Activation of the Cannabinoid CB1 Receptor May Involve a W6.48/F3.36 Rotamer Toggle Switch
Rajnish
Singh 1, Dow P. Hurst1, Judy Barnett-Norris 1,
Frank Guarnieri2 and Patricia H. Reggio 1
1Kennesaw State University, Department of Chemistry and Biochemistry,
1000 Chastain Rd., Kennesaw, Georgia 30144
2Mount Sinai School of Medicine New York, NY 10029
| References | Acknowledgements |
INTRODUCTION:
To date, two sub-types of the cannabinoid receptor, CB1 1 and
CB2 2 , have been identified. These receptors belong to the
rhodopsin (Rho) sub-family of G-protein coupled receptors (GPCRs). The
CB1 receptor has been shown to have a high level of ligand-independent
activation (i.e. constitutive activity). In contrast, rhodopsin exhibits
an exquisite lack of constitutive activity. 3
Activation
of GPCRs is accompanied by rigid domain motions and rotations of transmembrane
helices (TMHs) 3 and 6. At their intracellular ends, TMHs 3 and 6 in Rho
are constrained in a salt bridge by a E3.49 / R3.50/ E6.30 salt bridge
that limits the relative mobility of the cytoplasmic ends of TMH3 and
TMH6 in the inactive state 4 and acts like an “ionic lock”.
5,6 During activation, P6.50 of the highly conserved CWXP motif
in TMH6 of GPCRs, may act as a flexible hinge, permitting TMH6 to straighten
upon activation, moving its intracellular end away from TMH3 and upwards
towards the lipid bilayer. 7 Recent evidence in the literature
points to the importance of C6.47 and W6.48, both part of the CWXP motif,
to the conformational changes that TMH 6 may undergo. In the dark (inactive)
state of Rho, the beta-ionone ring of 11-cis-retinal is close to W6.48(265)
on TMH F and helps constrain it in a 
1
= g+ and a 2
= -70oconformation4. In the light activated state,
the beta-ionone ring moves away from TMH F and toward TMH D where it resides
close to A4.58(169). 8 This movement releases the constraint
on W6.48, making it possible for W6.48 to undergo a conformational change
(1 g+ 
trans ). 6 W6.48 in the b2 AR has been reported to undergo
the same transition.9
In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i-4 (F6.44) and i+3 (Y6.51), while in CB1 the residues i-4 and i+3 from W6.48 are leucines (L6.44 and L6.51) (Fig 1).
In the
work described here, we employ the method of Conformational Memories to
show that for CB1 TMH6 as an isolated helix, the W6.48 1
g+ trans transition
is correlated with the degree of kinking in TMH6 and consequently with
activation of CB1.
Further,
we show that as an isolated helix, TMH6 of CB1 appears to be pre-set by
sequence divergences from Rho at key positions, 6.44 and 6.51 ( L6.44,
L6.51 in CB1; F6.44, Y6.51 in Rho) to favor a W6.48 1
= trans state.
Finally,
in the context of the entire TMH bundle of CB1, we show that in the inactive
state of CB1, F3.36 (1
= trans) is strategically located to restrict the conformational freedom
of W6.48 and stabilize a W6.48 g+ 1
inactive state conformation. Further, we show that F3.36 must assume a
g+ 1 conformation in the
activated state to avoid steric clashes with W6.48 as TMHs 3 and 6 move
during activation.
METHODS:
Conformational Memories. In order to explore the relationship between
the rotamer state of W6.48 and the degree of kinking caused by the CWXP
motif in TMH6 of CB1, we used the Conformational Memories (CM) method,12
a method that employs multiple Monte Carlo/simulated annealing random
walks and the Amber* force field.13 The calculation is performed
in two phases. In the first phase, repeated runs of Monte Carlo/simulated
annealing are carried out to map the entire conformational space of the
helix. In the second phase, new Monte Carlo/simulated annealing runs are
performed only in the populated regions identified in the first phase
of the calculation.
The sequence of human wild type (WT) CB1 TMH 6 1 is illustrated in Figure 1. The human WT CB1 TMH6 and the L6.44F and L6.51Y mutants were built using Macromodel. 13 Table I lists the starting conformations for each study. The charges on all charged residues were reduced to one-third of their values to prevent artifacts during the CM runs.
Exploratory
Phase.
In the exploratory phase, a random walk was used to identify the region
of conformational space that is populated for each torsion angle studied.
Starting at a temperature of 2070 K, 20,000 steps were applied to the
rotateable bonds with cooling in 18 steps to a final temperature of 310K.
Trial conformations were generated at each temperature by randomly picking
3 torsion angles from the set of torsion angles for each helix, and changing
each angle by a random value within the range set in the calculation.
Accepted conformations were used to map the conformational space of each
TMH 6 by creating “memories” of values for each torsion angle that was
accepted. For WT CB1 and the L6.44F mutant TMH 6, 73 torsion angles were
allowed to vary during the CM runs. For the L6.51Y mutant TMH6, 74 torsion
angles were varied. The backbone 
’s
and 
’s for I6.46 through
P6.50 (i.e., the turn before P6.50) were allowed to vary ± 50o
from their minimized values. All other backbone torsions were allowed
to vary ± 10o. Side chain torsions were also allowed to vary
± 180o without constraints, with the following exceptions:
the 1 and 2
dihedrals on beta branched residues ( V, I or T) were excluded from all
the runs, except the 2
dihedrals for isoleucines; the 1
of Cys6.47 and W6.48 were allowed to vary ± 60o ; the 2
of W6.48 was allowed to vary ± 60o, while the 2
of C6.47 was not varied. For some of the runs 1
of C6.47 and Y6.51were constrained in either g+ or trans by not varying
these torsions in the CM runs.
Biased Annealing Phase. In the second phase of the CM calculation, the only torsion angle moves attempted were those that would keep the angle in the “populated conformational space” mapped in the Exploratory phase. The Biased Annealing phase began at a temperature of 722 K cooling to 310 K in 8 steps.
Receptor Model Construction. The model of the inactive R form of CB1 was created using the 2.8 Å crystal structure of bovine rhodopsin (Rho)4. Our CM study of CB1 TMH 6 revealed two distinct conformational families for TMH 6 that differed in the degree of kinking in the CWGP flexible hinge region of TMH 6. 11 A conformer from the more kinked CM family of CB1 TMH 6, capable of forming a salt bridge at the intracellular ends of TMHs 3 and 6, was used for our model of the inactive R state.
An active
R* CB1 model was created by modification of our Rho-based model of the
inactive R form of CB1. This R* model construction was guided by the biophysical
literature on the R to R* transition in Rho and the 
-2-adrenergic
receptor. The transition to the R* state is accomplished by the straightening
of TMH 6 such that the intracellular part of TMH 6 moves away from the
receptor core and upwards towards the lipid bilayer. 7 In the
active R* bundle, a TMH 6 conformer from the second major conformational
family was substituted for the TMH 6 conformer used in the inactive bundle
of CB1.
The energy of the CB1 R or R* TMH bundle complex was minimized using the AMBER* united atom force field in Macromodel 6.5. 13 A distance dependent dielectric, 8.0 Å extended non-bonded cutoff (updated every 10 steps), 20.0 Å electrostatic cutoff, and 4.0 Å hydrogen bond cutoff were used. The first stage of the calculation consisted of 2000 steps of Polak-Ribier conjugate gradient (CG) minimization in which a force constant of 225 kJ/mol was used on the helix backbone atoms in order to hold the TMH backbones fixed, while permitting the side chains to relax. The second stage of the calculation consisted of 100 steps of CG in which the force constant on the helix backbone atoms was reduced to 50 kJ/mol in order to allow the helix backbones to adjust. Stages one and two were repeated with the number of CG steps in stage two incremented from 100 to 500 steps until a gradient of 0.001 kJ/(mole Å 2) was reached.
ANALYSIS:
The proline kink, wobble and face shift angles for each set of 100
helices along with the average and standard deviation for each set of
100 helices was calculated using the Prokink program. 14 Using
the criterion that W6.48 undergoes a shift in its 1
( g+ trans) during
activation,17 the W6.48 1
rotamer states of the resultant helices were assessed according to the
percentages of each TMH6 (WT CB1 , L6.44F and L6.51Y) which exist in an
inactive (W6.48 g+ 1) vs.
an active (W6.48 trans 1)
W6.48 rotamer state.
RESULTS:
As indicated in Table 2, CM studies
identified helices with the W6.48 1
in both g+ and trans with a slight change in their relative populations
depending on the C6.47 1.
The Cys6.47 trans 1 run
exhibited a clear preference for the W6.48 in WT CB1 to adopt a trans
1. In this run, 63 out
of 100 helices had a W6.48 1
of trans; while, 37 helices out of 100 were found to have a W6.48 1
of g+ . Runs where Cys6.47 1
was set to g+ had helices with the W6.48 1
of trans in a slightly larger population.
In Figure
2, the CM output for WT CB1 has been combined (200 helices total)
and helices superimposed on their extracellular ends. It is very clear
that the population of W6.48 trans 1
(red) is slightly larger and that this 1
occurs predominantly in helices with reduced kink angles relative to the
W6.48 g+ 1 rotamer population
(purple).
There
is a correlation between the average proline kink value and the W6.48
rotamer state (Table 3). When 1
of W6.48 is g+, the average proline kink was greater (i.e., helices were
more kinked) than when 1
of W6.48 is trans (i.e., lower average proline kink resulting in less
kinked helices). The rotamer state of C6.47 did not influence the degree
of kinking found for the W6.48 rotamers.
Aromatic Mutation at 6.44: The L6.44F mutant has a reduced proline kink bend angle relative to WT (Table 4). The origin of this diminishment is evident from Fig. 3 (B-D).
Unlike
WT TMH 6, which was found to lack two helix backbone H-bonds in the CWXP
region, the L6.44F mutant lacks only one helix backbone H-bond. Also,
in contrast to the WT W6.48 1
population, the L6.44F mutant is decidedly shifted towards g+ (inactive
state; see Table 2). This is due to intrahelix,
aromatic-aromatic stacking interactions between F6.44 and W6.48 which
stabilize W6.48 in the g+ state (Fig. 4).
Aromatic
Mutation at 6.51. The L6.51Y mutant also has a reduced proline kink
bend angle relative to WT (Table 5). Additional
H-bonds (Fig. 3) results in a strengthening
of the backbone which prevents the large proline bend angles seen in WT
TMH 6. Also, the trans 1
rotamer of Y6.51 participates in aromatic stacking with W6.48 ( 1
= g+; Fig. 5) favoring an inactive conformation
of W6.48.
The
W6.48/F3.36 Toggle Switch in WT CB1
Models of
the CB1 inactive (R) and active (R*) TMH bundles illustrated here in Figure
6, show that in the inactive state, residues W6.48 (1
= g+) and F3.36 (1 = trans)
are engaged in a direct aromatic stacking interaction. In this interaction,
F3.36 appears to serve the function of the aromatic residues at 6.44 (1
= trans) and 6.51 (1 =
trans) in Rho which help stabilize W6.48 in its g+ 1
rotamer state. In addition, in the R state of WT CB1, F3.36 also can sterically
block W6.48 from changing its 1
in much the same way as the -ionone
ring of 11-cis-retinal blocks W6.48 in Rho. 4 In the active
state of CB1, F3.36 and W6.48 rotate past each other and F3.36 (1=
g+) and W6.48 (1 = trans)
are located too far apart in R* to interact with each other.
CONCLUSIONS:
CB1 TMH
6 is extremely flexible, largely due to the presence of the helix breaker,
Gly in the CWXP motif (CWGP in CB1) and the absence of aromatic residues
flanking this motif.
Activation
is accompanied by a 1 change
in W6.48 from g+ trans
and a 1 change in F3.36
from trans g+ (see
above). The W6.48/F3.36 interaction may act as the “toggle switch” for
CB1 activation, with W6.48 1
g+/F3.36 1 trans representing
the inactive and W6.48 1
trans/F3.36 1 g+ representing
the active state of CB1.
WT CB1
TMH6 has been “designed” for the ease of the W6.48 1
g+ trans rotamer shift
by the presence of leucines rather than aromatic residues at 6.44 and
6.51. While an aromatic residue at 6.44 tends to disfavor the W6.48 g+
trans transition and
an aromatic at 6.51 would require a concomitant movement of Y6.51 from
trans g+ when W6.48
changes from g+ to trans, the presence of leucines at 6.44 and 6.51 in
the CB1 WT sequence provide W6.48 with greater conformational mobility
and do not help to constrain W6.48 in its 1
g+ conformation.
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ACKNOWLEDGEMENTS:
Camille and Henry Dreyfus Foundation Scholar / Fellow Award (P.H.R. and
R.S.) and NIDA Grant RO1 DA03934 (P.H.R.)