynamics of membrane bound proteins. Our recently
published studies with the cytoplasmic domain of the bacterial inner membrane
osmosensor kinase EnvZ (EnvZc) have revealed the molecular basis for osmo-
sensing through modulation of helix-coil transitions in a critical four helix
bundle subdomain (Wang, LC, Morgan, L, Godakumbura, P, Kenney, LJ and
Anand, GS EMBO J. 31(11):2648-59, 2012). This study highlighted the impor-
tance of local folding-unfolding equilibria in the functioning of EnvZ signal-
ling. Most surprising was that a cytoplasmic deletion fragment of EnvZ
lacking the transmembrane helices was able to fully rescue the osmosensing
function in a strain lacking the envZ gene. This immediately raised the question
as to what is the function of membrane anchoring? Our preliminary results
indicate that the helix-coil transitions and associated local unfolding we ob-
served in EnvZc also occur in transmembrane helix regions in EnvZ. Confor-
mational dynamics of full-length EnvZ embedded within nanodiscs indicatethat the osmosensing core of EnvZ shows greater deuterium exchange com-
pared to EnvZc, suggesting a regulatory role of the membrane in modulating
the autokinase activity of EnvZ. Our results reveal that membrane anchoring
is responsible for ‘damping down’ the helix-coil transitions and likely increases
the sensitivity range for the receptor. These results are broadly relevant to all
receptors. Supported by Mechanobiology RCE and VA-1IOBX000372 to LJK.
Platform: Protein-Nucleic Acid Interactions II
2774-Plat
PICH:ADNATranslocase Essential for ResolvingAnaphase BridgedDNA
Andreas Biebricher1, Seiki Hirano2, Jacqueline Enzlin3, Erwin Peterman1,
Ian Hickson3, Gijs J.L. Wuite1.
1VU University Amsterdam, amsterdam, Netherlands, 2University of Oxford,
Oxford, United Kingdom, 3University of Copenhagen, Copenhagen,
Denmark.
PICH (PLK1-Interacting Checkpoint Helicase) is a recently identified member
of the RAD54 subgroup of SNF2 family proteins. PICH localizes to so-called
ultra-fine anaphase bridges (UFBs) in mitosis alongside a multi-protein com-
plex of DNA repair proteins including BLM, the Bloom’s syndrome gene prod-
uct. Very little is known about the function of PICH or how it is recruited
selectively to UFBs. Nevertheless, depletion of PICH results in genomic insta-
bility, including an elevated frequency of sister chromatid exchanges,
micronuclei and loss of heterozygosity. using a combination of microfluidics,
single-molecule fluorescence microscopy and optical tweezers, we have
defined the properties of PICH in an in vitro model of an anaphase bridge.
We show that PICH binds with a remarkably high affinity to dsDNA and
displays ATP-dependent dsDNA translocase activity. The application of
stretching forces to the DNA, which mimics the effects of the mitotic spindle
on a UFB, enhances the binding of PICH to dsDNA, but also serves to diminish
stretching-induced DNA melting. Based on our findings, we suggest that PICH
plays several roles in the development and processing of UFBs: (i) to recognize
and bind to dsDNA exposed by the mitotic spindle force-induced unwrapping
of nucleosomes, (ii) to help expel exclusively these unwrapped nucleosomes,
(iii) to stabilize stretched dsDNA, and (iv) to recruit the DNA repair machinery
required for UFB resolution in anaphase.
2775-Plat
Computational Study of RNA Translocation in a Hexameric Helicase
Wen Ma, Klaus Schulten.
Beckman Institute, University of Illinois, Urbana-Champaign, Urbana,
IL, USA.
Hexameric helicases are ATP-driven molecular motors that participate in im-
portant genetic processes. A particularly interesting helicase is E. coli transcrip-
tion termination factor Rho, which translocates towards the 3’-end of nascent
transcript. It is still an open question of how the ATP hydrolysis cycle is cou-
pled to RNA translocation in Rho. We present results from all-atom molecular
dynamics simulations that studied the conformation transitions and the corre-
sponding energy landscape in the hydrolysis cycle based on the available crys-
tal structure of Rho (Nathan D. Thomsen and James M. Berger. 2009, Cell,
139:523-534). We define collective variables involving the conformations of
key residues at the monomer-monomer interface in different ATP binding
states. The simulations reveal how interface conformational changes propagate
around the circular helicase and regulate RNA translocation along the central
channel in a collaborative manner. Monomers change their relative positions
along the translocation direction based on the ATP binding states. The sug-
gested allosteric inter-monomer communication in Rho is also revealed by net-
work analysis based on cross-correlation of protein motion. Lys326 in each
monomer is crucial in ratcheting the RNA and its movement is coupled to
the ATP binding state. Arg269 participates directly in linking monomer-
monomer communication with the ATP binding state. The simulations further
demonstrate the influence of different RNA sequences (poly(U) and poly(C)).
Our study, which elucidates the structure-function relationship in Rho, can be
extended to other hexameric helicase systems, such as E1 and DnaB, whose
crystal structures in complex with substrates are available.
2776-Plat
DNA Scanning Mechanism of a Translocating Motor Protein
Carolina Carrasco1, Neville Gilhooly2, Mark S. Dillingham2,
Fernando Moreno-Herrero1.
1Centro Nacional de Biotecnologı´a, Madrid, Spain, 2University of Bristol,
Bristol, United Kingdom.
Unrepaired DNA breaks can lead to genomic instability or cell death. For repair
by the ubiquitous homologous recombination pathway, broken ends are first
processed to produce a 3’-ssDNA overhang. In Bacillus subtilis, this reaction


VDAC1 Topology in the Outer Mitochondrial Membrane: The Final Answer 

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VDAC1 Topology in the Outer Mitochondrial Membrane: The Final Answer

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