Thursday, February 25, 2021
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924-Plat
Binding Mode of SARS-CoV2 Fusion Peptide to Human Cellular
Membranes
Defne Gorgun1,2, Muyun Lihan1,2, Karan Kapoor2,3, Emad Tajkhorshid1,2,3.
1
Center for Biophysics and Quantitative Biology, University of Illinois at
Urbana-Champaign, Urbana, IL, USA, 2NIH Center for Macromolecular
Modeling and Bioinformatics, Beckman Institute, Urbana, IL, USA,
3
Department of Biochemistry, University of Illinois at Urbana-Champaign,
Urbana, IL, USA.
Infection of human cells by the SARS-CoV2 relies on its binding to a specific
receptor and subsequent fusion of the viral and the host cell membranes. The
fusion peptide (FP), a short peptide segment in the spike protein, plays a central
role in the initial penetration of the virus into the host cell membrane, followed
by the fusion of the two membranes. Here, we use an extensive array of molec-
ular dynamics (MD) simulations taking advantage of the Highly Mobile Mem-
brane Mimetic (HMMM) model, to investigate the interaction of the SARS-
CoV2 FP with a lipid bilayer representing human cellular membranes at an
atomic level, and to characterize its membrane-bound form. Six independent
systems were generated by changing the initial positioning and orientation of
the FP with regard to the membrane, and each system was simulated in five in-
dependent replicas. In 60% of the simulations, FPs reached a stably membrane-
bound configuration where the peptide deeply penetrated into the membrane.
Clustering of the results reveals two major membrane binding modes, the
helix-binding mode and the loop-binding mode. Taken into account sequence
conservation among the viral FPs and the results of mutagenesis studies estab-
lishing the role of specific residues in the helical portion of the FP in membrane
association, we propose that the helix-binding mode is the biologically relevant
form. In this binding mode, the helix is stabilized in an oblique angle with
respect to the membrane with the N-terminus tilted towards the membrane
core. Analysis of the FP-lipid interactions shows the involvement of the fusion
active core residues of the helix in membrane binding. These results shed light
on a key step involved in the process of viral infection by SARS-CoV2 with
potential use in designing novel inhibitors.
925-Plat
Describing Antifungal Drug-Sterol Interactions Inside the Membrane: The
Role of Dynamics
Kevin J. Cheng1, Ashley M. De Lio2, Agnieszka Lewandowska2,
Corinne P. Soutar2, Martin D. Burke2, Chad M. Rienstra3,
Taras V. Pogorelov2.
1 Although the molecular mechanism is not completely deciphered yet, lipid-
Center for Biophysics and Quantitative Biology, University of Illinois at
Urbana-Champaign, Champaign, IL, USA, 2Department of Chemistry,
University of Illinois at Urbana-Champaign, Champaign, IL, USA,
3 are present. By means of atomic force microscopy (AFM), we investigated
Department of Chemistry, University of Wisconsin Madison, Madison, WI,
USA.
Amphotericin (AmB) is a potent and effective drug to combat life-threatening
fungal infections. It, however, remains extremely toxic to human cells. In worst
case scenarios, high dosages can lead to life-threatening complications such as
kidney damage or heart failure. It has been shown that its anti-fungal activity
originates from the binding to ergosterol in fungal cells. Since ergosterol shares
many chemical similarities with human cholesterol, the toxicity effects is pur-
ported to come from AmB’s non-specific binding to both sterols. A recent
experiment-based model demonstrated that AmB forms an aggregate structure
and extracts sterols; effectively acting as a sponge. In order to develop AmB de-
rivatives that maintain potency while reducing toxicity, a comprehensive under-
standing of the drug’s mechanism is necessary. The goal of our study is therefore
to characterize how both cholesterol and ergosterol bind to AmB at the atomistic
level. We are addressing this with all-atom molecular dynamics simulations using
an NMR-derived AmB lattice structure. We utilize the highly mobile-mimetic
(HMMM) model with an updated, cholesterol compatible in-silico solvent that
replaces phospholipid tails that thereby accelerates lipid dynamics. From these
simulations, we characterized distinct binding modes of each sterol with AmB,
while reporting key intermolecular interactions. We furthermore report on the
conformational states between bound and unbound sterol as well dynamics of
the AmB sponge. This study will inform future experiments that focus on the
design of AmB derivatives to kill yeast cells and not human cells. Such deriva-
tives have the potential to save lives by reducing AmB toxicity.
926-Plat
Ebola Virus Glycoprotein Interacts with Cholesterol to Enhance
Membrane Fusion and Cell Entry
Jinwoo Lee1, Alex J.B. Kreutzberger2, Laura Odongo2,
Elizabeth A. Nelson3, David Nyenhuis4, Volker Kiessling2, Binyong Liang2,
David S. Cafiso4, Judith M. White3, Lukas K. Tamm2.
191a
Department of Chemistry & Biochemistry, University of Maryland, College
Park, MD, USA, 2Department of Molecular Physiology and Biological
Physics, University of Virginia, Charlottesville, VA, USA, 3Department of
Cell Biology, University of Virginia, Charlottesville, VA, USA, 4Department
of Chemistry, University of Virginia, Charlottesville, VA, USA.
Cholesterol serves critical roles in enveloped virus fusion by modulating mem-
brane biophysical properties. The glycoprotein (GP) of Ebola virus (EBOV)
promotes fusion in the endosome after internalization of the virus by macropino-
cytosis. Fusion of EBOV requires the endosomal cholesterol transporter NPC1,
but the role of cholesterol in EBOV fusion is unclear. Here we show that the mem-
brane proximal external region and transmembrane domain (MPER/TM) of GP
interacts with cholesterol, and that cell entry of virus-like particles (VLPs) and
GP2-mediated fusion to supported membranes are enhanced with increasing
cholesterol in EBOV membranes. NMR-based experiments revealed that several
glycines, notably G660, in GP2 TM are critical for interaction with cholesterol.
Moreover, G660L MPER/TM shows a more open angle between MPER and
TM domains compared to WT. G660L particles show a higher probability of
hemifusion (vs. full fusion), correlating with a lower efficiency of cell entry by
G660L vs. WT VLPs. VLPs produced under cholesterol-lowering statin condi-
tions show less entry than VLPs from mock-treated cells. We propose that
cholesterol-TM interactions affect structural features of GP2 thereby facilitating
fusion and cell entry and that statins may be used as part of EBOV treatments due
to their ability to lower cholesterol in the viral membrane.
927-Plat
Nanomechanical Characterization of the Synergism of Antimicrobial
Peptides PGLa and Mag2 for Lipid Membrane Disruption
Fabio Perissinotto1, Sebastien Janel1, Javier Lopez-Alonso1,
Vincent Dupres1, Burkhard Bechinger2, Frank Lafont1,
Lorena Redonda-Morata1.
1
Institut Pasteur Lille, Center for Infection and Immunity of Lille, Lille
University, Lille, France, 2Membrane Biophysics and NMR, CNRS,
University of Strasbourg, Strasbourg, France.
Antimicrobial peptides (AMPs) are amphiphilic molecules used by eukary-
otic organisms as first defensive mechanism against infections. There is an
increasing interest in the study of these peptides, considered promising alter-
natives to antibiotics for the treatment of multi-resistance pathogens. AMPs
target the bacterial membrane leading to membrane lysis by pore formation
and bilayer disruption. Here we studied PGLa and Magainin 2 (Mag2), which
are known for a particular synergistic behavior in their antimicrobial activity.
mediated interactions are responsible for such synergism, directly correlated
to a change in the peptide orientation into the membrane when both peptides
the effect of both individual peptides compared with their equimolar mixture
on the structure, dynamics and nanomechanical properties of model lipid bi-
layers mimicking gram-negative bacterial cell membrane. Both individual
peptides and the mixture led to a lateral expansion and a drastic thinning
of the lipid bilayer, pointing out a deep structural reorganization of the mem-
brane. AFM-based Quantitative nanomechanical mapping allowed us to
investigate the membrane elastic constants and the resistance to failure.
The mechanical rupture of the lipid bilayer by the AFM tip has been used
extensively as a hallmark of its mechanical stability, usually by measuring
the forces required to break the bilayer probed at different velocities. Inter-
estingly, the elasticity of the membrane does not undergo significant changes
upon the addition of the peptides. However, we observed a major increase in
the breakthrough force, especially in the equimolar mixture of peptides. This
particular nanomechanical behavior may indicate a complex synergistic
mechanism between PGla and Mag2 to drive membrane disruption.
Platform: Ligand-gated Channels
928-Plat
Structural Basis of Functional Transitions in Mammalian NMDA
Receptors
Tsung-Han Chou1, Nami Tajima2, Annabel Romero-Hernandez1,
Hiro Furukawa1.
1
Keck Laboratory of Structural Biology, Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY, USA, 2Physiology and Biophysics, Case Western
Reserve University, Cleveland, OH, USA.
N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that
mediate calcium influx in excitatory synapses and play a crucial role in syn-
aptic plasticity, learning, and memory function. In mammalian brains, the ma-
jority of NMDARs are heterotetramers composed of two copies each of
192a Thursday, February 25, 2021
GluN1 and GluN2 subunits to form a functional channel. Activation of
NMDARs requires simultaneous binding of glycine and glutamate due to its
heterotetrameric assembly. Conversely, competitive inhibition of NMDARs
can be evoked by binding of either a glycine antagonist on GluN1 or a gluta-
mate antagonist on GluN2. Despite the extensive efforts on unraveling the
mechanism of channel gating and competitive inhibition of NMDARs, it re-
mains elusive due to the lack of detailed structural information. Here, we pre-
sent multiple cryo-EM structures in distinct ligand states at 4 Å or better. The
structures elucidate conformational transitions and inter-subunit/domain reor-
ientations across different ligand binding states, providing great insights of
ligand-gating and subunit dependent competitive inhibition. Also, the struc-
tures reveal that activation and competitive inhibition of NMDARs is
controlled by the tension of the linker between the ligand-binding domain
and the transmembrane ion channel of the GluN2 subunit. Our results provide
detailed mechanistic insights into NMDAR pharmacology, activation, and
inhibition.
929-Plat
Heterogeneous Populations of Ligand-Gated Ion Channel, GLIC,
Revealed by Cryo-Electron Microscopy and Simulations
Urska Rovsnik1, Yuxuan Zhuang1, Bjorn Forsberg1, Marta Carroni1,
Linnea Axelsson1, Christian Blau2, Rebecca J. Howard1, Erik R. Lindahl1.
1
Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden,
2
Theoretical and Computational Biophysics, KTH, Solna, Sweden.
Ligand-gated ion channels are critical mediators of electrochemical signal
transduction across evolution. Biophysical and pharmacological develop-
ment in this family relies on high-quality structural data in multiple, subtly
distinct functional states; however, structural data remain limited, particu-
larly for the native closed state. Here, we report cryo-electron microscopy
structures of the proton-gated Gloeobacter violaceus ligand-gated ion chan-
nel (GLIC) under resting and activating conditions (pH 7, 5, and 3). Pre-
dominant classes in all three conditions featured constricted pores,
consistent with a low maximal open probability; nonetheless, densities in
predicted gating regions were better defined at low pH, implicating key
electrostatic networks in channel activation. Molecular dynamics simula-
tions further reflected domain stabilization at lower pH, and greater align-
ment with previous X-ray structures. Further analysis of datasets collected
in each condition enabled the reconstruction of minority classes, including
an alternative low-pH state with an expanded pore. In addition to providing
new structures of closed GLIC in multiple conditions without crystalliza-
tion, these results offer dynamic insight into an ion channel’s heterogeneous
resting state, with activating conditions condensing the energy landscape on
a pathway towards gating.
930-Plat
Structures of Human Proton-Activated Chloride Channel (PAC) Reveal
Mechanism of pH Sensing and Gating
James Osei-Owusu1, Zheng Ruan2, Juan Du2, Wei Lu2, Zhaozhu Qiu1.
1
Physiology, Johns Hopkins University, Baltimore, MD, USA, 2Van Andel
Research Institute, Grand Rapids, MI, USA.
Severe local acidosis causes tissue damage and pain, and is one of the hall-
marks of many diseases including ischemia, cancer, and inflammation. A
family of chloride channels activated by extracellular acidic pH has been
implicated in acidosis-induced cell death by mediating Cl¯ influx and subse-
quent cell swelling. PAC currents are widespread and display characteristic
biophysical properties, including strong outwardly rectifying current-voltage
(I-V) relationship, time-dependent activation at positive membrane potentials,
and anion permeability sequence of I¯ > Br¯ > Cl¯. The molecular identity of
PAC was unknown until we recently identified a novel gene PACC1
(TMEM206) encoding the PAC channel. PAC shares no sequence homology
to other membrane proteins, representing a completely new ion channel fam-
ily. By performing cryo-EM and electrophysiology, we present two structures
of human PAC, a high-pH (pH 8) resting closed state and a low-pH (pH 4)
proton-bound non-conducting state, and reveal critical functional residues
involved in pH-dependent activation and ion selectivity. We show a unique
desensitization of PAC upon prolonged treatment at pH 4. The trimeric
PAC contains a transmembrane domain (TMD) and an extracellular domain
(ECD). We observed conformational change in the ECD-TMD interface and
the TMD between the pH 8 and pH 4 structures. A titratable residue H98
from the ECD-TMD interface is involved in PAC pH sensing. The selectivity
filter of PAC is located in the intracellular side of TM2, in which a charge
reversal mutant (K319E) converts PAC to a cation-selective channel. Our
data provide the structural basis to understand the proton-dependent gating
mechanisms of PAC channel.
931-Plat
Role of D-serine Binding to the NMDA Receptor Ligand-Binding Domain
Remy A. Yovanno, Albert Y. Lau.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins
University School of Medicine, Baltimore, MD, USA.
The N-methyl-D-aspartate receptor (NMDAR) is a ligand-gated ion channel
that promotes synaptic plasticity critical for learning and memory. NMDAR
activation is initiated by binding of two different neurotransmitter agonists
to the ligand-binding domains (LBDs). Specifically, the GluN2 subunit
binds glutamate, while the GluN1 subunit binds either glycine or D-serine.
While the mechanisms of glycine and glutamate binding to NMDAR LBDs
have already been determined, little is known about the mechanisms by
which D-serine can bind to and activate the receptor. To probe binding
mechanisms, we performed multi-microsecond molecular dynamics simula-
tions of the GluN1/GluN2A LBD dimer in the presence of freely diffusing
D-serine and glutamate and observed multiple binding and unbinding
events. While D-serine is primarily a GluN1 agonist, we surprisingly
observed that D-serine binds to both GluN1 and GluN2A NMDAR sub-
units. Computing interaction frequencies between LBD residues and D-
serine revealed residues important for agonist binding. However, we
observed that binding pathways are made up of sequences of interactions
that guide D-serine into the binding site. To identify properties of these
pathways, we developed a framework for analyzing binding pathways by
geometric and spatial similarity, which allowed us to cluster them accord-
ing to the region of the LBD with which D-serine interacts. This allowed us
to identify groups of residues that work together to contribute to particular
binding pathways. We determined that D-serine and glutamate bind by
similar pathways and found that D-serine is able to stabilize the active
LBD conformation. Lastly, we determined that N-linked glycans at physi-
ologically relevant glycosylation sites generally do not interfere with
agonist-binding pathways to modulate LBD activity. These insights high-
light the versatility of D-serine as an NMDAR agonist and its unique role
in receptor activation.
Platform: Cytoskeletal Assemblies, Cell
Mechanics, Mechanosensing and Motility
932-Plat
Patient-Derived Glioblastoma (GBM) Cells Exhibit Distinct Rheological
Profiles Associated With Altered Cytoskeleton Regulation
Amelia Foss, Woong Young So, Michael M. Gottesman, Kandice Tanner.
Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
Glioblastoma multiforme (GBM) is the most common adult tumor of the
central nervous system, with an average overall survival of only 15
months. Tumors are characterized by rapid proliferation, aggressive inva-
sion into the stroma, and nearly inevitable recurrence. Although advances
in stratification of GBM by molecular subtype have provided attractive
druggable targets, there has been no advance in standard of care for
over a decade. The high degree of heterogeneity among patients as well
as within an individual tumor have posed significant challenges to the
development of effective therapies. We hypothesized that this heterogene-
ity is also present in the form of mechanical phenotypes, as mechanical
properties influence tumor cell migration, cell cycle regulation, and ther-
apy resistance. To assess these phenotypes, we embedded patient-derived
GBM cells into 3D hyaluronic acid-based hydrogels to mimic the stromal
environment of the brain. Using our home-built optical trap-based active
microrheology system to determine intracellular viscoelastic properties,
we found that each GBM line has a distinct rheological profile. Cell lines
were further characterized by strong power law dependence describing
viscoelastic behavior within the cell. Single-cell analysis of power law
dependence was capable of describing intra-line heterogeneity, and was
able to identify subpopulations within the treatment resistant line. Prote-
omic analysis revealed that distinct mechanical profiles were associated
with alterations in cytoskeletal regulation pathways, particularly in actin-
and myosin-binding pathways. In particular, myosin class II proteins,
associated with cytoskeleton dynamics, cell adhesion, and tumor cell in-
vasion, were differentially abundant among lines, and serve as promising
targets for future investigation. Our work suggests that a deeper under-
standing of mechanical properties of GBM may serve as a valuable
dimension for further stratification of these tumors, and supports the
investigation of cytoskeleton regulation as a potential therapeutic
target.