Polymorphism of the signaling molecule c-di-GMP

Article abstract of DOI:10.1021/ja0613714

Using UV, CD, and NMR, we demonstrate that the important bacterial signaling molecule involved in biofilm formation, cyclic diguanosine monophosphate (c-di-GMP), exists as a mixture of five different but related structures in an equilibrium that is sensit

Full text of DOI:10.1021/ja0613714

Published on Web 05/04/2006
Polymorphism of the Signaling Molecule c-di-GMP
Zhaoying Zhang, Seho Kim, Barbara L. Gaffney, and Roger A. Jones*
Contribution from the Department of Chemistry and Chemical Biology, 610 Taylor Road,
Rutgers, The State UniVersity of New Jersey, Piscataway, New Jersey 08854
Received February 27, 2006; E-mail: jones@rutchem.rutgers.edu
Abstract: Using UV, CD, and NMR, we demonstrate that the important bacterial signaling molecule involved
in biofilm formation, cyclic diguanosine monophosphate (c-di-GMP), exists as a mixture of five different
but related structures in an equilibrium that is sensitive both to its concentration and to the metal present.
At the lower concentrations used for UV and CD work (0.05-0.5 mM), Li+, Na+, Cs+, and Mg2+ favor a
+
bimolecular self-intercalated structure, while K+, Rb+, and NH4 favor formation of one or more guanine
quartet complexes as well. At the higher NMR concentrations (∼30 mM), the bimolecular structures associate
and rearrange to a mixture of all-syn and all-anti tetramolecular and octamolecular quartet complexes.
With K+ the octamolecular complexes predominate, while with Li+ the tetramolecular and octamolecular
quartet complexes are present in approximately equal amounts, along with the bimolecular structure. We
also find that both guanine amino groups in c-di-GMP are essential for formation of the quartets, because
substitution of inosine for one guanosine allows formation of only the bimolecular structure. Further, two
molecules of c-di-GMP tethered together are constrained in such a way that limits their ability to form
these quartet complexes. The polymorphism we describe may provide different options for this signaling
molecule when performing its functions in a bacterial cell, with K+ and its own local concentration controlling
the equilibrium.
Introduction
The importance of cyclic diguanosine monophosphate, c-di-
GMP, 1 (Figure 1), as a ubiquitous intracellular bacterial
signaling molecule responsible for regulating biofilm formation,
cell differentiation, and a variety of pathogenic processes has
recently been recognized.1 Biofilms are complex, multicellular
aggregations of bacteria attached to surfaces and encased in a
matrix of polysaccharides.2 Formation of such communities is
an ancient adaptation that allows bacteria to survive a range of
environmental challenges in a protected state. The medical,
industrial, and environmental threat of biofilms for human life
is profound, because they are far more resistant than free-living
bacteria to antimicrobials as well as to natural host defense
mechanisms.3 Not only are antibiotics typically ineffective in
treating chronic infections protected by biofilms, but low doses
of aminoglycosides have recently been shown to actually induce
biofilm formation in Pseudomonas aeruginosa and Escherichia
coli through a mechanism controlled by c-di-GMP.4
Specific c-di-GMP cyclases with GGDEF domains in many
different bacteria are responsible for formation of c-di-GMP
from two molecules of GTP,5,6 and a crystal structure has been
Figure 1. Structures of c-di-GMP, 1, c-GMP-IMP, 2, and oligoethylene-
oxide tethered c-di-GMP, 3a (n ) 4) and 3b (n ) 12).
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determined for the cyclase from Caulobacter crescentus.7 c-di-
GMP levels in P. aeruginosa, E. coli, Vibrio cholerae,
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C.; O’Toole, G. A. Trends Microbiol. 2001, 9, 34-39.
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Salmonella enterica, and Yersinia pestis have been shown to
be inversely regulated by such cyclases along with specific
phosphodiesterases associated with EAL domains.8,9 Many
bacterial proteins contain both GGDEF and EAL domains,10
and in C. crescentus, a phosphodiesterase with an active EAL
domain has recently been found to be regulated by the binding
of GTP to its neighboring catalytically inactive GGDEF
domain.11 Thus, control of c-di-GMP concentrations in bacteria
is extremely complex and may also involve gradients as well
as spatial localization.12
demonstrated a monovalent metal ion-dependent polymor-
phism.24 We found evidence for a stacked structure with Li+
and Na+, and the suggestion of a quartet structure with K+.
We now describe UV and CD data for additional metals that
further define these forms, and we have initiated more detailed
structural studies using 2D NMR. We have also synthesized a
mixed dimer containing guanosine and inosine, c-GMP-IMP,
2 (Figure 1), and found that the elimination of one amino group
precluded quartet formation. Further, in an effort to evaluate
the consequences of molecular constraints on the behavior of
1, we have connected two units together with a flexible
oligoethylene oxide tether of two different lengths, 3a where n
) 4 and 3b where n ) 12 (Figure 1). This constraint also has
a significant effect on the equilibrium, shifting it substantially
to the self-intercalated structure. The new data support the
existence of five distinct but related complexes for 1 that exist
in an equilibrium that is sensitive to both concentration and the
metal present.
As an intracellular signaling molecule, c-di-GMP is known
to regulate cell mobility,5 production of adhesive components,8,13
and other features of multicellular behavior in response to
environmental cues.12 It also plays an important role in the
pathogenic virulence of Salmonella in mice14 and virulence gene
expression in V. cholerae.15 A group of extracellular signaling
molecules termed autoinducers also regulate biofilm formation
and virulence in bacteria through a process known as quorum
sensing.16 Examples include acyl homoserine lactones,17 modi-
fied oligopeptides,18 modified quinolones,19 and a possible
universal autoinducer, AI-2.20 The integration of these extra-
cellular signals with the c-di-GMP pathways is only now being
explored.21 To date, the molecular mechanisms for these
complex pathways remain unknown and represent a major
challenge. Although several crystal structures for c-di-GMP
published some time ago revealed a self-intercalated arrange-
ment of two molecules,22,23 structures in solution have not been
determined. Understanding the structure and behavior of c-di-
GMP in solution would provide valuable insight into how it
might interact with the multitude of other molecules involved
in its pathways.
Experimental Procedures
Synthesis. The detailed procedures for synthesis of c-GMP-IMP,
2, and the tethered c-di-GMP, 3a and 3b, are described in the Supporting
Information.
UV. UV melting curves were obtained on an Aviv 14 UV
spectrophotometer using 1, 2, 5, and 10 mm path length cells. After
preparation in the appropriate buffer, samples were degassed, heated
to 80 °C, allowed to cool slowly to room temperature, and then placed
in a refrigerator for at least 15 h. UV absorbance spectra were obtained
using samples in the 1 mm cells.
CD. CD spectra were obtained on an Aviv model 60DS CD
spectrometer using a 1 mm cell. The samples were prepared as de-
scribed for UV. For time-dependent CD experiments, data at a given
wavelength were acquired immediately after the temperature was
changed. The effective temperature change rate of the instrument was
10 °C/min.
1D NMR. The 31P NMR spectra were acquired on a Varian Mercury
300 MHz spectrometer and referenced to 10% phosphoric acid in D2O;
the 1H NMR spectra were acquired on a Varian Unity 400 MHz
spectrometer.
We recently reported a new method for the efficient synthesis
of c-di-GMP, 1, as well as UV, CD, and 1D NMR results that
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2D NMR. 2D NOESY (nuclear Overhauser effect spectroscopy),
HMBC (heteronuclear multiple bond correlation), and HMQC (hetero-
nuclear multiple quantum coherence) spectra were acquired at 25 °C
on a Varian Inova 500 MHz spectrometer. NOESY data were collected
by 4096 (t2) times 512 (t1) complex data points with spectral widths
of 8000 Hz in both dimensions and transformed to spectra with 2048
(D1) times 1024 (D2) real data points. The mixing time for NOESY
spectra was 150 ms, the number of scans per each t1 increment was
16, and the relaxation delay for each scan was 2 s. Natural abundance
1H-13C HMQC and HMBC were acquired by 2048 (t2) times 256 (t1)
complex points with spectral widths of 8000 Hz (t2) and 18000 Hz
(t1) and transformed to spectra with 2048 (D1) times 512 (D2) real
points. The DEPT transfer delay in HMQC was chosen as 0.5/1JHC
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1339-1344.
1
with JHC ) 150 Hz, and the transfer delay in HMBC was chosen as
2,3
0.5/2,3JHC
(
J
) 12 Hz), which is close to multiples of 0.5/1JHC to
HC
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K. M.; Meijler, M. M.; Lowery, C. A.; Boldt, G. E.; Janda, K. D. Chem.
Commun. 2005, 4863-4865.
suppress HMQC peaks. The HMBC pulse sequence and data collection
mode (phase sensitive mode) were the same as HMQC with decoupling
1
during H data acquisition. The number of scans was 32 for HMQC
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U.S.A. 1990, 87, 3235-3239.
and 256 for HMBC, and the relaxation delay for each scan was 1 s.
1
All 2D NMR spectra used 1 s H presaturation during the relaxation
delay to suppress water.
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J. M.; van der Marel, G. A.; A., v. B. J.; Wang, A. H.-J. FEBS Lett. 1990,
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Polymorphism of c-di-GMP
A R T I C L E S
Figure 3. CD spectra of 1 at 5 °C in 0.1 cm cells of (A) 0.384 mM Li+
form (red), 0.395 mM Na+ form (blue), and 0.437 mM Mg2+ form (green)
in 10 mM LiOAc with 0.1 M LiCl, 10 mM sodium phosphate with 0.1 M
NaCl, and 10 mM Mg(OAc)2 with 0.1 M MgCl2, respectively, all at pH
7.0, and (B) 0.409 mM K+ form (black), 0.383 mM Rb+ form (blue), 0.382
mM Cs+ form (green), and 0.378 mM NH4+ form (red) in 10 mM potassium
phosphate with 0.1 M KCl, 10 mM RbOAc with 0.1 M RbCl, 10 mM
CsOAc with 0.1 M CsCl, and 10 mM NH4OAc with 0.1 M NH4Cl,
respectively, all at pH 7.0.
2B, black), but with lower stabilities. Tmax values from first
derivative curves are 30 °C/54 °C for K+ and <5 °C/20 °C for
+
Rb+. Although the UV melting profile for the NH4 form
appears to show a single conformational transition (Figure 2B,
red), its first derivative curve shows two broad overlapping
maxima, with Tmax values of 10 °C/20 °C.
Figure 2. Normalized UV melting profiles (A) at 250 nm of four different
concentrations of the Mg2+ form of 1 in 10 mM Mg(OAc)2 with 0.1 M
+
MgCl2, pH 7.0, (B) at 250 nm of the K+ (black), Rb+ (blue), and NH4
The UV spectrum of the K+ form of 1 displays a distinct
shoulder between 295 and 305 nm that is not present in the
(red) forms of 1 in 10 mM potassium phosphate with 0.1 M KCl, 10 mM
rubidium phosphate with 0.1 M RbCl, and 10 mM ammonium phosphate
with 0.1 M NH4Cl, respectively, all pH 7.0, and (C) at 305 nm of three
different concentrations of the K+ form of 1 in 10 mM potassium phosphate
with 0.1 M KCl.
+
Li+, Na+, or Mg2+ forms, and is weak in the Rb+ and NH4
forms (Supporting Information). Because similar shoulders were
found to be characteristic of intramolecular guanine quartet
structures formed by a series of oligodeoxynucleotides,26 we
now report UV melting experiments at 305 nm for a series of
three K+ samples with different concentrations (Figure 2C). As
seen with the quartet-forming oligonucleotides,26 the profiles
at 305 nm display hyperchromic changes rather than the typical
hypochromic changes seen at 250 nm. Together, the UV results
support the presence of one or more additional structures for 1
with K+ and Rb+ that contain guanine quartets. Further, because
intramolecular guanine quartet oligonucleotides that exhibit
hyperchromic behavior all contain some guanines with syn
conformations,27 by extension, at least one of the quartet
structures of 1 in K+ and Rb+ may also contain some syn
guanines.
Results
1. c-di-GMP. UV Melts. UV melting profiles at 250 nm for
four different concentrations of 1 with Mg2+ (Figure 2A) show
single conformational transitions similar to those with Li+ and
Na+,24 but reflect a more thermally stable structure. First
derivative curves for the most concentrated sample with each
metal (Supporting Information) show one clear maximum, with
T
max values of 8 °C for Na+, 10 °C for Li+, and 45 °C for Mg2+
.
The concentration dependence of the transitions with these
metals indicates that the structured forms have a molecularity
of at least 2.25 Further, the significant hypochromicity of the
UV curves indicates that stacking of the guanines plays an
important role in the structure.
The UV melting profile at 250 nm for the Rb+ form of 1
(Figure 2B, blue) shows two broad conformational transitions,
somewhat similar to those of the K+ form24 (included in Figure
CD Spectra. A CD spectrum of the Mg2+ form of 1 (Figure
3A, green) closely resembles those of Li+ (red)24 and
Na+ (blue).24 Upon heating to 75 °C, the bands all quickly
(26) Mergny, J.-L.; Phan, A.-T.; Lacroix, L. FEBS Lett. 1998, 435, 74-78.
(27) Keniry, M. A. Biopolymers 2001, 56, 123-146.
(25) Marky, L. A.; Breslauer, K. J. Biopolymers 1987, 26, 1601-1620.
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Figure 5. Cartoons of proposed structures of 1.
Figure 4. Plots of molar ellipticity at a given wavelength as a function of
time after (A) cooling or (B) heating preequilibrated samples of the Rb+
form of 1 in a 0.1 cm cell.
orientations, while those with intermolecular quartets that are
all anti do not have this 300 nm band.27,33
We have previously reported the slow appearance and
disappearance of the 215 and 309 nm bands for the K+ form of
1 (∼20 h).24 We now report similar behavior for the Rb+ form,
where nearly 8 h is required for the full appearance of the 215
nm band upon cooling from 75 to 5 °C (Figure 4A, blue).
Further, a similar cooling curve for the 254 nm band (red)
displays a fast increase over minutes to a maximum value,
followed by a slow decrease to a somewhat smaller value. This
slow decrease of the 254 nm band correlates with the slow
increase of the 215 nm band and reflects the shifting of the
central positive band that presumably occurs upon quartet
formation. Our previously reported data for the K+ form at 250
nm upon cooling24 also show a fast increase followed by a
similar slow decrease that is less pronounced than with Rb+.
These results support the initial formation of a bimolecular
stacked structure in K+ and Rb+, which then undergoes a slow
association and rearrangement to one or more quartet structures.
We had already shown the slow loss of the 215 and 250 nm
bands upon heating the K+ form from 5 to 40 °C (∼20 h),
consistent with both bands being associated with the quartet
structure.24 Heating the Rb+ form from 5 to 40 °C also shows
a slow loss of the 215 and 254 nm bands (Figure 4B), but over
only about 10 min, consistent with the lower thermal stability
of the Rb+ quartet structure.
diminish to small residual values (not shown). The positive band
at 257 nm and the negative band at 281 nm form a pattern that
is inverted relative to right-handed DNA and RNA, but is
similar to CD spectra of cyclic dithymidine monophosphate,28
5′-GMP and 3′-GMP in 0.1 M K+,29 left-handed 5′-5′ GpG,29
left-handed Z DNA,30 and diadenosine and diguanosine 5′,5′′′-
oligophosphates.31 For the Li+, Na+, and Mg2+ forms of 1, the
pattern for the 257 and 281 nm bands is consistent with the
possibility of a left-handed alignment of the guanine transition
moments.
The CD spectrum that we previously reported for the K+ form
of 1 displays additional bands at 215 nm (strong) and 309 nm
(weak)24 (Figure 3B, black). Further, its central positive band
appears at 252 nm rather than 257 nm. We now report CD
spectra for Rb+ (blue), Cs+ (green), and NH4 (red) forms
+
(Figure 3B). The presence of a similar 215 nm band for the
Rb+ form (although only one-half the intensity), but none for
the Cs+ form, demonstrates the same dependence on ion size
shown by guanine quartets.27,32 The Rb+ form, but not the Cs+
form, also displays a shift to lower wavelength for the central
positive band, although only about one-half as much as for the
K+ form. The NH4 form shows a weak 215 nm band and a
+
small shift of its central band. Positive CD bands near 300 nm
have been observed for oligodeoxynucleotides with intra-
molecular quartet structures that have a mixture of syn and anti
NMR Spectra. We previously reported 1D 1H and 31P NMR
spectra for the Li+, Na+, and K+ forms of ∼35 mM samples of
1 in 0.1 M LiCl, NaCl, or KCl, respectively, at pH 7.24 The
data for the K+ form were consistent with two different types
of quartet structures that were both stable even at 55 °C: one
type (∼50%) displayed two guanine H8 resonances and two
upfield 31P resonances, and another type (∼44%) displayed only
(28) Cantor, C. R.; Fairclough, R. H.; Newmark, R. A. Biochemistry 1969, 8,
3610-3617.
(29) Chantot, J.-F.; Haertle, T.; Guschlbauer, W. Biochemie 1974, 56, 501-
507.
(30) Pohl, F. M.; Jovin, T. M. J. Mol. Biol. 1972, 67, 375-396.
(31) Scott, J. F.; Zamecnik, P. C. Proc. Natl. Acad. Sci. U.S.A. 1969, 64, 1308-
1314. Holler, E.; Holmquist, B.; Vallee, B. L.; Taneja, K.; Zamecnik, P.
Biochemistry 1983, 22, 4924-4933.
(32) Hardin, C. C.; Perry, A. G.; White, K. Biopolymers 2001, 56, 147-194.
Davis, J. T. Angew. Chem., Int. Ed. 2004, 43, 668-698.
(33) Jin, R.; Gaffney, B. L.; Wang, C.; Jones, R. A.; Breslauer, K. J. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 8832-8836.
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Polymorphism of c-di-GMP
A R T I C L E S
1
Figure 6. NMR spectra of a 37 mM sample of the K+ form of 1 in 0.1 M KCl in 90/10 H2O/D2O at 25 °C, with its 1D H spectrum at the top, a portion
1
of its superimposed H-13C HMBC/HMQC spectrum at the center, and a portion of its 1H-1H NOESY spectrum at the bottom. Regions of interest are
labeled to correspond with the text.
one H8 resonance and only one downfield 31P resonance. Two
additional minor (∼2% and 4%) H8 and 31P resonances were
also present. 31P NMR spectra at 5 °C for the Li+ and Na+
forms of 1 also displayed upfield and downfield resonances,
but in more complex patterns.24 These signals had significantly
diminished at 55 °C, leaving a prominent central resonance near
H-bonded amino NOE highlighted in red). The 2D NMR data
described below demonstrate that the larger complexes exist in
both all-syn and all-anti conformations.
The 1D spectrum of a 37 mM sample of the K+ form of 1 in
KCl at pH 7 at 25 °C is shown at the top of Figure 6, with a
1
portion of its superimposed H-13C HMBC/HMQC spectrum
1
1
-1.0 ppm. H NMR spectra at 5 °C for the Li+ form showed
at the center, and a portion of its H-1H NOESY spectrum at
six guanine H8 resonances. These results are consistent with
the presence of a group of structures having lower stabilities
than those of the K+ form. Despite their different stabilities,
the bottom. Region A in the NOESY spectrum shows a set of
H8 to H1′ cross-peaks. The three major H8 resonances show
cross-peaks to H1′ resonances, of which one is significantly
stronger than the other two. The different NOE intensities are
consistent with a syn guanosine conformation for the stronger
NOE and anti conformations for the weaker NOEs.35 The H8
resonances at 7.75, 8.20, and 7.95 ppm, and the H1′ resonances
at 6.05, 6.19, and 5.77 ppm, are therefore labeled Os, Oa, and
Oa′ for octamolecular/syn and anti, respectively. There are no
cross-peaks between the syn and anti resonances, while cross-
peaks are observed between the two anti resonances. Of the
two minor H8 resonances, one shows a stronger cross-peak to
the H1′ region than the other, again indicating syn and anti
conformations. The H8 resonances at 7.85 and 8.26 ppm and
the H1′ resonances at 6.12 and 5.96 ppm are therefore labeled
1
the Li+, Na+, and K+ forms in 90% H2O all displayed H
resonances near 11 ppm consistent with hydrogen-bonded
guanine H1s, as well as broad resonances near 9 and 6.5 ppm
consistent with hydrogen-bonded and non-hydrogen-bonded
guanine amino protons, respectively.34 As described more fully
in the Discussion, these previous 1D NMR results and new 2D
NMR data introduced below for both the K+ and the Li+ forms
of 1 support the presence in varying amounts of five different
but related structures shown in the cartoons at the bottom of
Figure 5: one bimolecular self-intercalated structure (shown in
more detail at the top left of Figure 5), two tetramolecular and
two octamolecular structures, all of which contain guanine
quartets (shown at the top right of Figure 5, with the H8 to
(35) Wang, Y.; de los Santos, C.; Gao, X.; Greene, K.; Live, D.; Patel, D. J. J.
Mol. Biol. 1991, 222, 819-832. Smith, F. W.; Feigon, J. Nature (London)
1992, 356, 164-1168. Wang, K. Y.; McCurdy, S.; Shea, R. G.; Swami-
nathan, S.; Bolton, P. H. Biochemistry 1993, 32, 1899-1904.
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Feigon, J. Biochemistry 1993, 32, 8682-8692.
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Zhang et al.
Ts and Ta, for tetramolecular/syn and anti, respectively. These
NOESY cross-peaks had the same positive sign as the auto-
peaks due to negative NOE enhancements and built up quite
fast at a mixing time of 150 ms, which indicates that the
molecular species giving rise to them are in the slow tumbling
regime.36 Because neither the monomeric (MW ) 689) nor the
dimeric (MW ) 1378) forms of 1 in water are large enough to
generate strong negative NOE enhancements, our data suggest
that the four species present in this sample with K+ are larger
than the bimolecular form.
Region B in the HMBC/HMQC confirms the assignments,
because the H1′ resonances labeled Os and Ts show stronger
cross-peaks to their C4 than to their C8 resonances, while the
H1′ resonances labeled Oa and Oa′ do not show cross-peaks to
their C4 but do to their C8 resonances. Such differences have
been shown to support a syn assignment for the former and an
anti assignment for the latter.37
The HMBC/HMQC spectrum also allows assignment of the
amido H1 resonances, as shown in Region C. The large
downfield H1 resonance at 11.27 ppm (Os) correlates only with
the syn H8 resonance Os, while the pair of large upfield H1
resonances at 11.18 ppm (Oa and Oa′) both correlate only with
the two anti H8 resonances, Oa and Oa′. From Region D in the
NOESY spectrum, the broad hydrogen-bonded amino reso-
nances can then be assigned in terms of syn and anti. The syn
H1 resonance Os correlates only with the more upfield
hydrogen-bonded amino resonance at 8.81 ppm (labeled s), and
the anti H1 resonances Oa and Oa′ correlate with the more
downfield hydrogen-bonded amino resonance at 9.45 ppm
(labeled a). The minor downfield H1 resonance at 11.42 ppm,
labeled Ts,a because it presumably represents these two
overlapping resonances, shows a cross-peak to the region of
the syn amino resonance at 8.81 ppm.
Because in a given quartet each hydrogen-bonded amino is
close to the H8 of its neighbor (Figure 5), the pattern of their
NOE cross-peaks can define the arrangement of syn and anti
conformations within each quartet.38,39 Region E in the NOESY
spectrum shows that the broad anti hydrogen-bonded amino
region (a) has cross-peaks only to the two anti H8 resonances
Oa and Oa′, and the broad syn hydrogen-bonded amino region
(s) has a cross-peak only to the single syn H8 resonance Os.
Thus, we conclude that with K+, c-di-GMP exists primarily as
two major different quartet-containing species, with the Oa/
Oa′ structure being all-anti and the Os quartet structure being
all-syn. The different intensities of the Os and Oa resonances
do not support the presence of a mixed species with one anti
quartet and one syn quartet. All-anti quartets are well known
in parallel guanine-containing oligodeoxynucleotides,27,32 and
all-syn quartets have been found in lipophilic guanine quartet
assemblies.38,40
Figure 7. NMR spectra of a 33 mM sample of the Li+ form of 1 in 0.1 M
1
LiCl in 90/10 H2O/D2O at pH 7 at 25 °C, with its H 1D spectrum at the
top, a portion of its superimposed 1H-13C HMBC/HMQC spectrum at the
center, and a portion of its 1H-1H NOESY spectrum at the bottom. Regions
of interest are labeled to correspond with the text.
spectrum at the bottom. Although the relative intensities of the
resonances are quite different from those of the K+ form, the
2D spectra are strikingly similar. Region A in the NOESY
spectrum shows significant cross-peaks from the two most
upfield H8 resonances (Ts at 7.83 and Os at 7.74 ppm) to H1′
resonances, but weak cross-peaks from the three more downfield
H8 resonances (Ta at 8.25, Oa at 8.19, and Oa′ at 7.95 ppm) to
H1′ resonances. These NOE cross-peaks were also positive with
strongly negative NOE enhancements, similar to those with K+.
However, no cross-peak is seen for the sixth resonance at 7.90
ppm, labeled Ba for bimolecular/anti. These results indicate the
presence of the same syn and anti structures as with the K+
form, although in more similar amounts, and with one additional
structure. The HMBC/HMQC spectrum again confirms these
assignments, as shown in Region B. Further, for the sixth H8
resonance at 7.90 ppm (Ba), a cross-peak is seen between its
corresponding H1′ resonance at 5.84 ppm to its C8, but not to
its C4 resonance, indicating this additional structure has an anti
conformation.
A 1D NMR spectrum of a 33 mM sample of the Li+ form of
1 in 0.1 M LiCl at pH 7 at 25 °C is shown at the top of Figure
Region C of the HMBC/HMQC spectrum again allows
assignment of the amido H1 resonances. The most downfield
H1 resonance at 11.38 ppm (Ta) correlates only with the H8
resonance Ta at 8.25 ppm; the center two H1 resonances at
11.36 and 11.23 ppm (Ts and Os) correlate with H8 resonances
Ts at 7.83 ppm and Os at 7.74 ppm; and the upfield H1
resonance at 11.14 ppm (Oa,a′) correlates with H8 resonances
Oa at 8.19 ppm and Oa′ at 7.95 ppm. From Region D in the
NOESY spectrum, the hydrogen-bonded amino resonances again
can be assigned in terms of syn and anti. The anti H1 resonance
1
7, with a portion of its superimposed H-13C HMBC/HMQC
1
spectrum at the center and a portion of its H-1H NOESY
(36) Cavanagh, J.; Fairbrother, W. J.; Palmer, A. G., III; Skelta, N. J. Protein
NMR Spectroscopy, Academic Press: San Diego, CA, 1996; pp 289-290.
(37) Zhu, G.; Live, D.; Bax, A. J. Am. Chem. Soc. 1994, 116, 8370-8371.
(38) Marlow, A. L.; Mezzina, E.; Spada, G. P.; Masiero, S.; Davis, J. T.;
Gottarelli, G. J. Org. Chem. 1999, 64, 5116-5123.
(39) Phan, A. T.; Patel, D. J. J. Am. Chem. Soc. 2003, 125, 15021-15027.
(40) Forman, S. L.; Fettinger, J. C.; Pieraccini, S.; Gottarelli, G.; Davis, J. T. J.
Am. Chem. Soc. 2000, 122, 4060-4067.
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Polymorphism of c-di-GMP
A R T I C L E S
Ta correlates with the more downfield hydrogen-bonded amino
region at 9.42 ppm (a); the syn H1 resonance Ts correlates with
the more upfield hydrogen-bonded amino region at 8.85 ppm
(s); the syn resonance Os correlates with the s amino region;
and the anti H1 resonance Oa correlates with the a amino region.
Region E in the NOESY spectrum shows two cross-peaks for
the broad syn hydrogen-bonded amino region (s), one to the
H8 resonance Os and one to the H8 resonance Ts. The anti
hydrogen-bonded amino (a) is apparently too weak to show
analogous cross-peaks. These data are again consistent with an
Oa/Oa′ quartet structure that is all-anti and an Os quartet
structure that is all-syn.
The NOESY data described above suggest that the two major
and the two minor species with K+ all have molecular weights
larger than the bimolecular form. To obtain additional informa-
tion, NMR diffusion experiments were performed. The data
(Supporting Information) show that, with K+, the translational
diffusion rates of the two major forms, Oa and Os, are similar
to each other, but smaller than those of the two minor forms,
Ta and Ts, which are also similar to each other. These results
demonstrate that Oa and Os have a higher molecular weight
than Ta and Ts. The measured diffusion rates of these four
species agree well with the published range of DNA diffusion
rates.41 Diffusion data for the Li+ form show a similar pattern
in that the rates for Ta and Ts are larger than those for Oa and
Os. The rate for the fifth form, Ba, was considerably larger
than any of the other species, demonstrating a smaller molecular
weight that is consistent with a bimolecular complex. The
absolute values of the rates for the analogous species in the
two samples are slightly different, perhaps because the samples
are not identical.
These new UV, CD, and NMR results together present a
compelling case for the presence of a mixture of five different
structures that 1 can adopt, whose ratio is both metal and
concentration-dependent. As discussed more fully below, a
bimolecular structure (Ba) is dominant with Li+, Na+, Cs+, and
Mg2+ at dilute concentrations of 1, but is accompanied by one
or more quartet structures with K+ and Rb+. At the higher NMR
concentrations, with K+ the bimolecular structures associate and
rearrange primarily to two stable higher molecular weight quartet
structures, one all-syn (Os) and one all-anti (Oa), with two minor
lower molecular weight quartet structures also present, Ts and
Ta. With Li+, the bimolecular structure (Ba) predominates, but
both quartet structures (Oa, Os, Ta, and Ts) are also present.
2. c-GMP-IMP, 2. We have now synthesized a mixed cyclic
dimer containing one guanosine and one inosine, 2 (Figure 1),
by a method similar to what we previously reported for 1.24 In
brief, an intermolecular coupling using phosphoramidite chem-
istry was followed by an H-phosphonate cyclization, with the
H-phosphonate group also serving as a 3′ protecting group
during the initial coupling. The detailed procedures are described
in the Supporting Information. We describe below UV, CD,
and NMR spectra for this molecule that has one fewer amino
group than 1.
Figure 8. (A) Normalized UV melting profiles at 250 nm of four different
concentrations of Li+ (blue) and K+ (red) forms of 2 in 10 mM LiOAc
with 0.1 M LiCl, and 10 mM potassium phosphate with 0.1 M KCl,
respectively, both pH 7.0, and (B) CD spectra at 5 °C in 0.1 cm cells of
0.537 mM Li+ form (blue) and 0.544 mM K+ form (red) of 2 in 10 mM
LiOAc with 0.1 M LiCl, and 10 mM potassium phosphate with 0.1 M KCl,
respectively, both at pH 7.0.
than the Li+ form of 1. The Tmax values of the most concentrated
samples of both forms of 2 were found to be 18 °C, while the
T
max value for the Li+ form of 1 was only 10 °C. These results
suggest that loss of one of the guanine amino groups prevents
formation of quartet structures in K+, but allows a bimolecular
stacked structure.
CD Spectra. CD spectra (Figure 8B) for the Li+ and K+
forms of 2 are nearly identical and display much lower
intensities than the Li+ form of 1, perhaps due to the different
absorption properties of inosine. The lack of any bands near
215 and 305 nm supports the absence of quartet structures.
NMR Spectra. The 1D NMR spectra for 61 mM samples of
the Li+ and K+ forms of 2 at 5, 30, and 55 °C (Supporting
Information) are much simpler than those for 1. Both 31P NMR
spectra of 2 at 5 °C show only two resonances near -1.5 ppm,
one for each of the different phosphates in the molecule. Neither
of the 1H NMR spectra in 90% H2O at 5 °C shows H1
resonances near 11 ppm or amino resonances near 9 and 6.5
ppm, demonstrating the exchange of these protons with solvent.
Further, single sharp resonances are present for the guanine H8
1
and the inosine H8 and H2. As seen in Figure 9, the H-1H
NOESY spectrum for 2 with K+ does not show cross-peaks
1
between the H8 and H1′ resonances, but the H-13C HMBC/
HMQC spectrum does show cross-peaks between the two H1′
resonances and their C8 resonances, but not their C4 resonances,
indicating anti conformations. Thus, absence of one amino group
precludes quartet formation but allows a bimolecular anti stacked
structure in which the amido and amino protons exchange
readily with solvent.
UV Melts. UV melting profiles (Figure 8A) at 250 nm for
different concentrations of 2 with both Li+ and K+ show single
concentration-dependent conformational transitions that are
identical for the two cations and reflect a more stable structure
3. Oligoethyleneoxide Tethered c-di-GMP, 3a and 3b. We
have also linked two molecules of 1 together with flexible
(41) Lapham, J.; Rife, J. P.; Moore, P. B.; Crothers, D. M. J. Biomol. NMR
1997, 10, 255-262.
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A R T I C L E S
Zhang et al.
Figure 10. (A) Normalized UV melting profiles at 250 nm of two different
concentrations each of the Li+ (dark and light green) and K+ (red and blue)
forms of 3a and 3b in 10 mM LiOAc with 0.1 M LiCl, and 10 mM
potassium phosphate with 0.1 M KCl, respectively, both pH 7.0, and (B)
CD spectra at 5 °C in 0.1 cm cells of 0.227 mM Li+ form (dark green) and
0.227 mM K+ form (red) of 3a in 10 mM LiOAc with 0.1 M LiCl, and 10
mM potassium phosphate with 0.1 M KCl, respectively, and 0.209 mM
Li+ form (blue) and 0.212 mM K+ form (light green) of 3b in 10 mM
LiOAc with 0.1 M LiCl, and 10 mM potassium phosphate with 0.1 M KCl,
respectively, all at pH 7.0.
Figure 9. NMR spectra of a 60.8 mM sample of the K+ form of 2 in
90/10 H2O/D2O at 25 °C, with its 1D spectrum at the top, a portion of its
superimposed 1H-13C HMBC/HMQC spectrum at the center, and a portion
1
of its H-1H NOESY NMR spectrum at the bottom.
oligoethyleneoxide tethers of 4 (3a) or 12 (3b) units (Figure
1), thereby constraining their interactions. Their syntheses and
1D NMR spectra are described in the Supporting Information,
and their UV and CD data are presented below.
solvent. Single sharp resonances are present for each of the two
types of guanine H8, one of which is closer to the tether and
the other farther away. The K+ form also shows two dominant
sharp H8 resonances, but a cluster of several smaller H8
resonances is also present. Further, there are several small H1
resonances near 11 ppm and amino resonances near 9 and 6.5
ppm. The UV, CD, and NMR results for 3a with Li+ are
consistent with the presence of only a unimolecular self-
intercalated structure related to the bimolecular self-intercalated
structure of 1, Ba. With K+, the results support the presence of
a mixture with the major component being the self-intercalated
structure, but with small amounts of quartet complexes also
present. Because the concentration of 3a with K+ (26 mM in
terms of guanine) is similar to that of 1 with K+ (37 mM in
guanine), we conclude that the tether makes formation of a
quartet structure significantly more difficult, but allows the self-
intercalated structure to readily form.
UV Melts. UV melting profiles (Figure 10A) at 250 nm for
two different concentrations each of 3a and 3b with Li+ and
K+ show hypochromic conformational transitions that have very
little concentration dependence. Further, for both tether lengths,
the K+ forms are only slightly more stable than the Li+ forms.
The transitions are thus primarily intramolecular and cation
independent. The structured forms of 3b have Tmax values of
24 °C with Li+ and 26 °C with K+, significantly lower than
those of 3a, which have Tmax values of 42 °C with Li+ and 44
°C with K+. The similar hypochromicity in both metals indicates
that the tethers allow intramolecular formation of related stacked
structures, while the lower stabilities with the longer tether may
be due to entropic factors.
CD Spectra. CD spectra (Figure 10B) for the Li+ and K+
forms of 3a and 3b are nearly identical to each other and similar
to the CD spectrum of 1 with Li+. The lack of any bands near
215 or 305 nm shows that the tether precludes formation of
guanine quartets at this concentration.
Discussion
NMR Spectra (Supporting Information). As with 2, the
1
1D H and 31P NMR spectra of a 15 mM sample of the Li+
On the basis of the UV, CD, and NMR data presented above
and in our earlier report,24 we propose that c-di-GMP can adopt
five different but related structures, all participating in an
equilibrium that is sensitive to both concentration of 1 and the
metal present. The cartoons at the bottom of Figure 5 depict
these five structures: an anti bimolecular self-intercalated
complex, syn and anti versions of a tetramolecular quartet
complex, and syn and anti versions of an octamolecular self-
intercalated quartet complex. The UV concentration dependence
form of 3a are much simpler than those of 1. The 31P NMR
spectrum shows only three resonances near -2.0 ppm, one for
each type of phosphate in the molecule. The 31P NMR spectrum
of a 13 mM sample of the K+ form also gives three dominant
resonances near -2.0 ppm, but smaller clusters of signals are
present upfield and downfield as well. The 1H NMR spectrum
of the Li+ form shows no H1 signals near 11 ppm or amino
signals near 9 and 6.5 ppm, signifying their exchange with
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Polymorphism of c-di-GMP
A R T I C L E S
and hypochromicity, as well as the strong CD spectra, demon-
strate the presence of intermolecular guanine stacking. At dilute
concentrations with Li+, Na+, Cs+, or Mg2+, 1 forms the self-
intercalated bimolecular complex that had been found in crystal
(bottom of Figure 5, right). The presence of inner and outer
guanosines would account for the two sets of NMR resonances
in the all-anti form, in which models show that one set of H8
atoms appears to be significantly closer to nearby phosphates
than the other. In the all-syn form, models show the H8 atoms
all appear to be in more similar environments, which may
explain the failure to observe more than one set of resonances
at the field strength we used. Presumably, these large octa-
molecular complexes would most easily form by gradual
association and readjustment of four of the self-intercalated
bimolecular structures.
structures reported for 1 in the presence of Mg2+ or Co2+ 22,23
,
as well as for 1 associated with its cyclase from Caulobacter
crescentus.7 At the higher NMR concentrations with Li+, single
H8 and 31P resonances are seen from this structure. Although
this complex has two different types of phosphates and guanines
(inner and outer), and the amido H1s and aminos of the inner
guanines may be oriented so as to form hydrogen bonds with a
phosphate of its partner, our data are consistent with this self-
intercalated structure being in fast exchange on the NMR time
scale with an unstructured form. Thus, no amido or amino
resonances are present and only single H8, H1′, and 31P
resonances are observed. The lack of positive NOE cross-peaks
and the relatively large translational diffusion rate support the
lower molecular weight of a bimolecular complex.
A slow equilibrium would exist among these five different
complexes at high concentrations of 1, with K+ highly favoring
the two octamolecular complexes. Because the smaller, less
easily dehydrated Li+ is less able to stabilize quartets, the
resulting mixture has approximately similar amounts of the
bimolecular, the two tetramolecular, and the two octamolecular
complexes. Although Li+ does not normally stabilize quartet
structures, in this case the presence of two aligned guanines in
one molecule presents unique circumstances. c-GMP-IMP (2)
is unable to form any of these quartet structures because the
lack of one amino prevents the full quartet hydrogen bonding.
The tethered c-di-GMP (3a, and presumably 3b) are restricted
in their abilities to form quartet structures with K+. An
equilibrium among these five structures for c-di-GMP in a
bacterial cell might provide a range of options for the actions
of this signaling molecule that would significantly depend on
its own concentration as well as local K+ concentrations, or
the concentrations of other as yet unidentified effectors. The
subcellular compartmentalization and gradients that are known
to affect the signaling ability of c-di-GMP12 may be taking
advantage of the equilibrium we describe here. As first proposed
by Wang,23 the tetramolecular complexes may participate in
biological pathways by intercalating aromatic groups between
the two quartets. We further speculate that the octamolecular
complexes may serve as a way to sequester c-di-GMP in an
inactive state, with the bimolecular complex providing the active
form for signaling. Additional work in our laboratory is
underway to further characterize the structural details of these
five different complexes, as well as explore in more depth the
equilibrium among them.
Our data indicate that the tethered c-di-GMP (3a and 3b) as
well as c-GMP-IMP (2) are also able to adopt this self-
intercalated structure, despite the constraints of the tethers and
lack of one amino, respectively. Their UV spectra show
significant hypochromicity, and the CD spectra of 3a and 3b
are nearly identical to those of 1. The smaller intensity of the
CD spectrum of 2 is most likely because of the altered
absorption properties of inosine. Both the 1D and the 2D NMR
data are consistent with the predominant presence of this self-
intercalated structure in the Li+ and K+ forms of 2 and 3a.
At low concentrations of 1 with K+, Rb+, and NH4+, our
UV and CD data strongly support the presence of one or more
additional structures containing guanine quartets. The UV melt
of the K+ form of 1 at 250 nm displays two transitions, and its
melt at 305 nm shows characteristics of oligodeoxynucleotides
containing quartets with syn conformations. The CD spectra
with these metals display new bands at 215 and 309 nm that
are slow to appear and diminish, consistent with a higher
molecularity. At the higher NMR concentrations of the K+ form,
1H and 31P NMR resonances distinct from those corresponding
1
to the bimolecular structure persist at 55 °C. The H NMR
spectra, both 1D and 2D, are consistent with two major
structures, both of which have similar and relatively high
molecular weights. One has two sets of H8, H1′, and 31P
resonances, to which we can assign anti conformations, and the
other has only one set to which we can assign the syn
conformation. The NMR spectra also show two minor structures
with similar and relatively smaller molecular weights that are
still greater than the bimolecular form, one of which is anti and
one of which is syn.
Conclusion
We have demonstrated that c-di-GMP, an important bacterial
signaling molecule involved in biofilm formation and other
pathogenic processes, can adopt a mixture of five different but
related structures in ratios that are sensitive to both its
concentration and the metal present. A bimolecular self-
intercalated structure is dominant at dilute concentrations with
Li+, Na+, Cs+, and Mg2+, while it is accompanied by one or
more quartet complexes with K+, Rb+, and NH4+. At higher
concentrations with K+ the bimolecular structures associate and
rearrange primarily to two stable octamolecular self-intercalated
quartet complexes, one all-syn and one all-anti. Two minor
tetramolecular quartet complexes are also present, one all-syn
and one all-anti. At the higher concentrations with Li+ the
tetramolecular and octamolecular quartet complexes (each with
syn and anti versions) are present in approximately equal
amounts, along with the bimolecular structure.
In Wang’s reports on the self-intercalated crystal structure
of 1 with Mg2+ and Co2+ 23
he had speculated about the
,
possibility of its rearrangement to a cage-like complex of four
molecules of 1 containing two parallel guanine quartets 7 Å
apart (bottom of Figure 5, center). Although he described only
an all-anti version, it might also occur in an all-syn version,
and our data support the presence of these two tetramolecular
quartet structures as minor components in the K+ form of 1 at
high concentrations. However, if two such all-anti or all-syn
quartet complexes (a total of eight molecules of 1) were able
to self-intercalate, they should be stabilized by favorable stacking
(3.5 Å apart) and additional interactions with the K+ ions
9
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A R T I C L E S
Zhang et al.
Acknowledgment. This work was supported by a grant from
NIH (EB002809). We thank Kenneth J. Breslauer, James Elliott,
and Jens Vo¨lker in this department for assistance with the CD
and helpful discussions.
spectra of 1 in Li+, Na+, K+, Rb+, NH4+, and Mg2+
;
translational diffusion rates for 1 with K+ and Li+; 1D NMR
of 1, 2, 3a, and 3b. This material is available free of charge via
the Internet at http://pubs.acs.org.
Supporting Information Available: Synthesis of 2, 3a, and
3b; first derivative curves of UV melting profiles; full UV
JA0613714
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7024 J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006