Structural Basis for the High-Affinity Inhibition of Mammalian
Membranous Adenylyl Cyclase by 2?,3?-O-(N-Methylanthraniloyl)-
Melanie Hu ¨bner, Anshuman Dixit, Tung-Chung Mou, Gerald H. Lushington, Cibele Pinto,
Andreas Gille, Jens Geduhn, Burkhard Ko ¨nig, Stephen R. Sprang, and Roland Seifert
Department of Pharmacology and Toxicology, University of Regensburg, Regensburg, Germany (M.H.); Molecular Modeling
and Graphics Laboratory, University of Kansas, Lawrence, Kansas (A.D., G.H.L.); Center for Biomolecular Structure and
Dynamics, University of Montana, Missoula, Montana (T.-C.M., S.R.S.); Department of Pharmacology and Toxicology,
University of Kansas, Lawrence, Kansas (C.P., A.G.); Institute of Organic Chemistry, University of Regensburg, Regensburg,
Germany (J.G., B.K.); and Institute of Pharmacology, Medical School of Hannover, Hannover, Germany (R.S.)
Received February 20, 2011; accepted April 14, 2011
2?,3?-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most po-
tent inhibitor of mammalian membranous adenylyl cyclase
(mAC) 5 (AC5, Ki, 1 nM) yet discovered and surpasses the
potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:
1156–1165, 2009). AC5 inhibitors may be valuable drugs for
treatment of heart failure. The aim of this study was to elucidate
the structural basis for the high-affinity inhibition of mAC by
MANT-ITP. MANT-ITP was a considerably more potent inhibitor
of the purified catalytic domains VC1 and IIC2 of mAC than
MANT-GTP (Ki, 0.7 versus 18 nM). Moreover, there was con-
siderably more efficient fluorescence resonance energy transfer
between Trp1020 of IIC2 and the MANT group of MANT-ITP
compared with MANT-GTP, indicating optimal interaction of
the MANT group of MANT-ITP with the hydrophobic pocket.
The crystal structure of MANT-ITP in complex with the Gs?-
and forskolin-activated catalytic domains VC1:IIC2 compared
with the existing MANT-GTP crystal structure revealed only
subtle differences in binding mode. The higher affinity of
MANT-ITP to mAC compared with MANT-GTP is probably due
to fewer stereochemical constraints upon the nucleotide base
in the purine binding pocket, allowing a stronger interaction
with the hydrophobic regions of IIC2 domain, as assessed
by fluorescence spectroscopy. Stronger interaction is also
achieved in the phosphate-binding site. The triphosphate group
of MANT-ITP exhibits better metal coordination than the
triphosphate group of MANT-GTP, as confirmed by molecular
dynamics simulations. Collectively, the subtle differences in
ligand structure have profound effects on affinity for mAC.
Mammals express nine membranous AC isoforms (ACs
1–9) that play an important role in transmembrane signal
transduction (Sunahara et al., 1996; Tang and Hurley, 1998).
ACs are activated by the G-protein Gsvia numerous recep-
tors for hormones and neurotransmitters and catalyze the
production of the second-messenger cAMP. Studies with
AC5(?/?) mice indicate that potent and selective AC5 inhib-
itors may be valuable drugs for the treatment of heart fail-
ure, aging, bone loss, anxiety, and acute and chronic pain
(Chester and Watts, 2007; Rottlaender et al., 2007; Kim et
al., 2008; Okumura et al., 2009).
Previous studies from our group showed that 2?,3?-O-(N-
methylanthraniloyl) (MANT)-substituted purine nucleotides
are competitive AC inhibitors and inhibit AC isoforms differ-
entially (Gille and Seifert, 2003; Gille et al., 2004). Moreover,
MANT nucleotides are fluorescence probes of conformational
changes in the catalytic site of AC. Crystal structures of the
purified catalytic subunits of Gs?-bound mammalian AC [C1
subunit of AC5 (VC1) and C2 subunit of AC2 (IIC2) in com-
plex with MANT-GTP and MANT-ATP] were resolved (Mou
This work was supported by the National Institutes of Health National
Institute of Diabetes and Digestive and Kidney Diseases [Grant DK46371] (to
S.R.S); the Deutsche Forschungsgemeinschaft [Grant Se 529/5–2] (to R.S.);
and the Elite Network of Bavaria (graduate research scholarship to M.H.).
Article, publication date, and citation information can be found at
The online version of this article (available at http://molpharm.
aspetjournals.org) contains supplemental material.
ABBREVIATIONS: AC, adenylyl cyclase; MANT, 2?,3?-O-(N-methylanthraniloyl); FS, forskolin; GTP?S, guanosine 5?-[?-thio]triphosphate; MES,
2-(N-morpholino)ethanesulfonic acid; PDB, Protein Data Bank; GBSA, Generalized Born, augmented by solvent-accessible surface; FRET,
fluorescence resonance energy transfer; ?ex, excitation wavelength; ?em, emission wavelength; mAC, mammalian membranous adenylyl cyclase;
VCI and IIC2, the N- and C-terminal catalytic domains, respectively, from canine AC5 and rat AC2 expressed as soluble proteins.
Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 80:87–96, 2011
Vol. 80, No. 1
Printed in U.S.A.
et al., 2005, 2006). Those studies revealed a tripartite binding
pocket for MANT nucleotides in the catalytic core consisting
of sites for the base, the MANT-substituted ribosyl group,
and the polyphosphate chain. The combined analysis of en-
zymatic, fluorescence spectroscopy, crystallographic, and mo-
lecular modeling data revealed that the MANT group con-
tributes most to the AC binding energy, whereas the base
makes the smallest contribution. This is reflected by the fact
that MANT nucleotides exhibit up to 17,000-fold higher af-
finity for AC than their nonsubstituted parent nucleotides
and that purine and pyrimidine nucleotides exhibit similar
affinity for AC (Gille et al., 2004, 2005). On the basis of those
data, we proposed that ACs exhibit a high degree of confor-
mational flexibility, allowing the catalytic site to accommo-
date structurally diverse bases (Mou et al., 2006; Wang et al.,
During the course of our subsequent systematic studies on
2?,3?-O-ribosyl-substituted nucleotides as mAC inhibitors
(Go ¨ttle et al., 2009; Suryanarayana et al., 2009), we seren-
dipitously identified MANT-ITP as the most potent mAC
inhibitor known so far. Specifically, MANT-ITP inhibits AC5
with a Kivalue of 1 nM and mouse heart AC (predominantly
representing AC5) with a Kivalue of 4 nM. Compared with
MANT-GTP, MANT-ITP possesses a 55-fold higher affinity
for recombinant AC5. The base hypoxanthine differs chemi-
cally from the base guanine by the absence of an NH2group
at C2 of the purine ring (Fig. 1). Accordingly, hypoxanthine
cannot to form a hydrogen bond with the backbone oxygen of
Ile1019 (Mou et al., 2005). On the basis of analogous studies
with GTP, ITP, and XTP on GTP-binding proteins (Seifert et
al., 1999; Gille et al., 2003; Gille and Seifert, 2004), one would
also expect that the missing hydrogen bond should actually
reduce affinity of mAC for MANT-ITP. This notion is further
supported by the fact that MANT-XTP, bearing a keto group
at C2 of the purine ring (Fig. 1), binds to mAC with much
lower affinity than MANT-GTP because of electrostatic re-
pulsion of the keto group of the xanthine group by the back-
bone oxygen of Ile1019 (Mou et al., 2005).
Therefore, the aim of our present study was to elucidate
the structural basis for the high-affinity interaction of mAC
with MANT-ITP, using the VC1:IIC2 heterodimer as an es-
tablished model of the mAC catalytic domain. First, we de-
termined the affinities of VC1:IIC2 for MANT-GTP, MANT-
ITP, and MANT-XTP in enzyme activity assays (Gille et al.,
2004). Second, we performed fluorescence studies with the
three MANT nucleotides (Mou et al., 2006). Third, we ana-
lyzed the crystal structure of VC1:IIC2 in complex with
MANT-ITP and compared this structure with the known
VC1:IIC2 structure in complex with MANT-GTP (Mou et al.,
2005). Last, we conducted molecular dynamics simulations to
further evaluate the structural requirements for the high-
affinity interaction of MANT-ITP with AC5 in comparison to
MANT-GTP (Wang et al., 2007). Here, we show that even
subtle differences in ligand structure have profound effects
for interaction with mAC.
Materials and Methods
Materials. MANT-ITP was synthesized according to Hiratsuka
(1983) with the modifications described by Taha et al. (2009).
MANT-GTP and MANT-XTP were obtained from Jena Bioscience
(Jena, Germany). [?-32P]ATP (800 Ci/mmol) was purchased from
PerkinElmer Life and Analytical Sciences (Boston, MA). Alumi-
num oxide 90 active, neutral (activity 1, particle size 0.06–0.2 mm)
was purchased from Merck (Darmstadt, Germany). Bovine serum
albumin, fraction V, highest quality, was from Sigma-Aldrich (St.
Louis, MO). MnCl2tetrahydrate (highest quality) was from
Merck. FS was from LC Laboratories (Woburn, MA).
AC Activity Assay. AC activity was determined essentially as
described in the literature (Mou et al., 2005). In brief, reaction
mixtures contained 100 ?M ATP/Mn2?, 10 mM MnCl2, and MANT
Fig. 1. Structure of MANT nucleoside 5?-triphosphates
(NTPs). Represented are MANT-ITP, MANT-GTP, and
MANT-XTP, the MANT nucleotides used for enzymatic
studies, fluorescence spectroscopy, crystallography, and
structure activity evaluation. The MANT group isomer-
izes between the 2? and 3?-O-ribosyl function. Note the
different substitution of the C2 carbon atom of the
purine ring in the various nucleotides.
Hu ¨bner et al.
nucleotides at concentrations from 0.1 nM to 1 mM as appropriate to
obtain saturated inhibition curves. In addition, assay tubes con-
tained VC1 (8 nM) and IIC2 (40 nM). For experiments with Gs?-
GTP?S, tubes contained VCI (3 nM), IIC2 (15 nM) and Gs?-GTP?S
(51 nM). Reactions were conducted in the presence of 100 ?M FS.
After a 2-min preincubation at 30°C, reactions were initiated by
adding 20 ?l of reaction mixture containing (final) 1.0 ?Ci/tube
[?-32P]ATP, 0.1 mM cAMP, and 100 mM KCl in 25 mM HEPES/
NaOH, pH 7.4. Reactions were conducted for 10–20 min at 30°C
and were terminated by the addition of 20 ?l of 2.2 N HCl. Denatured
protein was precipitated by a 1-min centrifugation at 25°C and
15,000g. Sixty-five microliters of the supernatant fluid were applied
onto disposable columns filled with 1.3 g of neutral alumina.
[32P]cAMP was separated from [?-32P]ATP by elution of [32P]cAMP
with 4 ml of 0.1 M ammonium acetate, pH 7.0. Recovery of
[32P]cAMP was ?80% as assessed with [3H]cAMP as standard.
[32P]cAMP was determined by liquid scintillation counting using
Ecolume scintillation cocktail (Thermo Fisher Scientific, Waltham,
MA). Competition isotherms were analyzed by nonlinear regression
using the Prism 4.0 software (GraphPad Software, San Diego, CA).
Fluorescence Spectroscopy. All experiments were conducted
using a Cary Eclipse fluorescence spectrophotometer equipped with
a Peltier-thermostated multicell holder at 25°C (Varian, Palo Alto,
CA). Measurements were performed in a quartz fluorescence micro-
cuvette (Hellma, Plainview, NY). The final assay volume was 150 ?l.
Reaction mixtures contained a buffer consisting of 100 mM KCl, 10
mM MnCl2, and 25 mM HEPES/NaOH, pH 7.4. Steady-state emis-
sion spectra were recorded at low speed with ?ex? 350 nm (?em?
370–500 nm) and ?ex? 280 nm (?em? 300–500 nm) with various
MANT nucleotides (1 ?M each) in the absence and presence of 5 ?M
VC1 plus 25 ?M IIC2 without and with 100 ?M FS. Fluorescence
recordings were analyzed with the spectrum package of the Cary
Eclipse software (Varian, Walnut Creek, CA). Baseline fluorescence
(buffer alone) was subtracted. Figure 2 shows superimposed original
fluorescence recordings representative for two to three independent
experiments with at least two different batches of VC1:IIC2.
Preparation of Proteins. The catalytic subunits VC1, IIC2, and
GTP?S-activated Gs? were expressed in Escherichia coli BL21(DE3)
cells containing pREP4 plasmid. The plasmids encode the wild-type
C1a domain of canine AC 5 (residues 364–580), the C2a domain of rat
AC 2 (residues 874-1018), and bovine Gs? (residues 1–396). VC1 and
Gs? constructs were expressed with a hexahistidine tag at their N-
and C-termini, respectively. They were purified and stored as de-
scribed previously (Tesmer et al., 2002). Gs? was further activated by
incubation with 500 ?M GTP?S and 2 mM MgCl2at 30°C for 2 h, and
digestion of the complex with trypsin was necessary for crystalliza-
tion work. The smaller fragment, containing residues 39–387 was
further purified using a nickel-nitrilotriacetic acid-NTA column fol-
lowed by MonoQ anion exchange chromatography.
Complex Formation and Crystallization with MANT-ITP.
To form a stable heterotrimeric complex, the recombinant proteins
were mixed in the following order VC1-IIC2–Gs?–GTP?S in a molar
ratio 1.5:1:1. Thereafter, forskolin (200 ?M) was added to further
stabilize the complex. The protein mixture was incubated on ice for
at least 30 min and then applied to tandem-arranged Superdex 75
and 200 gel filtration columns (GE Healthcare, Chalfont St. Giles,
Buckinghamshire, UK). Only the fractions containing the complex
were collected and concentrated to ?9.5 mg/ml in a buffer of 20 mM
HEPES, pH 8.0, 1 mM EDTA, 2 mM MgCl2, 5 mM dithiothreitol, 100
mM NaCl, 200 ?M 7-acetyl-7-[O-(N-methylpiperazino)-?-butyryl)]-
forskolin, and 500 ?M GTP?S. The concentrated protein solution was
used to grow crystals via the sitting-drop method with a reservoir
solution of 7.2% (m/v) polyethylene glycol 8000, 0.5 M NaCl, and 0.1
M MES, pH 5.4, at 16°C for 3 to 4 weeks. Large crystals were soaked
with 2 mM MANT-ITP and 3 mM MnCl2for at least 2 h at room
temperature and then harvested in a cryoprotectant consisting of 9%
(m/v) polyethylene glycol 8000, 30% (m/v) polyethylene glycol 400,
0.1 M MES, pH 5.4, 0.5 M NaCl, 20 mM HEPES, pH 8.0, 1 mM
EDTA, 2 mM dithiothreitol, 200 ?M 7-acetyl-7-[O-(N-methyl-piper-
azino)-?-butyryl)]-forskolin, 100 ?M GTP?S, 2 mM MANT-ITP, and
3 mM MnCl2. The cryoprotected crystals were mounted in 0.1- to
0.2-mm loops and stored in liquid nitrogen.
Structure Determination and Model Refinement. Diffraction
data sets were collected at the Stanford Synchrotron Radiation
Lightsource SSRL-SMB-MC 9-1 beamline (Stanford, CA) by the os-
cillation method (1°/frame, 60 s/frame). The incident beam wave-
length was 0.9795 Å. The images were processed using the HKL2000
package (Otwinowski and Minor, 1997). Because of anisotropy of the
plate-type crystal, data with l index ?21 were excluded from the data
set. Structures were determined by molecular replacement using the
structure of the Gs?-GTP?S:VC1:IIC2 complex [Protein Data Bank
(PDB) code 1AZS] as the initial phasing model (Tesmer et al., 1997).
Atomic positions and thermal parameters of the mAC structure were
refined by Refmac5.5 using the CCP4 program suite (Collaborative
Computational Project Number 4, 1994). MANT-ITP and metal ions
in the structure were located in the weighted |Fo|?|Fc| omit map
computed with phases from the refined model. The model was iter-
atively improved by manual refitting into weighted 2|Fo|?|Fc|map
using the computer graphics program Coot (Emsley and Cowtan,
2004) and subsequent refinement cycles with CCP4. The refined
crystal structure was visualized with PyMOL (DeLano, 2002). Coor-
dinates for the MANT-ITP:Mn2?structure were deposited in the
Protein Data Bank with the code PDB 3G82.
Molecular Dynamics Simulations. Atomic coordinates of pro-
tein-ligand complexes for MANT-ITP and MANT-GTP interacting
with VC1:IIC2 were extracted from structures PDB 3G82 and PDB
1TL7. Ligand parametrization for FS, MANT-GTP, and MANT-ITP
was performed by extracting the relevant ligands from the crystal
structures and editing them via SYBYL (Tripos Inc., St. Louis, MO)
to ensure proper representation of valence and bond types. Thereaf-
ter, the antechamber (Wang et al., 2001) module of AMBER10
(http://www.ambermd.org) was used to assign Austin model 1-bond
charge correction (AM1-BCC) charges to the ligands and calculate
force field parameters for them. To avoid excessively large computa-
tional expense, only the VC1:IIC2 portion of each crystal structure
(plus ligand and cofactor) was retained; bound Gs?-GTP?S was re-
moved. The tleap module of AMBER10 was used for the preparation
of topology and coordinate files for the protein-ligand complexes
using the ff99SB force field parameters for protein and the ante-
chamber-calculated parameters for ligands. The parameters for
Mn2?were obtained from Bradbrook et al. (1998). The structures
PDB 3G82 and PDB 1TL7 were separately solvated in water boxes
with buffering distance of 10 Å. Assuming normal charge states of
ionizable groups corresponding to pH 7.0, Na?ions were added to
achieve charge neutrality and to mimic the biological environment
Our primary simulation engine for probing the differences in the
dynamic nature of the two complexes was NAMD (Phillips et al.,
2005; http://www.ks.uiuc.edu/Research/namd/), which was chosen
because of its excellent parallel scalability, which enabled us to
perform the simulations in an expeditious manner on 32 nodes of a
large Linux cluster. To relieve severe steric contacts and instances of
higher energy conformations that might destabilize the molecular
dynamics integrator at later stages, the system was subjected to
initial minimization of 2 ? 104steps wherein the protein backbone
was held fixed (to relax surrounding water molecules and computa-
tionally specified hydrogen positions), followed by 2 ? 104steps of
minimization without positional constraints (i.e., to allow the system
to relax freely). The resulting low-energy (?0 K) model was then
equilibrated to room temperature (?300 K) by gradually increasing
the system temperature in increments of 20 K up to a target tem-
perature of 300 K. At each of the 15 temperature increments, 1.5 ?
104dynamics steps (30 ps) were run while employing a restraint of
10 kcal ? mol?1? Å?2on protein ? carbons (C?) to avoid any prospect
of unrealistic denaturation behavior. Thereafter, the system was
equilibrated for 15 ? 104steps (300 ps) at 310 K (approximate
Crystal Structure of Adenylyl Cyclase with MANT-ITP
physiological temperature) under conditions of constant volume and
temperature, and then for a further 15 ? 104steps (300 ps) at 310 K
using a Langevin piston (constant pressure and temperature) to
achieve uniform pressure of 1 bar. Finally the restraints were re-
moved and the system was equilibrated for 5 ? 106steps (1 ns) to
prepare the system for final analysis. For the latter, a constant
pressure and temperature simulation was run on the equilibrated
structure for 9.6 ns, keeping the temperature at 310 K and pressure
at 1 bar using Langevin piston coupling algorithm. The integration
time step of the simulations was set to 2.0 fs, the SHAKE algorithm
was used to constrain the lengths of all chemical bonds involving
hydrogen atoms at their equilibrium values, and the geometry was
restrained rigidly via the SETTLE algorithm. Nonbonded van der
Waals interactions were treated via a switching function at 10 Å and
reaching zero influence at a distance of 12 Å, and the Mn2?cofactors
were subjected to a soft harmonic constraint (1 kcal/mol) to encour-
age adherence to equilibrium approach distances relative to the
ligand phosphate oxygens as determined from an earlier simulation.
The particle-mesh Ewald algorithm as implied in NAMD was used to
handle long-range electrostatic forces.
To probe relative contributions to the total free energy of the
complexes, we employed the GBSA (Generalized Born, augmented by
solvent-accessible surface) method (Weiser et al., 1999; Bashford and
Case, 2000; Onufriev et al., 2000). For this, the trajectory obtained
from the molecular dynamics run was converted into individual
coordinate files at 100-ps intervals excluding the initial 1.0 ns of the
simulation. Thus, 86 frames were used in GBSA calculation for each
complex. AMBER parameters (using bonding radii for GBSA calcu-
lation) were then generated from the resulting structures using tleap
and were subsequently applied in GBSA calculation. The radii for
λex= 350 nm
λex =280 nm
λex= 280 nm
λex= 350 nm
λex= 280 nm
λex= 350 nm
Fig. 2. Fluorescence emission spectra of MANT-GTP,
MANT-ITP and MANT-XTP. Emission at ?ex? 280 nm
(?em? 300–500 nm) and at ?ex? 350 nm (?em? 370–500
nm) are represented. Experiments were conducted at 25°C.
Addition of MANT nucleotides (1 ?M), blue lines; subse-
quent addition of VC1 (5 ?M) and IIC2 (25 ?M), green
lines; subsequent addition of FS (100 ?M), red lines.
Dashed lines in A to C represent endogenous tyrosine/
tryptophan fluorescence of VC1:IIC2. Reaction mixtures
contained a buffer of 100 mM KCl, 10 mM MnCl2, and 25
mM HEPES/NaOH, pH 7.4. Three independent experi-
ments with at least two different batches of VC1:IIC2 were
performed. A and B, MANT-GTP; C and D, MANT-ITP; E
and F, MANT-XTP. Fluorescence intensities are shown in
arbitrary units (a.u.). For FRET (A, C, and E, ?ex? 280
nm), the fluorescence observed with VC1:IIC2 was set to
100%. In direct fluorescence experiments (B, D, and
F, ?ex? 350 nm), the fluorescence observed with MANT
nucleotides alone was set to 100%.
Hu ¨bner et al.
Mn2?was specified as being the same as Mg2?, because GBSA
parameters for Mn2?are unavailable in AMBER. The change of
conformational entropy was not considered. Apart from a number of
settings chosen specifically for this analysis, default GBSA parame-
ters were employed [implicit Generalized Born (IGB) ? 2 to specify
the Onufriev, Bashford and Case (OBC) model, GBSA ? 1 to choose
the linear combination of pairwise overlaps (LCPO) method for
solvent-accessible surface area calculation, external dielectric
(EXTDIEL) ? 78.50 to set the solvent dielectric for room-
temperature water, internal dielectric (INTDIEL) ? 1.0 to specify
a solute dielectric for the system, and surface tension
(SURFTEN) ? 0.0072 to describe the water surface tension].
Enzymatic Studies. Table 1 lists the Kivalues of MANT-
GTP, MANT-ITP, and MANT-XTP for inhibition of VC1:IIC2
under maximally stimulatory conditions (i.e., in the presence
of Gs?-GTP?S) as well as under submaximally stimulatory
conditions (i.e., in the absence of Gs?-GTP?S). The latter
reflects the assay conditions for the fluorescence spectroscopy
studies. In accordance with the data for AC5 and mouse
heart AC (Go ¨ttle et al., 2009), under maximally stimulatory
conditions, MANT-ITP was a considerably more potent in-
hibitor of VC1:IIC2 than MANT-GTP, which, in turn, was
much more potent than MANT-XTP. The omission of Gs?-
GTP?S reduced the overall MANT nucleotide potencies by 4-
to 10-fold, but the rank order of affinity of nucleotides was
preserved. Collectively, the enzyme inhibition studies with
purified catalytic subunits of mAC confirmed the exception-
ally high affinity of MANT-ITP for the catalytic site previ-
ously reported for AC5 and mouse heart AC (Go ¨ttle et al.,
Fluorescence Spectroscopy Studies. To elucidate fur-
ther differences in the interaction of MANT nucleotides with
VC1:IIC2, we exploited the fluorescence properties of these
nucleotides (Jameson and Eccleston, 1997) and the ability of
the MANT group to bind to a hydrophobic pocket in the
interface of VC1:IIC2 (Mou et al., 2005, 2006). The emission
spectra of nucleotides at ?ex? 350 nm for direct excitation of
the MANT group (Jameson and Eccleston, 1997), and at
?ex? 280 nm for analysis of FRET between Trp1020 in IIC2
and the MANT group were determined (Mou et al., 2005).
Fluorescence studies were performed in the presence of a
5-fold excess of VC1 relative to MANT nucleotides to ensure
quantitative ligand binding to the catalytic site.
At ?ex? 280 nm, MANT nucleotides were only minimally
excited, whereas at ?ex? 350 nm, they showed substantial
intrinsic fluorescence signals with an emission peak at ?450
nm (Fig. 2, blue tracings). The dashed black lines indicate the
endogenous tryptophan fluorescence of VC1:IIC2 at ?ex?
280 nm (i.e., the fluorescence in the absence of MANT nucle-
otide). After the addition of VC1:IIC2, at ?ex? 280 nm,
MANT-ITP exhibited a much higher basal FRET signal, as
revealed by a second emission peak at ?em? 420 nm, than
MANT-GTP (Fig. 2, A and C, green tracings). At ?ex? 350
nm, the interaction of MANT-ITP with VC1:IIC2 resulted in
considerably higher increases in fluorescence compared with
MANT-GTP (Fig. 2, B and D, green tracings). The blue shift
of the fluorescence emission (Mou et al., 2005) was similar for
MANT-ITP and MANT-GTP. FS (100 ?M) increased basal
FRET and direct fluorescence with MANT-GTP more effec-
tively than with MANT-ITP, but the absolute FRET with
MANT-ITP was still considerably larger than with MANT-
GTP. These data suggest that the MANT group of MANT-ITP
binds to the hydrophobic pocket in mAC more effectively
than MANT-GTP. Compared with MANT-GTP and MANT-
ITP, MANT-XTP exhibited only minimal FRET and direct
fluorescence (Fig. 2, E and F), reflecting suboptimal binding
of the xanthine ring to mAC and suboptimal insertion of the
MANT group into the hydrophobic pocket (Mou et al., 2005).
Crystallographic Studies. To better understand the
high inhibitory potency of MANT-ITP at ACs 1, 2, and 5
(Go ¨ttle et al., 2009), as well as at the catalytic domains
VC1:IIC2 (Table 1), crystallographic studies were conducted.
Crystals of FS-VC1:IIC2-Gs?-GTP?S were soaked with 2 mM
MANT-ITP and 3 mM MnCl2. The structure of the MANT-
ITP:Mn2?complex was determined at a resolution of 3.1 Å by
molecular replacement using the structure of the Gs?-
GTP?S:VC1:IIC2 complex as the initial phasing model (Tes-
mer et al., 1997) (PDB code 1AZS). Crystallographic data
collection and refinement statistics are summarized in Table 2.
Interactions between Gs?-GTP?S and the pseudosymmetric
VC1 and IIC2 catalytic domains center largely on IIC2, as
described previously (Tesmer et al., 1997). The two domains
form a very large interface, facilitating the binding of MANT-
ITP at the catalytic site, and FS at the pseudo–dyad-related
site. The structure was very similar to the corresponding
complex with MANT-GTP (Fig. 1) (Mou et al., 2005) (PDB
code 1TL7). Superimposing the two structures revealed that
the overall placement of VC1 and IIC2 did not differ greatly
from each other with the root-mean-square deviation less
than 0.5 Å (Fig. 3C).
MANT-ITP was modeled into the continuous |Fo|-|Fc|
map in the binding pocket. The electron density was more
consistent with 3?-O-MANT-ITP than the 2?-O-MANT isomer
(Fig. 3A). Our previous crystallographic studies with MANT-
GTP and MANT-ATP gave similar results (Mou et al., 2005;
200). Difference electron density peaks corresponding to the
two Mn2?ions are observed; wherein the A site exhibits
lower occupancy than the B site, suggesting that the Mn2?
ion is bound more tightly at the B site, as observed in other
mAC crystal structures (Mou et al., 2005, 2006).
The overall conformation of MANT-ITP bound to VC1:IIC2
is similar to that observed for MANT-GTP. The ligands are
similar with respect to interactions with protein residues and
metal ion coordination (Fig. 3, B and D). The |Fo|-|Fc|
electron density for MANT-ITP is well defined and similar to
Inhibitory potencies of MANT-NTPs on the catalytic activity of VC1:
Catalytic activities of VC1:IIC2 were determined as described under Materials and
Methods. Reactions were conducted in the presence of 10 mM MnCl2and 100 ?M FS
in the absence or presence of Gs?-GTP?S. Data were analyzed by nonlinear regres-
sion to calculate Kivalues. The catalytic activity of C1/C2 in the presence of Mn2??
FS ? Gs?-GTP?S with 100 ?M ATP as substrate was 2700 ? 350 nmol ? mg?1? min?1
and in the presence of Mn2?? FS, the activity was 300 ? 110 nmol ? mg?1? min?1.
The Kmvalues for VC1:IIC2 were reported previously (Mou et al., 2005) for each
experimental condition (430 and 620 ?M, respectively) and were used to calculate Ki
values from IC50values. Data are the mean values ? S.D. of two to four independent
experiments performed in duplicates with at least two different batches of protein.
? FS ? Gs?-GTP?S
18 ? 6.0
0.7 ? 0.1
1,200 ? 370
130 ? 20
7.0 ? 3.2
4,600 ? 510
Crystal Structure of Adenylyl Cyclase with MANT-ITP
that observed for MANT-GTP, indicating no obvious differ-
ence in the conformation of the two ligands. One noticeable
difference is that the hypoxanthine ring of MANT-ITP lacks
an amino group at the C2 position (Fig. 1) that could form a
hydrogen bond with the side chain of Ile1019 of IIC2 domain
(MANT-GTP 2.6 Å; Fig. 3D). Despite the absence of this
interaction, MANT-ITP binds with higher affinity to VC1:
IIC2 than MANT-GTP, as indicated above (Table 1). The
absence of the C2-amino group allows a higher degree of
mobility of the purine moiety than in the case of MANT-
GTP (Fig. 3D). The purine ring of MANT-ITP is somewhat
more deeply inserted in the mAC binding site than that of
In the crystal structure, the MANT group of MANT-ITP
and MANT-GTP form similar interactions at the ?4?-?1 do-
main interface. The aryl function of MANT engages in hy-
Val1024, Val1026, and Trp1020 (Fig. 4). The increase in the
fluorescence signal for MANT-ITP may be due to changes of
the relative positions of Trp1020 and the MANT group (Fig.
2). However, electron density is weak for the MANT moiety,
suggesting that it is poorly ordered within the binding site
(Fig. 3A). The oxygen of the carbonyl group of MANT-ITP is
closer to Asn1025 than that of MANT-GTP (Fig. 4). However,
in this orientation, the side chain of Asn1025 does not form
an H-bond with the carbonyl group of MANT-ITP.
A stronger interaction of MANT-ITP with the phosphate
binding site is supported by two observations. The side chain
Lys1065 of IIC2 interacts with the ?- and ?-phosphates of
MANT-ITP, whereas the amino group of Lys1065 in the
MANT-GTP structure is oriented only toward the ?-phos-
phate (Fig. 3C). The amino group of Lys1065 is also closer to
the oxygen of the ?-phosphate of MANT-ITP (2.9 Å) compared
with the oxygen of the ?-phosphate of MANT-GTP (3.1 Å).
Of particular interest are the differences between the
MANT-ITP and MANT-GTP complexes with respect to coor-
dination of the Mn2?ions by the nucleotide ? and ? phos-
phates and the side chain of Asp396. The carboxylate group
of Asp396 coordinates the two metal ions, together with the
nucleotide phosphates and Ile397 and Asp440 of VC1 (Fig.
3B). Diffuse electron density in the region of the ?-phosphate
is consistent with conformational heterogeneity of the ligand
and, consequently, its ligation of the Mn2?ions at the A and
B sites (Fig. 3A). This may account in part for differences in
metal coordination for MANT-ITP and MANT-GTP. The
Mn2?ions in the MANT-ITP structure form close contacts
with the ?- and ?-phosphates, whereas in the MANT-GTP
structure, the Mn2?ions interact predominantly with the
?-phosphate. The ?-phosphate group of MANT-ITP also
seems to be more tightly coordinated, because of the shorter
(2.8 Å) contact with the carbonyl group of Ile397, than is the
case for MANT-GTP (3.4 Å). The phosphate site plays a
crucial role for binding affinity because phosphate group
removal dramatically reduces inhibitor potency (Gille et al.,
Molecular Dynamics Simulations. Our molecular dy-
namics simulations corroborate the finding that MANT-ITP
has affinity for VC1:IIC2 higher than that of MANT-GTP.
Specifically, the GBSA free energy analysis determined a
total free energy for the MANT-ITP/VC1:IIC2 system of
?99.71 ? 10.29 kcal/mol (mean ? S.D., as derived from
sampling 86 time steps), whereas the MANT-GTP/VC1:IIC2
system complex had a free energy of only ?69.60 ? 8.94
kcal/mol. Nearly all of this difference can be accounted for in
terms of two factors: the NT-ITP complex was predicted
to derive a substantial advantage from electrostatics
(?951.66 ? 36.75 versus ?847.41 ? 38.66 kcal/mol for
MANT-GTP) but to incur a penalty in terms of a less favor-
able solvation profile (890.24 ? 30.79 versus 819.68 ? 34.53
kcal/mol). To ascertain the source of these interaction differ-
ences, we performed distance analysis over a set of ?9600
sample conformers (taken from each 1000 time steps in the
9.6-ns simulation) to identify any specific receptor-ligand
interatomic distances that differed significantly from one
ligand to the other. The most important distinction was
found not in comparing the hypoxanthine and guanine rings
(the only true chemical difference between the two ligands),
but rather in the interactions between the Mn2?ions of the
receptor and an oxygen on the ?-phosphate group of the
ligand triphosphate moiety. Specifically, although both li-
gands orient one ?-, one ?-, and one ?-phosphate oxygen in
close association to one or more of the metal ions, the dy-
namic conformation of MANT-ITP enables a second ? oxygen
to remain significantly closer to a Mn2?ion (2.36 ? 0.08 Å)
than is the case for MANT-GTP (3.29 ? 0.65 Å). The sub-
stantially shorter mean distance between the MANT-ITP ?
oxygen and the nearest Mn2?suggests a significantly stron-
ger electrostatic interaction (Fig. 5). Specifically, a twist in
the orientation of the nucleobase propagates through the
ribosyl moiety (in ways that do not substantially affect the
interactions of these groups) to the triphosphate chain.
The triphosphate group is pulled approximately 0.7 Å closer
to the nucleobase, which has the important effect of position-
ing both ? oxygens of MANT-ITP (rather than just one for
MANT-GTP) in an orientation that permits interaction with
Mn2?ions. It is noteworthy that the much greater S.D.
Summary of crystallography data collection and refinement statistics
Cell constants (Å)
No. of crystals
Resolution range for refinement (Å)c
Total reflections used
No. of protein atoms
No. water molecules
No. ligand atoms
rmsd bond length (Å)
rmsd bond angle (°)
Average B-factor (Å2)
rmsd, root mean square deviation.
aNumbers in parentheses correspond to the statistical data from the highest
bRsym? ?h?i| I(h) - I(h)i| / ?h?iI(h)i, where I(h) is the mean intensity after
cBecause of anisotropy, data with an l index greater than 21 were omitted from
dRwork? ?h||Fo(h)|-|Fc(h)||/?h|Fo(h)|, Fo(h) and Fc(h) are the observed and
calculated structure factors, respectively.
e5.1% of the complete data set was excluded from refinement to calculate Rfree.
Hu ¨bner et al.
observed for the ? oxygen to Mn2?interaction in MANT-GTP
complex suggests that the manganese ion can interact more
readily with solvent in the latter case, which is a plausible
cause for the more favorable solvation energy for the MANT-
GTP complex. Supplemental Fig. 1 and Supplemental Tables
1 to 3 provide further details on the differences in interac-
tions of MANT-GTP and MANT-IP with VC1:IIC2.
We developed a tripartite pharmacophore model for mAC
with a binding site for the base, the (substituted) ribosyl
group, and the polyphosphate chain (Mou et al., 2005, 2006).
In a previous study, we reported that MANT-GTP?S and
MANT-inosine 5?-[?-thio]triphosphate are similarly potent
inhibitors of various mACs (Gille et al., 2004). Omission of
the MANT group reduces inhibitor affinity by several orders
of magnitude, highlighting the importance of the MANT-
binding site for high inhibitor affinity (Gille et al., 2004). It is
noteworthy that unsubstituted inosine 5?-[?-thio]triphos-
phate is a more potent mAC inhibitor than GTP?S, whereas
GTP and ITP exhibit similar affinity (Gille et al., 2005). In
contrast, MANT-ITP is a considerably more potent mAC
inhibitor than MANT-GTP (Go ¨ttle et al., 2009). These data
indicate that subtle structural changes in nucleotide inhibi-
tors (exchange guanine and hypoxanthine; exchange ?-phos-
phate and ?-thiophosphate) substantially change the relative
contributions of the three binding subsites in mAC for inhib-
Fig. 3. Binding mode of MANT-ITP and two Mn2?ions in the catalytic site. MANT-ITP and two metal ions are bound in the cleft between the soluble C1a
and C2a domains. VC1 and IIC2 are colored wheat and light pink, respectively. MANT-ITP is shown as stick model, carbon atoms are cyan, nitrogen atoms
are dark blue, oxygen atoms are red, and phosphorus atoms are green. The two Mn2?ions are shown as orange spheres. A, difference electron density for
3?-O-MANT-ITP and Mn2?
the |Fo|-|Fc| electron density for the two Mn2?ions contoured at 5 ?. The coordinates for the ligands were omitted from the phasing model. The secondary
structure elements of the complex are labeled as defined previously (Tesmer et al., 1997). B, detailed view of substrate binding site of VC1:IIC2 with
MANT-ITP:Mn2?. The catalytic site of VC1:IIC2 shows MANT-ITP, A- and B- site of two Mn2?ions and the protein residues that are responsible for ligand
interaction. The interaction among protein residues and MANT-ITP, Mn2?are shown as dashed gray lines. C, superimposed crystal structures of
shown as a transparent yellow stick model (Protein Data Bank code 1TL7) (Mou et al., 2005). The protein residues are in almost identical conformation, and
the inhibitors are situated in the substrate binding pocket in a similar fashion. D, superimposed purine binding site of 3?-O-MANT-ITP and 3?-O-MANT-
GTP. The interaction of the hypoxanthine ring and guanine ring of MANT-ITP and MANT-GTP are shown as dashed black and olive green lines,
respectively. The distances of hydrogen bond between the hypoxanthine ring and surrounding protein residues of MANT-ITP are indicated in
Ångstroms. The hydrogen bond between Ile1019 and the amino group of MANT-GTP is missing in the MANT-ITP structure. Lys938 and the
oxygen of the hypoxanthine ring are further apart. The hypoxanthine ring has less binding constraint in the purine binding pocket in comparison
to the guanine ring of MANT-GTP.
.The lime green wire represents the |Fo|-|Fc| electron density for MANT-ITP contoured at 2.5 ?. The blue wire corresponds to
Crystal Structure of Adenylyl Cyclase with MANT-ITP
itor affinity. The main goal of the present study was to
elucidate the structural basis for the exceptionally high af-
finity of mAC for MANT-ITP (Go ¨ttle et al., 2009).
Our data suggest that a balance of binding energies among
the three pharmacophores in the mAC binding site (Mou et
al., 2006) considerably affects the affinity of mAC for MANT-
ITP versus MANT-GTP. The MANT-ITP structure shares the
common features with previously published mAC structures
in complex with 2?,3?-substituted purine and pyrimidine nu-
cleotide inhibitors, where the base, triphosphate, and 2?,3?-
ribose substituents reside in three distinct grooves of the
substrate binding site (Mou et al., 2005, 2006). The overall
conformations of MANT-ITP and MANT-GTP show only min-
imal differences. This result is not surprising. The crystal
structures were derived with a racemic mixture of 2?-O- and
3?-O-MANT-ITP. Both structures favor the 3?-O-MANT iso-
mer for binding to the catalytic center as shown with the
MANT-GTP and MANT-ATP crystals (Mou et al., 2005,
A very intriguing finding was the subtle difference in bind-
ing mode of MANT-ITP versus MANT-GTP at the purine-
binding site in comparison with the exceptionally high inhib-
itory potency of the nucleotide. Usually, high inhibitory
potency is accomplished through strong binding of the inhib-
itor to the active site of the enzyme. In this case, there were
actually fewer protein-ligand interactions because of a miss-
ing hydrogen bond in hypoxanthine compared with guanine
(Figs. 1 and 3D). However, a gain in affinity is related not
only to the number of hydrogen bonds but also to hydrophobic
interactions, residual mobility of the ligand, and partial sol-
vation of the binding pocket (Gohlke and Klebe, 2002). The
loss of hydrogen bonds does not necessarily lead to a decrease
in binding affinity of a ligand to a protein as assessed by
molecular thermodynamic and crystallographic studies of
thermolysin inhibitors (Morgan et al., 1991). Binding of those
inhibitors is dependent not only on hydrogen bonding but
also on metal coordination and higher ligand basicity (Gro-
belny et al., 1989). For binding of MANT-ITP, the absence of
the C(2) amino group eliminates a potential hydrogen bond
but at the same time reduces spatial constraints at the pu-
rine binding site, thereby allowing other protein-nucleotide
interactions to be optimized. Our crystallographic studies
and molecular dynamics simulations clearly show that posi-
tioning of the ?-oxygen of the triphosphate chain of MANT-
ITP allows for more favorable interactions with Mn2?than
the triphosphate chain of MANT-GTP, providing a straight-
forward explanation for the observed difference in affinity
(Figs. 3 and 5).
Direct fluorescence experiments and FRET studies de-
tected stronger hydrophobic interaction of the MANT group
of MANT-ITP with the hydrophobic pocket compared with
that of MANT-GTP (Fig. 2). In contrast, binding of MANT-
XTP does not result in an increase in direct fluorescence or
FRET. Compared with MANT-ITP and MANT-GTP, MANT-
XTP is at least 60-fold less potent at VC1:IIC2 (Table 1).
MANT-XTP bears an oxygen at the C2-position of the purine
ring (Fig. 1) that interacts unfavorably with Asp1018 in the
base-binding pocket. This may move the MANT group away
from the hydrophobic pocket, leading to a strong decrease in
inhibitory potency and fluorescence emission. As suggested
for metal-phosphate interactions, alleviation of binding con-
straints at the purine-binding pocket may allow MANT-ITP
to form more favorable nonpolar interactions with the MANT
binding site than is possible for MANT-GTP, resulting in
larger fluorescence signals.
MANT-ITP is the most potent competitive inhibitor of
membranous ACs known so far (Table 1) (Go ¨ttle et al., 2009).
MANT-ITP is most useful as fluorescence probe for biophys-
ical (Fig. 2) and crystallographic studies (Figs. 3 and 4) to
obtain detailed molecular information on ligand/receptor in-
teractions. Another application of MANT-ITP is to use this
Fig. 4. MANT-binding site. A detailed view of the MANT-binding site is
depicted. MANT-ITP is shown as a stick model; carbon atoms are cyan,
nitrogen atoms are dark blue, oxygen atoms are red and one phosphorus
atom is displayed in green. VC1 and IIC2 are colored wheat and light
pink, respectively. MANT-GTP is shown as a transparent yellow stick
model. The carbonyl group of MANT-ITP is in closer contact to Asn1025
but does not interact with the side chain of Asn1025 in this orientation.
Apart from this, no conformational differences between MANT-ITP and
MANT-GTP are detected. However, MANT-ITP might exert stronger
hydrophobic interactions due to changes of the relative positions of
Trp1020 and the MANT group.
Fig. 5. Comparison of the binding of MANT-ITP and MANT-GTP by
molecular dynamics simulations. Overlaid graphical representations of
the terminal (t ? 9.6 ns) time steps for the MANT-GTP (CPK-colored
sticks with green carbons and orange phosphorus atoms) and MANT-ITP
(CPK-colored sticks with cyan carbons and tan phosphorus atoms) inter-
acting with the VC1:IIC2 receptor (pale green ribbons) and its cofactor
Mn2?ions (magenta spheres for MANT-GTP simulation and purple for
MANT-ITP). Additional details on differences in the interactions of
MANT-GTP and MANT-ITP with VC1:IIC2 are provided in Supplemen-
tal Fig. 1 and Supplemental Tables 1 to 3.
Hu ¨bner et al.
ligand in screening programs for the development of AC
inhibitors, avoiding the use of the classic radioactive AC
assay with [?-32P]ATP as substrate. Specifically, upon bind-
ing to the substrate-binding site, nonfluorescent inhibitors
would quench the large basal or FS-stimulated direct fluo-
rescence or FRET signals. This assay is even feasible to
obtain information on relative inhibitor affinity through the
analysis of cumulative concentration/quench curves in a sin-
gle cuvette (Geduhn et al., 2011). Fluorescence assays with
MANT-ITP could also be useful to identify allosteric AC
However, the usefulness of MANT-ITP per se as starting
point for the development of AC isoform-specific inhibitors,
particularly AC5 inhibitors for the treatment of heart failure,
aging, bone loss, anxiety, and acute and chronic pain (Ches-
ter and Watts, 2007; Rottlaender et al., 2007; Kim et al.,
2008; Okumura et al., 2009) is limited for several reasons.
First, MANT-ITP per se is membrane-impermeant and
would have to be delivered as a prodrug (Laux et al., 2004;
Hu ¨bner et al., 2011). Second, the selectivity of MANT-ITP for
AC5 relative to other AC isoforms is only very moderate
(Go ¨ttle et al., 2009), because the catalytic site of the mem-
branous AC isoforms is highly conserved (Mou et al., 2005).
Third, one should also keep in mind that hypoxanthine-based
nucleotides bind not only to mACs but also to other signal-
transducing proteins, including soluble guanylyl cyclase and
G-proteins (Seifert et al., 1999; Gille et al., 2003, 2004, 2005;
Hu ¨bner et al., 2011). Accordingly, pleiotropic effects unre-
lated to direct mAC inhibition could arise (Hu ¨bner et al.,
2011). Despite these reservations regarding MANT-ITP, the
long-term goal of obtaining isoform-specific AC inhibitors is
not elusive. Specifically, we have shown that certain bis-
MANT-substituted nucleotides are very potent inhibitors of
the Bordetella pertussis AC toxin CyaA (Geduhn et al., 2011),
with high selectivity relative to mammalian ACs. The iden-
tification of Bis-MANT nucleotides as potent and selective
CyaA inhibitors resulted from a relatively small medicinal
chemistry program and not from an extensive high-through-
put screening effort. Unfortunately, Bis-MANT nucleotides
are not very potent inhibitors of mACs because the catalytic
site of these ACs is not spacious enough.
In conclusion, our data confirm the three-site pharmaco-
phore model already postulated in previous studies (Mou et
al., 2005, 2006). Our data also show that small differences in
ligand structure can have a profound impact on interactions
with mAC. The one missing opportunity for hydrogen bond-
ing in MANT-ITP relative to MANT-GTP enhances mobility
of the ligand in the catalytic site, thereby facilitating hydro-
phobic interactions of the MANT group with surrounding
amino acids and optimal positioning of the triphosphate
chain to divalent cations. Together, these factors result in
exceptionally high-affinity binding of MANT-ITP for mAC.
We thank the staff at the Stanford Synchrotron Radiation Light-
source SMB-MC 9-1 beamline (Stanford, CA) for their assistance
with data collection. Thanks are also due to the reviewers for a
helpful critique that stimulated us to resolve the mAC:MANT-ITP
crystal structure and to conduct molecular dynamics simulations.
Participated in research design: Hu ¨bner, Dixit, Mou, Lushington,
Pinto, Gille, Geduhn, Ko ¨nig, Sprang, and Seifert.
Conducted experiments: Hu ¨bner, Mou, Pinto, Gille, and Seifert.
Contributed new reagents or analytic tools: Geduhn and Ko ¨nig.
Performed data analysis: Hu ¨bner, Dixit, Mou, Lushington, Pinto,
Gille, Geduhn, Sprang, and Seifert.
Wrote or contributed to the writing of the manuscript: Hu ¨bner,
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Hu ¨bner et al.