From the Center for Neurobiology and Behavior, the § Department of Pharmacology, and the ¶ Howard Hughes Medical Institute, Columbia University, New York, New York 10032
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of cyclic nucleotide-gated channels is
thought to involve two distinct steps: a recognition event in which a
ligand binds to the channel and a conformational change that both opens the channel and increases the affinity of the channel for an agonist. Sequence similarity with the cyclic nucleotide-binding sites of cAMP-
and cGMP-dependent protein kinases and the bacterial
catabolite activating protein (CAP) suggests that the channel ligand
binding site consists of a -roll and three
-helices. Recent
evidence has demonstrated that the third (or C)
-helix moves
relative to the agonist upon channel activation, forming additional
favorable contacts with the purine ring. Here we ask if channel
activation also involves structural changes in the
-roll by
investigating the contribution of a conserved arginine residue that, in
CAP and the kinases, forms an important ionic interaction with the cyclized phosphate of the bound ligand. Mutations that conserve, neutralize, or reverse the charge on this arginine decreased the apparent affinity for ligand over four orders of magnitude but had
little effect on the ability of bound ligand to open the channel. These
data indicate that the cyclized phosphate of the nucleotide approaches
to within 2-4 Å of the arginine, forming a favorable ionic bond that
is largely unaltered upon activation. Thus, the binding site appears to
be polarized into two distinct structural and functional domains: the
-roll stabilizes the ligand in a state-independent manner, whereas
the C-helix selectively stabilizes the ligand in the open state of the
channel. It is likely that these distinct contributions of the
nucleotide/C-helix and nucleotide/
-roll interactions may also be a
general feature of the mechanism of activation of other cyclic
nucleotide-binding proteins.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyclic nucleotides regulate the activity of a diverse family of
proteins involved in cellular signaling. These include a transcription factor (the bacterial catabolite activating protein, CAP), the cAMP-
(PKA)1 and
cGMP-dependent protein kinases (PKG) and the cyclic
nucleotide-gated (CNG) ion channels involved in visual and olfactory
signal transduction (1, 2). Despite obvious divergence among the
effector domains of these proteins, the cyclic nucleotide binding (CNB)
sites appear to share a common architecture. Solution of the crystal
structures of CAP (3) and a recombinant bovine PKA RI subunit (4) has demonstrated that their CNB sites are formed from an
-helix (A
helix), an 8-stranded
-roll, and two more
-helices (B and C),
with the C-helix forming the back of the binding pocket. Six residues
are invariant among all members of the CAP and kinase families: three
glycines involved in turns between strands of the
-roll, an arginine
and a glutamate, each of which contact the cyclic nucleotide, and an
alanine whose function is uncertain (1) (see also Fig. 1). Strikingly,
these six residues are conserved in the CNG channels. Thus, it has been
suggested that the invariant residues play important and conserved
roles in the folding/function of the CNB sites of these diverse
proteins (1-6). Interestingly, only three of these residues (two
glycines and the arginine) appear to be conserved among the more
distantly related voltage-gated channels that bear the CNB site motif
and whose gating may be modulated by direct binding of cyclic
nucleotide (KAT1 (7, 8), AKT1 (9, 10), and dEAG (11, 12) see Fig.
1).
Surprisingly, this structural similarity of the CNB site does not
appear to be reflected in the conformation of the bound agonist. Thus,
the crystal structures reveal cAMP binds in an anti conformation to CAP
(3) but in a syn conformation to PKA RI (4), although this may not
reflect the conformation of the ligand bound to the proteins in
solution (1, 2, 19). While experiments with cyclic nucleotide analogs
and modeling, based upon the CAP and PKA R1
structures, have been
used to investigate the conformation adopted by agonists in other CNB
sites, this issue is unresolved (1-6). This uncertainty, coupled with
the lack of a crystal structure for any of the CNB proteins, in either the absence of bound agonist or presence of antagonist, leaves an
important question unresolved: what are the structural changes that
take place within these binding sites that result in the activation of
each of the CNB proteins?
By employing site-directed mutagenesis and patch clamp recording of CNG ion channels, it is possible to separate the coupled processes of ligand binding from activation, permitting a dissection of the molecular contributions of protein-ligand interactions to each of these events. Such studies have demonstrated that residues within the C-helix selectively contribute to channel activation (20, 21). Indeed, an aspartic acid residue (Asp604) in the bovine rod subunit 1 (bRET1 (16)) C-helix appears to interact with the purine ring of cGMP selectively when the channel is open (21). That is, the binding energy of this interaction predominantly serves to stabilize ligand binding in the active conformation of the binding site, thereby leading to stabilization of the active (open) state of the channel. However, the state dependence of interactions between the cyclic nucleotide and other residues in the binding site are less well defined.
Here we ask whether regions other than the C-helix of the CNB site are
likely to be altered upon channel activation and thereby contribute to
the increased affinity of the open channel for agonist. Studies of
cyclic nucleotide analogs bearing sulfur substitutions on one or
another of the exocyclic oxygens of the cyclized phosphate raise the
possibility that residues in the -roll may also contribute to
activation gating. These data show that, in the kinases, the equatorial
sulfur-substituted derivative (Rp-cAMPS) is an antagonist, whereas the
axial sulfur-substituted compound (Sp-cAMPS) is an agonist (22-24), a
profile that appears to be reversed in CAP (4, 25). CNG channels formed
from the catfish olfactory neuron subunit 1 (fOLF1 (17)) show an
identical pharmacological profile to that of the kinases (26). By
contrast, in CNG channels formed from bRET1, both cGMP derivatives are
agonists and both cAMP derivatives are antagonists (26). Since the
exocyclic oxygen atoms interact with residues in the
-roll, these
data raise the possibility that large and possibly divergent structural
changes may take place in the
-roll of each of the CNB proteins upon
activation (1-6).
We have focused upon the conserved arginine residue in the
-roll (Arg559 in bRET1, Arg529 in fOLF1, see
Fig. 1). The homologous residue forms an ionic bond with the cyclized
phosphate of the nucleotide in both CAP and the RI
subunit of PKA
(1-4), which suggests that this residue is well placed to detect any
significant rearrangement between the ligand and the
-roll upon
activation. We have previously reported that substitution of this
conserved arginine with the polar but uncharged glutamine residue leads
to a 27-fold increase in the K1/2 (agonist
concentration producing half-maximal activation) in a chimeric channel
(ROON-S2, see "Experimental Procedures" and Fig. 1). Despite this
reduction in sensitivity to ligand, there is no apparent change in the
ability of bound ligand to activate the ROON-S2 channel, as determined
from the maximum open probability (Pmax) of the
channel in the presence of a saturating concentration of ligand (27).
These data suggest that either the conserved arginine contacts the
bound agonist in a state-independent manner (that is, it interacts
equally well with ligand in the open and closed states of the channel)
or that the polar glutamine residue is able to substitute effectively
for the arginine to maintain any state-dependent contacts.
Here we explore further the role of Arg559 by studying a
wide range of mutations that conserve, neutralize, or reverse the
charge of this residue. Such mutations are tolerated and cause a
progressive decrease in the affinity for agonist with little or no
detectable change in the ability of bound agonist to activate the
channel. These data are consistent with the formation of a
state-independent, electrostatic interaction between this arginine and
the cyclized phosphate of the ligand, although they also reveal an
unexpected steric influence of chain length.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular Biology--
Point mutations were made by a polymerase
chain reaction/subcloning strategy, and the resulting cDNA was
verified by dideoxy chain termination sequencing of the polymerase
chain reaction fragment (17, 20). The amino acids swapped in the
construction of the chimeras are given in the legend to
Fig. 1. The majority of these experiments
were performed in the background of two chimeric channels for technical
reasons. The chimera RO133 is comprised of bRET1 whose pore-forming
P-region has been replaced with the corresponding amino acids from
fOLF1 (28). Channels formed from this chimera have cyclic
nucleotide-gating properties identical to those of bRET1 but have the
large single channel conductance of fOLF1 channels, facilitating
measurements of single channel currents and, hence,
Pmax (28). The other construct we utilized was a
double chimera, ROON-S2 (27), in which we replaced both the P-region
and amino-terminal N-S2 domain of bRET1 with sequences from fOLF1. This
construct has both a large single channel conductance and a very high
sensitivity to cGMP (due to the presence of the fOLF1 N-S2 domain and
the bRET1 CNB domain (20, 27)). The high apparent affinity of this
parent chimera permitted us to study ligand-dependent
gating of CNB-site point mutants whose apparent affinities for cGMP
were shifted by up to 4 orders of magnitude. In the parent bRET1 and
fOLF1 backgrounds, such mutations shift the dose-response curve of the
channel into a cGMP concentration range that is greater than 100 mM and thus unmeasurable. We have previously shown that the
only effect of introducing the fOLF1 N-S2 domain in the bRET1
background is to increase the efficacy with which bound ligands
activate this construct; the selectivity of the bRET1 CNB site for
ligand is not compromised (20, 27). Throughout the text, the invariant
arginine in 7 is identified according to the numbering sequence of
either bRET1 (Arg559) for those constructs that contain the
bRET1 CNB site (bRET1, RO133, and ROON-S2) or of fOLF1
(Arg529). In constructs where this residue is mutated, the
identity of amino acids substituted for the arginine is shown by a
letter after a slash mark in the construct name (single letter amino acid code); constructs with no slash mark and letter contain an arginine.
|
Electrophysiological Recordings--
Inside-out patches were
obtained from Xenopus oocytes 1-7 days after injection with
cRNA (Message Machine, Ambion). In most experiments, recordings were
performed with symmetrical solutions (67 mM KCl, 30 mM NaCl, 10 mM EGTA, 1 mM EDTA, 10 mM HEPES, pH adjusted to 7.2 with KOH). Na-cGMP was
included in the intracellular solution by iso-osmolar replacement of
NaCl. In some experiments, we completely replaced the KCl and NaCl with
100 mM Na-cGMP. Data were acquired using an Axopatch 200A
patch clamp amplifier (Axon Instruments) and then digitized (Macintosh
Centris 650 personal computer; ITC-16 interface and PULSE software,
Instrutech Corp.) following low pass filtering (8 pole Bessel filter,
Frequency Devices 902). Single channel recordings were filtered at 4 kHz and digitized at 20 kHz. Macroscopic currents were filtered at 1 kHz and digitized at 2 kHz. All data were acquired at a holding potential of 80 mV.
Data Analysis--
K1/2 was estimated from
fits to the Hill equation, Popen = Pmax/[1 + (K1/2/[A])h], where
K1/2 is the apparent affinity, [A] is
the agonist concentration, h is the Hill coefficient, and
Popen is the observed open probability at a
given concentration of cGMP. For all constructs except ROON-S2/D, this
was determined from patches containing many channels and calculated
according to Popen = (IcGMP/Imax)Pmax,
where IcGMP is the macroscopic current at a
given concentration of cGMP and Imax is the
maximal current at a saturating concentration of cGMP, measured in the
same patch. As such macroscopic recordings were never obtained for
ROON-S2/D, all open probabilities were determined from single channel
recordings, as described below. For the following constructs,
Pmax was determined from single channel patches
in the presence of a saturating concentration of cGMP (given in
parentheses): ROON-S2 (0.3 mM), ROON-S2/K (3 mM), ROON-S2/Q (3 mM), ROON-S2/N (30 mM), RO133 (3 mM), fOLF1 (3 mM),
and fOLF1/Q (30 mM). For each patch, 20-40 s of continuous
recording was accumulated into an all points amplitude histogram, such
as those shown in Fig. 2. As these histograms included all open and
closed events, the area of the closed peak represents the closed
probability (Pclosed) and
Pmax is equal to 1 Pclosed. However, for ROON-S2/L, ROON-S2/E, ROON-S2/D, and RO133/Q, 30 mM cGMP was not saturating.
Higher concentrations of cGMP caused the maximal current to decrease, possibly due to desensitization. Accordingly for these four constructs, we first normalized the dose-response data by the open probability directly measured with 30 mM cGMP.
Pmax was then obtained by fitting the Hill
equation to the normalized data. This introduced only a minor
correction for ROON-S2/L, ROON-S2/E, and RO133/Q. The correction was
larger for ROON-S2/D, which had the most displaced dose-response curve.
For ROON-S2/L and ROON-S2/D, this procedure can lead to
Pmax values that are slightly larger than 1, reflecting the error inherent in this procedure given that the observed
open probabilities are so close to 1 originally. Where appropriate, the
values for Popen (with 30 mM cGMP)
are reported in the legends to Figs. 4 and 6, in addition to the
estimated value of Pmax. This small error will
not significantly affect our estimates of K1/2.
Throughout the manuscript, data are given as mean ± S.E. or mean ± range for those cases in which n = 2. Fits
are weighted to the reciprocal of the standard deviation of the mean
data.
Determination of Electrostatic Distance--
Our goal in these
experiments is to dissect out the contribution of the conserved
arginine in 7 to ligand binding and channel activation. Although
K1/2 values depend, in general, on both ligand affinity and the coupled gating reaction, for those mutations that do
not alter channel gating (Pmax), changes in
K1/2 must reflect a selective change in ligand
affinity. Since the Arg559 mutations studied here do not
alter Pmax, we have used the observed changes in
K1/2 with the various Arg559 point
mutants to calculate the change in free energy of the actual binding
reactions. Thus, the change in free energy of binding,
(
G), upon changing charge at Arg559 is
given by,
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
Fits of the Monod-Wyman-Changeux Gating Model-- We have previously shown (20, 27) that the simplest kinetic scheme that describes the equilibrium gating properties of CNG channels is the cyclical allosteric model of Monod, Wyman, and Changeux (18) (Fig. 1D). According to the model, the channel undergoes an allosteric transition between the closed (C) and open (O) state in the absence of ligand, with an equilibrium constant L0 (equal to [C]/[O]). Agonists activate the channel by binding more tightly to the open state than to the closed state (dissociation constants KO and KC, respectively), thereby shifting the equilibrium from the closed state to the open state by the term (KO/KC)n (where n is the number of agonists bound to the channel). To partition the effects of mutating the conserved arginine between ligand-binding reactions and the channel-opening reaction, the increase in channel open probability as a function of ligand concentration was fit to the MWC model.
![]() |
(Eq. 4) |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fundamental effects on single channel function of replacing Arg559 of the chimeric CNG channel, ROON-S2, with either a neutral (ROON-S2/Q) or acidic (ROON-S2/E) amino acid residue are illustrated in Fig. 2. ROON-S2, ROON-S2/Q, and ROON-S2/E require progressively greater concentrations of cGMP to open. Whereas 0.003 mM cGMP is sufficient to cause ROON-S2 to be open for more than half the time, even a 10-fold higher concentration of cGMP (0.03 mM) activates ROON-S2/Q to only a relatively low extent. ROON-S2/E exhibits almost no openings, even at 0.3 mM cGMP. Despite this >1000-fold reduction in sensitivity to ligand, the open probability obtained at saturating concentrations of cGMP (Pmax) for all three constructs is very close to 1 (top traces). These data thus suggest that neutralization and reversal of the charge at position 559 leads to a progressive decrease in the sensitivity of the channel to cGMP. A concern in all mutagenesis experiments is that the observed effects are due to a global disruption of the structure and function of the protein. As the ion conducting pore of the CNG channels is largely formed from the loop between the 5th and 6th transmembrane domains (28), with no detectable contribution from the carboxyl terminus, we determined the single channel conductance properties of each of these mutants. Despite the large change in ligand sensitivity, the representative single channel traces (Fig. 2) reveal that the current flow through the open channel for the two point mutants is indistinguishable from that of ROON-S2. The open states of all three channels are characterized by pronounced open channel noise, which is readily seen by comparison with the base-line noise when the channels are closed (lower traces of each pair). This excess noise is due to the rapid, partial block and unblock of the open channel by external protons (28, 30).
|
The similarity of the open channel current properties among the constructs is confirmed from the all-points current amplitude histograms (Fig. 2, right panels). These data are well fitted by four Gaussian functions. The Gaussian function near 0 pA reflects the closed state of the channel. The other three Gaussian components reflect current flow through the open channel, with the largest current component corresponding to the fully open channel (no proton block), the intermediate component corresponding to channels occupied by a single proton, and the small component corresponding to channels occupied by two protons (28, 30). Neither the amplitude (the current value at the peak of each of the fitted Gaussian functions) nor the occupancy (the relative areas under the three open state Gaussian functions) of these open channel current states were significantly affected by the mutations at Arg559 (see also Fig. 5). The small variations in the shapes of the amplitude histograms probably reflect small variations in pH or temperature, and hence the proton block, among the recordings. The fact that the open channel characteristics are unchanged by the point mutations suggests that they do not cause a generalized disruption in channel structure.
To interpret the effect of these mutations quantitatively, we measured Popen over a broad range of cGMP concentrations and fit the dose-response relationships by the Hill equation. As is seen in Fig. 3, the effect of these mutations was to cause essentially parallel shifts in the dose-response curves toward greater concentrations of ligand. Thus, the slope of the relationships and the Pmax values were largely unaltered while the K1/2 for activation of ROON-S2, ROON-S2/Q, and ROON-S2/E by cGMP increased from 1.8 ± 0.3 µM (n = 10) to 50 ± 8 µM (n = 8) and 3379 ± 1005 µM (n = 5), respectively. That is, neutralization of Arg559 resulted in a 28-fold increase in the K1/2 value, whereas charge reversal increased further the K1/2 value by 68-fold.
|
We next asked if the chemical identity of the residue at position 559 was important or if the altered activation of the mutant channels was simply a consequence of the change in charge on the side chain. To investigate this, we constructed a more extensive series of mutations in the ROON-S2 background, generating channels with basic (arginine or lysine), neutral (glutamine, asparagine, or leucine), or acidic (glutamate or aspartate) residues at position 559. The gating properties of each construct were then determined.
Fig. 4A shows that
Pmax for all of the constructs was 0.98, indistinguishable from the parent chimera ROON-S2. Together, the data
in Figs. 2, 3, and 4A show that neither the charge nor chemical identity of the side chain of residue 559 has a detectable influence upon the ability of bound ligand to open the channel.
|
In contrast, a plot of K1/2 versus charge on the side chain of residue 559 reveals that there are both electrostatic and steric effects of side chain substituents upon the apparent affinity for ligand (Fig. 4B). Thus, introducing the charge-conserving lysine (ROON-S2/K) residue resulted in a decrease in apparent affinity. Surprisingly, the 70-fold increase in K1/2 was larger than the 28-fold increase seen upon neutralization with glutamine. Lysine has two important differences when compared with arginine. First, it is the equivalent of one methylene bridge shorter, and second, it has a point charge on a primary amine, whereas arginine has the charge delocalized over the guanidinium group. As there are no other amino acids with basic side chains, it is not possible to distinguish between the steric effect of shortening the side chain from an effect of alteration in local field strength.
Mutation of Arg559 to neutral and acidic amino acids does permit us to address this question further. Replacement of Arg559 with an asparagine (which is one methylene bridge shorter than glutamine, but otherwise identical), to generate the ROON-S2/N mutant, gives rise to a far more pronounced increase in K1/2 (392-fold) than does replacement with glutamine (ROON-S2/Q). This result suggests that chain length or the exact location of the polar groups, in addition to charge, is an important determinant of ligand affinity. The importance of side chain polarity is demonstrated upon introduction of the non-polar residue leucine, which increased the K1/2 by 882-fold, a more pronounced modification than that seen with either of the polar substitutions or with lysine. Leucine is effectively an asparagine in which the carbonyl oxygen and amino group of the side chain have been replaced by methyl groups and which has a volume intermediate between that of glutamine and arginine.
The importance of charge at position 559 was further explored by reversing the sign of the charge by introduction of either glutamic or aspartic acid. The K1/2 values of these two mutants was increased by 1877 and 5489 fold, respectively. The magnitudes of these increases in K1/2 are consistent with the generation of a repulsive interaction between the acidic side chain of the amino acid and the cyclized phosphate of the cyclic nucleotide. However, here again we see that amino acid residue with shorter side chain produced a more pronounced increase in K1/2.
A linear regression through the plot of log(K1/2) values versus charge at position 559 (solid line in Fig. 4B) yields a slope corresponding to a 19.5-fold increase in K1/2 for an elementary change in charge (the mean value of K2/K1, Equations 1 and 3 under "Experimental Procedures"). Assuming a coulombic interaction between the residue at position 559 and a single negative charge on cGMP, this relationship yields an approximate distance of 2.4 Å between the ligand and the charge at position 559 (determined from Equation 3, under "Experimental Procedures"). Approximate upper and lower bounds for this value are obtained from the largest and smallest changes in K1/2 observed upon reversal of charge. The 5489-fold increase in K1/2 upon replacing arginine by aspartate is equivalent to a distance of 1.7 Å, whereas the 27-fold increase in K1/2 upon replacing lysine by glutamate indicates a slightly longer distance of 4.4 Å. The electrostatic nature of this interaction is supported by the roughly similar fold increase in K1/2 seen upon changing the residue at position 559 either from a glutamine to a glutamate or from an asparagine to an aspartate. In each case, chain length is held essentially constant while a negative charge is introduced by conversion of the amide to the acid (Fig. 4B). Taken together, the data in Fig. 4 are consistent with the formation of a state-independent ionic bond between the side chain of residue 559 and the cyclized phosphate of the nucleotide.
Despite the pronounced effect of these point mutations on cGMP sensitivity, the conductance properties of the mutant channels are essentially identical to the parent chimera, ROON-S2. This is evident in Fig. 5, a two-dimensional plot of conductance versus fractional occupancy of the three open channel conductance states (unprotonated, singly and doubly protonated, see Fig. 2). The variability in the amplitude of the largest conductance state among the different mutants is not correlated with ionic charge at position 559. Rather, it is likely to reflect a technical difficulty in fitting this infrequently occupied conductance level next to the two dominant conductance levels, which represent the partially and fully protonated states. Taken together, the lack of an effect of the mutations upon either the single channel conductance or Pmax indicate that the mutations of Arg559 result in a discrete disruption of the binding site that selectively lowers the apparent affinity for ligand.
|
As these experiments were performed in the background of ROON-S2, a chimeric channel with unusually high apparent affinity, we were concerned that the introduction of either the fOLF1 P-region or N-S2 domains may have altered the normal interaction between Arg559 and the ligand. To address these concerns, we tested the effect of one of the Arg559 point mutations in the backgrounds of both bRET1 and the chimera RO133 (bRET1 with the fOLF1 P-region). Fig. 6A shows that the mutation R559Q in the R0133 background increased the K1/2 of the resulting construct (RO133/Q) 42-fold with no change in Pmax. A similar decrease in the apparent affinity was observed upon introduction of the R559Q mutation in bRET1 (52 ± 6 µM, n = 9 to 2793 ± 368 µM, n = 2; data not shown). The qualitative and quantitative similarity of the R559Q mutation in bRET1, RO133, and ROON-S2 demonstrate that the selective change in apparent affinity upon mutation of Arg559 is an intrinsic property of the bRET1 CNB site.
|
What is the basis for this selective increase in K1/2? To address this, we have utilized the cyclic allosteric model of Monod, Wyman, and Changeux (18) (Fig. 1D), which we have previously demonstrated to be the simplest kinetic scheme that adequately describes CNG channel activation (20, 27). Based upon this model, an increase in K1/2 can be produced either by a reduction in affinity for ligand (increase in KO or KC) or from an increase in the allosteric equilibrium constant, L0, between the open and closed state of the channel (L0 = [C]/[O]). However, we have previously shown, using measurements of agonist-free openings, that the arginine to glutamine mutation does not alter L0 in ROON-S2 (27). Moreover, a change in L0 in any of the mutants would not only increase K1/2 but would also significantly reduce Pmax, which was not observed (Figs. 2A, 4A, and 6A). Rather, these data suggest that mutation of Arg559 lowers the apparent affinity by specifically decreasing the absolute affinity of the CNB site for ligand.
To investigate the effect of the arginine to glutamine mutation
quantitatively, we fit cGMP dose-response curves with the MWC model to
determine KO and KC for RO133 and RO133/Q (Fig. 6A), constructs that allow us to determine
Pmax with a high degree of accuracy. Plotting
these equilibrium constants as free energy terms (G =
RT ln(1/K)) shows that the mutation reduces the absolute
binding affinities of both the open (KO) and closed
(KC) states of the channel
(Fig. 7A). Indeed, the change
in free energy of cGMP binding between the wild-type channel and the
glutamine mutant (
(
G), filled circles in
each plot) shows that KO and KC
are destabilized equivalently, by 2.21 and 2.27 kcal
mol
1, respectively. These amounts are consistent with the
disruption of an ionic bond.
|
Is this state-independent interaction between the conserved arginine in
7 and the cyclic nucleotide a common characteristic of all CNB
domains or do marked structural rearrangements occur around the
homologous arginine in the
-roll of other binding domains? To
address this, we have investigated the effects of mutation of the
homologous arginine to a glutamine in fOLF1. This comparison is
particularly interesting given the differential handling of the Rp- and
Sp-cyclic nucleotide analogs by bRET1 and fOLF1 (26), which suggests
that the
-roll portion of the binding site of fOLF1 may differ
significantly from that of bRET1. Fig. 6B compares
dose-response curves for fOLF1 and fOLF1/Q. Although there is a shift
in the fOLF1/Q dose-response curve to higher concentrations, this
effect is less marked (7.6-fold) than is seen in the bRET1 CNB site
(28-54-fold, depending upon the channel background). Surprisingly, the
fOLF1/Q mutant shows a higher Pmax compared with
wild-type fOLF1, despite the decrease in cGMP sensitivity.
These data raise two questions. First, to what extent do these
differences in gating properties between bRET1 and fOLF1 result from a
fundamental difference in the mechanistic behavior of their binding
sites? Second, how can a mutation destabilize binding but increase
efficacy? To investigate these questions, we fit the dose-response data
for fOLF1 and fOLF1/Q with the MWC model and determined
KO and KC for these two channels. This analysis reveals that the impact of the R to Q mutation on ligand
binding in fOLF1 is, in fact, very similar to the effect observed in
the bRET1 CNB site. Thus, the destabilization of KO (1.57 kcal mol1) and KC (1.90 kcal
mol
1) in fOLF1/Q are similar in sign and magnitude to the
changes seen in the bRET1 background. The less marked shift in the
dose-response curve and the increase in Pmax
seen with the arginine to glutamine mutation in fOLF1 arise from small
quantitative differences in the magnitude of the effect of the mutation
upon binding of agonist to the open and the closed states of the
channel, not from a qualitatively different utilization of the binding
energy.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Here we have investigated which regions of CNB sites contribute to
activation and, in particular, whether there is likely to be a
significant change in the interaction between the -roll of the CNB
site and the ligand upon activation. Our studies focused on an arginine
residue in the
-roll that is conserved among diverse CNB proteins
and that makes an ionic interaction with the cyclized phosphate of
cyclic nucleotides in both the bacterial CNB protein CAP as well as in
the regulatory subunit of cAMP-dependent protein kinase (1-4) (see also Fig. 1).
Mutations of this conserved arginine, in the background of the chimeric
CNG channel ROON-S2, to a series of residues that conserve, neutralize,
or reverse its charge, caused a progressive decrease in apparent
affinity of the channel for ligand. Although an unexplained steric
effect of chain length contributed to this decrease, the clear
dependence of the K1/2 values on charge at position 559 strongly supports the formation of an ion pair between
Arg559 and the cyclized phosphate. This result is
consistent with the x-ray crystallographic structural studies of PKA
RI and CAP (1-4). Indeed, the estimate of the electrostatic
distance between Arg559 and the cyclized phosphate from
these experiments (1.7-4.4 Å) is close to that predicted from the
crystal structures of CAP (3.1-3.5 Å, (3)) and PKA RI
(<3.3 Å,
(4)). Despite the large changes in ligand sensitivity with the
Arg559 mutants (spanning nearly four orders of magnitude),
the ability of the bound ligand to activate the channel (as determined
from Pmax) was virtually unaltered. These data
suggest that Arg559 plays an important role in stabilizing
cyclic nucleotide and that these interactions do not contribute to
channel activation. The absence of an effect of the mutations on the
single channel conductance or on Pmax shows that
these mutations are unlikely to cause a global disruption of the
protein.
This surprising result, that the Arg559 point mutants have large effects on ligand sensitivity but little effect on activation gating, can be readily explained within the context of the MWC allosteric reaction scheme (18). According to this scheme, a concerted allosteric conformational change in the channel both opens the channel pore and alters the binding site, causing the ligand affinity of the open state to be considerably higher than the ligand affinity of the closed state (dissociation constants KO and KC, respectively). By measuring ligand-independent openings, we previously determined the allosteric equilibrium constant between closed and open channels in the absence of agonist, L0, for both bRET1 and fOLF1 (27). We found that a 20-fold difference in L0 between bRET1 and fOLF1, which contributes to physiologically important differences in ligand gating (20, 27, 31), was localized to the amino-terminal N-S2 domain (27). Since this region of the channel interacts with the carboxyl terminus (32, 33) and is involved in subunit assembly in the homologous voltage-gated K channels (34-38), we have postulated that channel activation involves a change in quaternary structure.
Whereas the difference in the allosteric transition between fOLF1 and
bRET1 is localized to the amino terminus of the channel, the postulated
increase in ligand affinity of the open state of these channels is
mediated, at least in part, by interactions of the cyclic nucleotide
with the C-helix of the carboxyl terminus CNB domain (20, 21). In
particular, an aspartate residue in the C-helix of bRET1,
Asp604, has been shown to make important contacts with cGMP
in the open state, but not closed state, of the channel (21). These
results suggested a model of channel gating in which the allosteric
transition that opens the channel is coupled to a change in the
orientation of the C-helix relative to the roll, leading to an
enhancement of C-helix/ligand contacts. According to this model, the
-roll would provide a relatively stable structure that is involved
in the initial binding of ligand, which orients the nucleotide within the binding pocket. The lack of effect of mutation of
Arg559 on ligand-dependent gating is consistent
with this hypothesis.
A quantitative analysis of the effect of mutating arginine 559 to
glutamine was performed by fitting the MWC model to the cGMP
dose-response data. This was done in the background of a chimeric
channel, RO133 (bRET1 with the fOLF1 P region), because the gating
properties and large single channel conductance of this construct
facilitated accurate determination of Pmax, and hence, the channel activation parameters (28). This analysis shows that
the R559Q mutation decreases the affinity of the open (KO) and closed (KC) state of the
channel for ligand by an identical amount. From these data we can
conclude that there is no significant structural rearrangements between this deep part of the -roll and the ligand upon channel activation. Conversely, we can also conclude that all bonds between the protein and
the ligand that are made more favorable when the channel goes from the
closed to the open state, and stabilize the latter, are unaffected by
the electrostatic and steric effects of substitutions at position
559.
Does this analysis of the interaction between Arg559 in
bRET1 and the cyclic phosphate hold true for other cyclic nucleotide binding pockets? Although mutation of the conserved arginine in CAP (to
lysine, histidine, glutamine, or leucine) and PKA (to either lysine or
tryptophan) has been shown to interfere with ligand-dependent activation, it has been difficult in these
molecules to separate out effects of binding from activation (1, 25, 39-43). To address this question, we therefore constructed the homologous mutation in the fOLF1 CNB domain. This is particularly interesting given the different actions of Rp- and Sp-substituted ligands in fOLF1 and bRET1 (26). In the background of the olfactory channel, mutation of the homologous arginine (Arg529)
actually enhanced Pmax despite a decrease in
ligand sensitivity. This result suggested that there might be a
qualitatively different interaction between cGMP and the -roll of
the fOLF1 binding site compared with the cGMP/bRET1
-roll
interaction. However, a fit of the MWC model showed that these
differences can be explained by relatively small quantitative changes,
amounting to only an ~0.3 kcal mol
1 difference between
the effects of the R529Q substitution on KO and
KC, in which closed state binding is decreased to a
slightly greater extent than open state binding. Such small changes
(equivalent to a fraction of a hydrogen bond) may readily be explained
by indirect effects of the R529Q mutation on the orientation of the
bound ligand rather than a large scale change in the structure of the
fOLF1
-roll during channel activation.
The state-independent interaction with the conserved arginine in 7
in the CNG channels is in contrast to results suggesting that the
neighboring residue, Thr560 in bRET1, may contribute to
activation gating (21, 44). Thus, the mutation T560A produces a
somewhat greater decrease in binding to the open state compared with
the closed state, resulting in a 6-7-fold decrease in
Pmax. Although this suggests that there might be
a state-dependent interaction between Thr560
and ligand, this effect on gating could also be due to an indirect effect of the mutation, either by altering the conformation of the
binding pocket or the orientation of the ligand in the binding site.
For example, the T560A mutation might slightly decrease the ability of
bound ligand to form optimal contacts with the C-helix in the open
state. Although there are many possible interpretations for mutations
that alter gating, only one interpretation is consistent with the
profound state-independent changes in ligand binding seen with the wide
range of Arg559 mutations, that this region of the channel
does not alter its contacts with ligand during gating.
The data presented here, taken together with previous results, suggest
that the CNB site of both fOLF1 and bRET1 CNG channels comprises two
distinct structural and functional domains. The -roll forms
state-independent contacts with ligand that are important for
stabilizing the ligand in the binding pocket, whereas the C-helix makes
state-dependent contacts that increase ligand affinity upon
channel activation and stabilize the channel in its open state (1-4,
20, 21). Based on the qualitatively similar effects of the mutation in
bRET1 (R559Q) and fOLF1 (R529Q), our data suggest that these proteins
undergo a common structural change upon activation despite their
different patterns of activation with Rp- and Sp-cyclic nucleotide
analogs (26). The distinct pharmacology of these two proteins probably
reflects relatively small variations in the conformation of the binding
pockets or the bound ligand rather than qualitatively different
mechanisms of activation. Given the sequence similarity among CAP, the
kinases, and the CNG channels and the similar effects seen in each upon
mutation of the conserved arginine residue, we expect that the CNB
sites of these diverse proteins share a similar functional organization
that underlies the mechanism of ligand activation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Jose Ramirez-Latorre, Pierre Paoletti, and Edgar Young for insightful and critical help in preparation of this manuscript and Huan Yau and John Riley for expert technical assistance.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Columbia University,
722 West 168th Street, New York, NY 10032. Tel.: 212-543-5259; Fax:
212-795-7997; E-mail: grt1{at}columbia.edu.
1 The abbreviations used are: PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; CNG, cyclic nucleotide gated; CNB, cyclic nucleotide binding; CAP, catabolite activating protein; Rp- and Sp-, phosphorothioate derivatives of cyclic nucleotides; MWC model, model of Monod, Wyman, and Changeux.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|