TY - JOUR
T1 - A tethered ligand assay to probe SARS-CoV-2:ACE2 interactions
AU - Bauer, Magnus S.
AU - Gruber, Sophia
AU - Hausch, Adina
AU - Gomes, Priscila S.F.C.
AU - Milles, Lukas F.
AU - Nicolaus, Thomas
AU - Schendel, Leonard C.
AU - Navajas, Pilar López
AU - Procko, Erik
AU - Lietha, Daniel
AU - Melo, Marcelo C.R.
AU - Bernardi, Rafael C.
AU - Gaub, Hermann E.
AU - Lipfert, Jan
PY - 2022/4/5
Y1 - 2022/4/5
N2 - Severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) infections are initiated by attachment of the
receptor-binding domain (RBD) on the viral Spike protein to
angiotensin-converting enzyme-2 (ACE2) on human host cells. This
critical first step occurs in dynamic environments, where external
forces act on the binding partners and avidity effects play an important
role, creating an urgent need for assays that can quantitate SARS-CoV-2
interactions with ACE2 under mechanical load. Here, we introduce a
tethered ligand assay that comprises the RBD and the ACE2 ectodomain
joined by a flexible peptide linker. Using magnetic tweezers and atomic
force spectroscopy as highly complementary single-molecule force
spectroscopy techniques, we investigate the RBD:ACE2 interaction over
the whole physiologically relevant force range. We combine the
experimental results with steered molecular dynamics simulations and
observe and assign fully consistent unbinding and unfolding events
across the three techniques, enabling us to establish ACE2 unfolding as a
molecular fingerprint. Measuring at forces of 2 to 5 pN, we quantify
the force dependence and kinetics of the RBD:ACE2 bond in equilibrium.
We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical
stability, larger binding free energy, and a lower dissociation rate
compared to SARS-CoV-1, which helps to rationalize the different
infection patterns of the two viruses. By studying how free ACE2
outcompetes tethered ACE2, we show that our assay is sensitive to
prevention of bond formation by external binders. We expect our results
to provide a way to investigate the roles of viral mutations and
blocking agents for targeted pharmaceutical intervention.
AB - Severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2) infections are initiated by attachment of the
receptor-binding domain (RBD) on the viral Spike protein to
angiotensin-converting enzyme-2 (ACE2) on human host cells. This
critical first step occurs in dynamic environments, where external
forces act on the binding partners and avidity effects play an important
role, creating an urgent need for assays that can quantitate SARS-CoV-2
interactions with ACE2 under mechanical load. Here, we introduce a
tethered ligand assay that comprises the RBD and the ACE2 ectodomain
joined by a flexible peptide linker. Using magnetic tweezers and atomic
force spectroscopy as highly complementary single-molecule force
spectroscopy techniques, we investigate the RBD:ACE2 interaction over
the whole physiologically relevant force range. We combine the
experimental results with steered molecular dynamics simulations and
observe and assign fully consistent unbinding and unfolding events
across the three techniques, enabling us to establish ACE2 unfolding as a
molecular fingerprint. Measuring at forces of 2 to 5 pN, we quantify
the force dependence and kinetics of the RBD:ACE2 bond in equilibrium.
We show that the SARS-CoV-2 RBD:ACE2 interaction has higher mechanical
stability, larger binding free energy, and a lower dissociation rate
compared to SARS-CoV-1, which helps to rationalize the different
infection patterns of the two viruses. By studying how free ACE2
outcompetes tethered ACE2, we show that our assay is sensitive to
prevention of bond formation by external binders. We expect our results
to provide a way to investigate the roles of viral mutations and
blocking agents for targeted pharmaceutical intervention.
KW - AFM
KW - force spectroscopy
KW - host–pathogen interactions
KW - magnetic tweezers
KW - SARS-CoV-2
KW - COVID-19
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U2 - 10.1073/pnas.2114397119
DO - 10.1073/pnas.2114397119
M3 - Article
C2 - 35312342
SN - 0027-8424
VL - 119
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 14
M1 - e2114397119
ER -