Nailing Down the Structure
of the ß2
by Jeremy Cherfas
Top Ten Papers
K. Takahashi, et al.,
"Induction of pluripotent stem
cells from adult human
fibroblasts by defined
131(5): 861-72, 30 November
2007. [Kyoto U., Japan; CREST,
Kawaguchi, Japan; Gladstone
Inst. Cardio. Dis., San
Francisco, CA] *243MG
V. Cherezov, et al.,
structure of an engineered
1258-65, 23 November 2007.
[Scripps Res. Inst., La Jolla,
CA; Stanford U., CA] *233JG
Intl. HapMap Consortium (K.A.
Frazer, et al.), "A
second generation human
haplotype map of over 3.1
million SNPs," Nature,
449(7164): 854-61, 18 October
2007. [72 institutions
The ENCODE Project Consortium
E. Birney, et
al.), "Identification and
analysis of functional elements
in 1% of the human genome by
the ENCODE pilot project,"
799-816, 14 June 2007. [80
institutions worldwide] *178FV
I.I. Ivanov, et al.,
"The orphan nuclear receptor
ROR[gamma]t directs the
differentiation program of
IL-17+ T helper
cells, Cell, 126(6):
1121-33, 22 September 2006.
[Howard Hughes Med. Inst., New
York U., NY; Schering-Plough
BioPharma, Palo Alto, CA]
D.F. Easton, et al.,
"Genome-wide association study
447(7148): 1087-93, 28 June
2007. [87 institutions
A. Barski, et al.,
"High-resolution profiling of
histone methylations in the
human genome," Cell,
129(4): 823-37, 18 May 2007.
[NHLBI, NIH, Bethesda, MD; U.
Calif., Los Angeles] *172FA
S.G.F. Rasmussen, et
al., "Crystal structure of
450(7168): 383-8, 15 November
2007. [Stanford U., CA; MRC
Lab. Molec. Bio., Cambridge,
U.K.; Europ. Synchroton Radiat.
Fac., Grenoble, France; Argonne
Natl. Lab., IL] *231AM
M. Wernig, et al.,
reprogramming of fibroblasts
into a pluripotent ES-cell-like
448(7151): 318-24, 19 July
2007. [5 U.S. institutions]
T.S. Mikkelsen, et
al., "Genome-wide maps of
chromatin state in pluripotent
and lineage-committed cells,"
553-60, 2 August 2007. [6 U.S.
Brian Kobilka, of Stanford University School of Medicine, was a
member of the team that cloned the
ß2-adrenergic receptor, a molecule that
spans the cell membrane and that responds to the presence of
adrenaline by triggering diverse components of the "fight or
flight" response. More than 20 years later, he is one of the lead
authors on two Top Ten papers that describe the detailed molecular
structure of ß2AR, as the receptor is
known. Getting from there to here required a succession of
technical and conceptual breakthroughs that have paved the way to a
far greater understanding of the most common family of
trans-membrane signal receptors.
There are about 1,000 G protein-coupled receptors (GPCRs) that
respond to a huge range of stimuli, from the light that activates
the visual pigment rhodopsin to hormones and small molecules such as adrenaline. As Kobilka
and his colleagues note in the authors’ summary of one of
the highly cited papers (#2), "drugs that act on GPCRs command
more than 50% of the current market for human therapeutics, with
annual revenues in excess of $40 billion." But those drugs often
have untoward side effects; asthma drugs, for example, can make
the heart beat too fast if the dose is not carefully controlled.
Part of the reason is that drug design is difficult because the
structure and function of the receptors are not well understood.
And that is not surprising.
Rhodopsin, which was characterized around a decade ago, is unusual
in being physically quite stable, which means it is relatively easy
to make the crystals needed to determine its structure.
ß2AR is much "wobblier" and is in any
case hard to crystallize because the surface of the molecule that
sits within the membrane tends to be hydrophobic and thus to steer
clear of the close molecular contacts that are essential to crystal
formation. It also seems likely that
ß2AR, being a trans-membrane protein,
needs to be within a membrane to exhibit its true shape.
Kobilka’s team adopted two approaches to the problem. In
both, they stabilized the outside of the receptor by binding it
with the beta-blocker carazolol. For the inside, one group bound
one of the intracellular loops to a monoclonal antibody. The other
genetically engineered the ß2AR molecule,
replacing the same intracellular loop with a small protein derived
from T4 bacteriophage. The antibody and the T4 lysozyme both
encouraged the formation of a crystal lattice. Of course there is
more to mapping the molecular structure than just having the
crystals, but without the crystals nothing is possible. To have two
different sorts of crystal is fortunate indeed.
"We didn't know which [method] was going to work, so we tried
both," Kobilka recently told The Scientist (23: 51,
2009). "And they ended up both working at about the same time."
The paper by Rasmussen et al., at #8, reported the
structure based on the monoclonal antibody and was published in
Nature a week before Cherezov et al., at #2,
published in Science with a higher-resolution version
derived from the engineered ß2AR. (A
third paper by the team, in the same issue of Science
[D.M. Rosenbaum, et al., 318(5854): 1266-73, 2007], just
missed the current Top Ten with 22 citations this period.) The two
structures are all but identical. Perhaps the biggest surprise is
that an ionic lock, which holds the intracellular parts of the
molecule together in inactivated rhodopsin (and which may be partly
responsible for that molecule’s stability) is broken in
ß2AR, even though the agonist carazolol
is blocking the receptor and thus might be expected to have locked
the structure. If just one of the structures had demonstrated a
broken lock, it might have been dismissed as an artefact, but given
that it shows in both structures, even though they had different
intracellular components and different lipid supports, the
suggestion is that this is an important aspect of
There are other aspects of the structure that suggest ways in which
the receptor actually works. For example, there is a channel
through the middle of the molecule that seems to be filled with
water. This could provide space for the components of the receptor
to move around, reacting to different signal molecules by adopting
different positions and shapes and thus possibly helping to trigger
subtly different responses within the cell.
Other researchers have already made use of the
ß2AR structure, and even more so the
insights that went into determining it, to pursue their own GPCRs
with considerable success. Kobilka’s group is in hot pursuit
of the structure of active ß2AR, bound
not by a blocker but by its correct agonist adrenaline. That may
reveal details of how exactly it activates the G protein, and will
probably require the crystallization of an even more complex
three-part molecule—signal, receptor, and G protein
responder. That is unlikely to take another 20
Dr. Jeremy Cherfas is Science Writer at Bioversity
International in Rome, Italy.
KEYWORDS: G PROTEIN-COUPLED RECEPTORS, GPCRS, BETA-2
ADRENORECEPTOR, BRIAN KOBILKA, BETA2 AR.