Crystal
Structure of the
Beta-2 Adrenergic Receptor-Gs Protien Complex
Andrew Maurer '14 and Sam McQuiston '14
Contents:
I.
Introduction
The
Beta-2 adrenergic
receptor-Gs protien
complex has been used as a model system for
G-protien coupled receptors(GPCRs) since its discovery over 40 years
ago. As a trans-membrane receptor, the primary function of the
Beta-adrenergic receptor is to bind to extracellular adreniline and in
turn activate the Gs subunit, which then dissociates and continues the
messaging cascade. The exact structure of the receptor complex remained
a
mystery
for many years due to the instability of the complex while in the
detergent solution required for x-ray crystallography. This problem was
worked around by binding a nanobody, Nb35,
,
to the Gs subunit
and a lysosome,
T4L
,
to B2AR which both increased the stability of the
complex in the x-ray chromotography solution.
II.
General Structure
The Beta-2
Adrenergic Receptor(B2AR)
is
a single poly-peptide chain which
weaves inside and outside of the cellular membrane. Its secondary
structure is constituted by 8 α helices, 7 of which are
trans-membrane and one
which runs parallel to the intracellular face of
the cell membrane.
It
also contains three
extra cellular loops, three intracellular loops, an extracellular
N-terminus and intracellular C-terminus. The agonist binding site lies
within the trans-membrane section of the protein.
The Gs subunit
is
a heterotrimic
G-protein, with the subunits, Gαs,
Gβ
and
Gγ. The Gαs,
which
binds to GDP and GTP, is the primary functional subunit of the Gs
protein which binds to and activates the adenyl-cyclase channel,
continuing signal transduction. It is further divided into two
sub-units
,
GαsRas
and GαsAH
which clamp onto the guanine-nuceloside when it is bound.
III.
Agonist Binding and B2AR Conformation Shift
The
agonist binding site
in
the B2AR protein consists of the residues D130,
V131, F288, F289, and N312.
The primary interactions of this binding
site are Hydrogen bonding between the D130 and N312 hydrogen acceptors
and the agonist's proton donors, primarily in the form of hydroxyl
groups. Additionally, the F288 and F289 groups help to stabilize the
agonist in the binding site by providing non-polar-phenyl stacking
interactions which hold the agonist in place.
The
binding of the agonist to this site results in a conformational shift
in the trans-membrane
helix(TM) 3 and TM 6 of B2AR
, which
is
hypothesised to
activate the Gs subunit. However, this conformational shift also
results in the weakening of the association of between these helices,
which makes them vulnerable to outside attack. This is thought to be
one of the primary reasons for the instability of B2AR in detergent
solution. The reason for Gs activation is not certain because the
B2AR-Gs complex does not include a nucleotide and thus activation can
not be observed.
Additional
structural changes in B2AR include an eight residue shift in the TM5
helix and a change in in
secondary structure of the second
intracellular loop(2ICL) into
an α-helix
structure.
IV.
B2AR-Gs Binding
The
formation of the nucleotide-free B2AR-Gs protein complex first
requires the carboxy terminus of the α5-helix in to shift
away from the β6-strand in Gs (shown in next section). This
shift allows for proper Gs
interactions with B2AR. Gαs is composed of two GαsRas
domains
which activity bind to
B2AR
The
B2AR-GαsRas interface is the primary binding site for the
B2AR-Gs protein complex with a total buried surface of 2,576 A2 .
A
rotation
of GαsRas causes a conformational change in both
B2AR and GαsRas allowing for the stabilization and bond
formation between them. The interaction is formed by
ICL2
,
TM3
,
TM5
,
and
TM6
of
B2AR and
α5-helix
,
αN-β1
junction
,
the
top of β1-strand
,
and
α4-helix
of
GαRas.
There are many specific interactions which hold B2AR and Gs together.
1.) ARG 131, ALA 134,
ILE
135, and THR 136
of
TM3
interact with TYR 391, HIS 387,
GLN
384, and ARG 380
of the
α5-helix
in
a D/ERY motif.
2.) SER 143
of
ICL2
and ALA 39
of αN-β1
junction
..
3.) ARG 239
of TM5
and THR 350
of
α4-helix
.
4.) ALA 271,
THR
274, and LEU 275
of
TM6
and LEU 393
and GLU 392
of
α5-helix
.
Combined these interactions hold the B2AR-Gs complex together
.
It
is unknown
when GDP is released during the building of the B2AR-Gs
protein complex. Surprisingly there is no interaction
between B2AR and Gβϒ
,
the second subunit of Gs.
V.
Conformational Change
Allows Binding of Gs-Nucleotide
A major
conformational shift in the Gs sub-unit allows
GαsRas and GαsAH
to
bind to the Guanine nucleotide
.
GαsAH
subunit
displaces,
rotating 127
degrees, from a non-nucleotide bound state to a nucleotide
bound state. The lack of the
stability in the bond with guanine in between
the GαsRas
and GαsAH
subunits is responsible for the flexibility of the GαsAH
subunit when in the non-nucleotide-bound state.
While
the GαsRas
subunit is less flexible than its counterpart, it still undergoes
significant conformational shift. The
α5
helix
shifts
6Å towards the receptor and is rotated so the carboxyl
terminal end
is
pointed into the B2AR core in non-nucleotide-bound
state. In addition, the beta6 alpha5 loop, which interacts with the
guanine ring in the active site when bound, is displaced away
from
the binding pocket.
Although
not totally understood, the β1-α1 loop, a
P-loop motif, is directly involved in the nucleotide binding
.
The
interactions between GαsRas
and Gβγ
appear to
be unaffected by the binding of guanine nucleotides. However, because a
crystal structure of GDP-bound Gs heterotrimer has not yet been
reported and the Nb35 binds to the junction of GαsRas
and Gβ
and could be responsible for the relative stability of the interface,
the veracity of this phenomenon in biological systems can't be certain.
VI.
References
Ghanouni,
Pejman, Jacqueline J. Steenhuis,
David L.
Farren, and Brain K. Kobika. "Agonist-induced Conformational Changes in
the G-protein-coupling Domain of the β2 Adrenergic Receptor." Proceedings
of the Nation Academy of Sciences of the United States of America
98.11
(2001): 5997-6002. Web.
Kobilka,
B., and G. Schertler. "New
G-protein-coupled Receptor Crystal Structures: Insights and
Limitations." Trends
in Pharmacological Sciences 29.2
(2008): 79-83. Web.
Rasmussen,
Søren G. F., Brian T.
DeVree, Yaozhong Zou,
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Seok
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Joseph
A. Lyons, Martin Caffrey, Samuel H. Gellman, Jan Steyaert, Georgios
Skiniotis,
William I. Weis, Roger K. Sunahara, and Brian K. Kobilka. "Crystal
Structure of the β2 Adrenergic Receptor–Gs Protein
Complex." Nature
477.7366 (2011): 549-55. Web.
Rasmussen,
Søren G. F., Hee-Jung Choi,
Daniel M.
Rosenbaum, Tong Sun Kobilka, Foon Sun Thian, Patricia C. Edwards,
Manfred
Burghammer, Venkata R. P. Ratnala, Ruslan Sanishvili, Robert F.
Fischetti,
Gebhard F. X. Schertler, William I. Weis, and Brian K. Kobilka.
"Crystal
Structure of the Human β2 Adrenergic G-protein-coupled
Receptor." Nature
450.7168 (2007): 383-87. Print.
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