Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the optogenetics protein RhoGC

Article abstract of DOI:10.1074/jbc.M117.812685

RhoGC is a fusion protein from the aquatic fungus Blastocladiella emersonii, combining a type I rhodopsin domain with a guanylyl cyclase domain. It has generated excitement as an optogenetics tool for the manipulation of cyclic nucleotide signaling pathways. To investigate the regulation of the cyclase activity, we isolated the guanylyl cyclase domain from Escherichia coli with (GCwCC_Rho) and without (GC_Rho) the coiledcoil linker. Both constructs were constitutively active but were monomeric as determined by size-exclusion chromatography and analytical ultracentrifugation, whereas other class III nucleotidyl cyclases are functional dimers. We also observed that crystals of GC_Rho have only a monomer in an asymmetric unit. Dimers formed when crystals were grown in the presence of the non-cyclizable substrate analog 2′,3′-dideoxyguanosine- 5′-triphosphate, MnCl₂, and tartrate, but their quaternary structure did not conform to the canonical pairing expected for class III enzymes. Moreover, the structure contained a disulfide bond formed with an active-site Cys residue required for activity. We consider it unlikely that the disulfide would form under intracellular reducing conditions, raising the possibility that this unusual dimer might have a biologically relevant role in the regulation of full-length RhoGC. Although we did not observe it with direct methods, a functional dimer was identified as the active state by following the dependence of activity on total enzymeconcentration. The low affinity observed for GC_Rhomonomers is unusual for this enzyme class and suggests that dimer formation may contribute to light activation of the full-length protein.

Full text of DOI:10.1074/jbc.M117.812685

JBC Papers in Press. Published on November 8, 2017 as Manuscript M117.812685
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.812685
Structure and monomer/dimer equilibrium of guanylyl cyclase
Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of
the optogenetics protein RhoGC
Ramasamy P. Kumar†, Benjamin R. Morehouse†, Josiane Fofana, Melissa M. Trieu, Daniel H. Zhou,
Molly O. Lorenz, and Daniel D. Oprian*
Department of Biochemistry, Brandeis University, Waltham, MA 02454
Author Information
† These authors contributed equally to this work
Corresponding Author
*Department of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02454. Telephone:
781-736-2322. Fax: 781-736-8487. E-mail: oprian@brandeis.edu.
Funding
This work was supported by National Institutes of Health Grants T32GM007596 (B.R.M. and D.H.Z.)
and EY007965 (D.D.O.).
Running title
Structure and monomer/dimer equilibrium of guanylyl cyclase
Keywords
Blastocladiella emersonii, microbial rhodopsin, guanylate cyclase, cyclic GMP (cGMP), class III
nucleotidyl cyclase, crystal structure, disulfide, optogenetics.
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and monomer/dimer equilibrium of guanylyl cyclase
ABSTRACT
RhoGC is a fusion protein from the
fungus Blastocladiella emersonii,
for this enzyme class and suggests that dimer
formation may contribute to light activation of the
full-length protein.
aquatic
combining a type I rhodopsin domain with a
guanylyl cyclase domain. It has generated
excitement as an optogenetics tool for the
manipulation of cyclic nucleotide signaling
pathways. To investigate the regulation of the
cyclase activity, we isolated the guanylyl cyclase
The rhodopsin-guanylyl cyclase fusion
protein RhoGC was first identified in the
phototactic fungus Blastocladiella emersonii by
Avelar et al.(1). It is a unique protein composed of
a type I (microbial) rhodopsin(2,3) domain fused
to a guanylyl cyclase catalytic domain showing
homology to the rod outer segment guanylyl
cyclase from vertebrate retina. It was the first
retinylidene protein described that is directly
coupled to an enzyme. The protein is localized to
the B. emersonii eyespot and is essential for
zoospore phototaxis, a pathway known to signal
through the second messenger cGMP. RhoGC
contains four domains (Fig. 1): an intracellular N-
terminal domain of unknown function (N-term), a
type I rhodopsin domain (rho), a coiled-coil
domain (CC), and a guanylyl cyclase domain
(GC).
domain from Escherichia coli with (GCwCCRho
)
and without (GCRho) the coiled-coil (CC) linker.
Both constructs were constitutively active but
were monomeric as determined by size-exclusion
chromatography and analytical ultracentrifugation,
whereas other class III nucleotidyl cyclases are
functional dimers. We also observed that crystals
of GCRho have only a monomer in an asymmetric
unit. Dimers formed when crystals were grown in
the presence of the noncyclizable substrate analog
2’,3’-dideoxyguanosine-5’-triphosphate (ddGTP),
MnCl2, and tartrate, but their quaternary structure
did not conform to the canonical pairing expected
for class III enzymes. Moreover, the structure
contained a disulfide bond formed with an active-
site Cys residue required for activity. We consider
it unlikely that the disulfide would form under
intracellular reducing conditions, raising the
possibility that this unusual dimer might have a
biologically relevant role in the regulation of full-
length RhoGC. While we did not observe it with
direct methods, a functional dimer was identified
as the active state by following the dependence of
activity on total enzyme concentration. The low
affinity observed for GCRho monomers is unusual
RhoGC is of considerable interest as an
optogenetic tool for control of cyclic nucleotide
signaling pathways(4,5), and has now been joined
by another type I rhodopsin fusion protein,
RhoPDE(6,7), with potential as an optogenetic
tool. The gene for RhoPDE was found in the
genome of the choanoflagellate Salpingoeca
rosetta and encodes a fusion of the rhodopsin
domain with a cGMP specific phosphodiesterase
catalytic domain.
We
have
developed
an
expression/purification system and performed
preliminary characterization for RhoGC in
2

Structure and monomer/dimer equilibrium of guanylyl cyclase
preparation of studies to elucidate the mechanism
Ni-affinity chromatography, and show here that
the isolated protein is constitutively active in
catalyzing the formation of cGMP. It is a
monomer in solution, crystallizes with a monomer
in the asymmetric unit, but has a non-linear
dependence of activity on enzyme concentration
that is consistent with transient formation of an
active state dimer. Crystals of a dimeric form of
GCRho were obtained from crystallization trials in
the presence of the non-cyclizable substrate analog
2’,3’-dideoxyguanosine-5’-triphosphate (ddGTP),
Mn2+, and added potassium sodium tartrate, but
the two protomers are arranged in a head-to-head
conformation distinctly different from the
canonical head-to-tail wreath-like structures first
described for adenylyl cyclases(13,15) and then
later for the guanylyl cyclases(9-11). We
anticipate that both monomer and dimer structures
reported here for GCRho will help guide
mutagenesis experiments focused on the
mechanism by which light controls activity of the
cyclase domain in full-length RhoGC.
by which light controls guanylyl cyclase activity
in the enzyme(8). We now turn our attention to the
individual domains to more fully identify their
functional contribution to the full-length RhoGC.
We begin here with the guanylyl cyclase domain,
GCRho
.
Crystal structures of GC catalytic domains
have been determined for bacterial(9), algal(10),
and human(11) proteins (see Fig. S1 for sequence
alignment). GC domains from the cyanobacterium
Synechocystis PCC6803 (GCCya2)(9) and the
unicellular algae Chlamydomonas reinhardtii
(GCCYG12)(10) were crystallized as homodimers,
whereas the GC domain (GCHum) from the human
soluble guanylyl cyclase, sGC(11), was
crystallized as both homodimer and heterodimer,
although the enzyme is active only in the
heterodimeric form. The active sites form at the
dimer interface where catalytic residues are
contributed by both monomers. Hence,
dimerization is necessary for catalysis in this class
of enzymes(12,13). All three GC structures solved
so far are the substrate free form and reported to
be an inactive ‘open’ conformation(11).
Comparing the ‘open’ GC structures with the
active ‘closed’ conformation of the adenylyl
cyclase catalytic domain(14), AC, revealed that
the protomers in the inactive GC dimers are
rotated by 7-8° for GCCya2 and GCCYG12, and by
26° for GCHum relative to the monomers in the
active AC structure.
RESULTS
Expression and purification of GCRho and
GCwCCRho
Conventional PCR was used to construct
genes for the isolated guanylyl cyclase domain,
with (GCwCCRho, residues 397-627) and without
(GCRho, residues 443-627) the connecting linker
(CC, residues 397-442), from full-length RhoGC
(Fig. 1). Both constructs contained a His6-tag on
the C-terminus for purification of the protein by
We have expressed the catalytic domain of
RhoGC (GCRho) in E. coli, purified the protein by
3

Structure and monomer/dimer equilibrium of guanylyl cyclase
Ni-affinity chromatography. Initial tests in E. coli
domain, as has been suggested for other systems,
but this is clearly not the case if GCwCCRho is
compared to GC in the presence of Mg2+, where
GCwCCRho displays the higher activity. At 10 µM
enzyme concentration, the activities were as
follows: GCRho with Mn2+ = 21 ± 1 µM/s;
GCwCCRho with Mn2+ = 1.0 ± 0.1 µM/s;
GCwCCRho with Mg2+ = 0.71 ± 0.06 µM/s; and
GCRho with Mg2+ = 0.064 ± 0.002 µM/s.
showed that the proteins were expressed to high
level in T7-express cells under standard induction
conditions (1 mM IPTG at OD600 followed by
incubation for 3 hours at 37 °C), but neither
protein was found in the soluble fraction after cell
lysis and centrifugation. Subsequent attempts
varying induction conditions, growth and
expression temperature, and a change of cell lines
met with limited success. In the end, coexpression
with the molecular chaperone proteins GroEL and
GroES(16) dramatically increased the amount of
both GCwCCRho and GCRho in the soluble fraction
such that we could purify (Fig. 2A) on average
The dependence of activity on GTP
concentration for GCRho in the presence of MnCl2
is presented in the plot of Fig. S2, where the data
are fit with a Hill-type equation using the
parameters: S0.5 = 0.9 ± 0.1 mM, Vmax = 16 ± 1
µM/s, and n = 1.2 ± 0.3.
about
50
mg
of
protein
(determined
spectrophotometrically using an extinction
coefficient ε280nm = 31,065 M-1cm-1) per liter of cell
All known class III cyclases function as
dimers(12,17,18). They can be either hetero- or
homodimers, with active sites formed at the
dimeric interface. Given the robust constitutive
activity of both GCwCCRho and GCRho, we
anticipated that the proteins were assembled as
homodimers in solution. Contrary to expectations,
both proteins ran as monomers by size exclusion
chromatography (SEC) on a Superdex-200 column
(Fig. 2C), with molecular mass estimates for the
peak elution fractions from standards within 5%
error of the calculated molecular mass of
monomeric GCRho (21.5 kDa) and GCwCCRho
(26.5 kDa).
culture,
a
quantity sufficient to undertake
biochemical and structural characterization of the
proteins.
GCwCCRho and GCRho both displayed
constitutive guanylyl cyclase activity that varied
depending on whether Mn2+ or Mg2+ was used for
the metal ion. As is shown in Fig. 2B, GCRho
displayed much higher activity in the presence of
Mn2+ than in Mg2+, consistent with observations
from other isolated guanylyl(9-11,16) and
adenylyl cyclase(12) domains. However, this is
not the case when the CC linker was included.
GCwCCRho displays about the same activity in
Mn2+ as in Mg2+, and both are significantly less
active than the GC domain alone in Mn2+. These
data seem to suggest that the role of CC might be
to attenuate the constitutive activity of the GCRho
Structure of GC
Initial crystallization trials were conducted
with GCwCCRho
.
After three months, a small
rectangular shaped crystal appeared in a PEG3350
4

Structure and monomer/dimer equilibrium of guanylyl cyclase
solution. The structure of the enzyme was
Comparison with other guanylyl cyclases
determined to 1.6 Å resolution by molecular
replacement using human soluble guanylyl cyclase
(PDB entry 2WZ1) as a search model. The protein
had crystallized in space group P22121 with one
molecule in the asymmetric unit. Although the
full-length GCwCCRho construct was purified and
used in the crystallization trials, electron density
was not observed for the CC linker, likely as a
result of proteolysis or disorder in the crystal.
Repeated trials in the presence of sodium azide to
prevent bacterial growth did not yield crystals, and
further attempts to optimize crystallization
conditions were performed with GCRho alone.
Crystals of GCRho formed in the same
crystallization conditions after about three weeks,
still in space group P22121 with one molecule in
the asymmetric unit, and the structure was
determined to 1.13 Å resolution and refined to an
Rwork and Rfree of 0.188 and 0.195, respectively.
Importantly, crystal packing did not reveal dimer
formation with symmetry-related molecules.
Synechocystis guanylyl cyclase Cya2. The
catalytic domain (GCCya2) of the Cya2 guanylyl
cyclase from the cyanobacterium Synechocystis
PCC6803 crystallized as a homodimer, and its
structure was determined at 2.3 Å resolution(9). A
superpose of the GCRho monomer and molecule A
of GCCya2 shows that the two proteins are very
similar in overall fold with a rmsd for Ca atoms of
0.9 Å. The a3-b4 loop in GCRho is four residues
shorter than the corresponding loop in GCCya2, and
the a3 helix is two turns longer in GCCya2. This
structural feature appears to be unique to GCCya2
since the respective region in Chlamydomonas(10)
and human sGCs(11) were similar to that of the
GCRho monomer. The GCRho b4-b5 and b7-b8
loops are each one residue shorter than in GCCya2
whereas GCCya2 has an extended C-terminus.
,
Chlamydomonas reinhardtii guanylyl
cyclase CYG12. The catalytic domain (GCCYG12
)
of the soluble guanylate cyclase from the green
algae Chlamydomonas reinhardtii crystallized as a
homodimer, and its structure was solved at 2.6 Å
The overall structure of the GCRho
catalytic domain has five alpha helices and eight
beta strands (Fig. 3) as has been reported
previously for other GC(9-11) and AC
structures(13,15). Clear electron density was
observed for residues T443 through K626, with
the exception of two missing loop regions not
modeled in the final structure: G559-N562 in the
b4-b5 loop, which has a major role in dimer
formation, and V608-K616 in the b7-b8 loop,
which carries one of the eight conserved catalytic
residues, K612.
resolution(10).
A
superpose of the GCRho
monomer and molecule A of GCCYG12 shows that
the two proteins are very similar in overall fold
with a rmsd for Ca atoms of 1.2 Å. The a2-b2 and
a5-b7 loops in GCRho are one and two residues
shorter, respectively, than the corresponding
regions in GCCYG12. The C. reinhardii protein also
has an additional short a6 helix which is not
observed in other GC structures.
Human soluble guanylyl cyclase. The
catalytic domain (GCHum) of the human soluble
5

Structure and monomer/dimer equilibrium of guanylyl cyclase
guanylate cyclase crystallized as a heterodimer,
and its structure was solved at 1.6
resolution(11). superpose of the GCRho
observed, corresponding to dimer, monomer, and
free nucleotide (Fig. 4B). The percentage of dimer
in the SEC experiment was less than that observed
in AUC likely due to slight differences in sample
preparation.
Å
A
monomer with the a-subunit of the human enzyme
shows that the two proteins are very similar in
overall fold with a rmsd for Ca atoms of 1.2 Å but
that they differ in conformation of the a3-b4 loop.
In addition, the a1-a2, a2-b2 and a6-b7 loops in
GCRho are five, three and eight residues shorter
Crystal structure of the dimer
GCRho crystallized in space group C2221
from PEG3350 in the presence of 1 mM ddGTP,
10 mM MnCl2, and 200 mM potassium sodium
tartrate additive. The structure was solved to 1.7
Å resolution by molecular replacement using the
GCRho monomer (Fig. 3) as search model and
yielded two molecules in the asymmetric unit (Fig.
5A). The final model was refined to Rwork and Rfree
of 0.164 and 0.196, respectively. Clear electron
density was observed for residues M442 to M627
in molecule A and A445 to K626 in molecule B.
The b7-b8 loop region residues E610-G615 in
molecule A and K612-K614 in molecule B were
highly disordered and not modeled in the final
structure.
than respective regions in GCHum
.
Dimeric GCRho
Given that the active state of GCRho was
expected to be a dimeric enzyme, we explored
other crystallization conditions that might trap a
dimer in the asymmetric unit. Since the enzyme
displayed significant guanylyl cyclase activity
when assayed in solution (Fig. 2B), we reasoned
that substrate and metal ion might facilitate
formation of the dimer. We used the substrate
analog 2’,3’-dideoxyguanosine 5’-triphosphate
(ddGTP) which lacks the ability to be cyclized due
to the absence of a hydroxyl group at the 3’
position of the sugar ring. GCRho was mixed with
ddGTP and Mn2+ and then monitored for
Superposition of the GCRho monomer with
both monomers of the dimer showed that there are
no significant structural changes in the monomer
units, with a rmsd for Ca atoms of 0.4 Å and 0.3
Å for monomer A and monomer B, respectively,
although minor changes were observed in part of
the dimer interface around the a1 helix (N460-
S469) and b4-5 loop (L558-H564). Nonetheless,
the monomer units do not form the signature head-
to-tail wreath-like conformation observed in other
GC(9-11) and AC(13-15) dimeric structures.
Instead, the two monomers are in a head-to-head
formation
of
dimer
by
analytical
ultracentrifugation (AUC). Sedimentation velocity
profiles showed the existence of two separate
sedimenting species consistent with the molecular
mass expected for monomeric and dimeric GCRho
(Fig. 4A). Notably, GCRho sediments exclusively
as monomer in the absence of ddGTP and Mn2+.
When a similar sample was applied to the
Superdex-200 column three major species were
6

Structure and monomer/dimer equilibrium of guanylyl cyclase
arrangement. Superposition of the GCRho dimer
catalytically important residues D457, D501, and
R545(12-14) from both monomers A and B (Fig.
5D). The density was further resolved into two 22
σ Fo – Fc peaks and an overlapping, elongated ~ 8
σ Fo – Fc peak. Both 22 σ peaks were modelled
with a Mn2+ ion. The 8 σ peak electron density
was not fit well by ddGTP, cGMP, or PPi, but was
with the active conformation of the mammalian
AC heterodimer ((14); PDB entry 1CJU), using
monomer A as a reference, shows that monomer B
of GCRho is rotated by 101° and displaced by 6.3 Å
with respect to AC monomer B (Fig. 5B). The
large rotation of monomer B compared to the AC
structure suggests that the GCRho dimer is non-
functional.
modeled well by the tartrate additive. Mn2+ is
A
hexa-coordinate with D457 Od2 of molecule B,
D501 Od1 of molecule A, O2 and O6 atoms of the
tartrate molecule, and two water molecules as
The GCRho dimer has a buried surface area
of
1273
Å2
and
geometric
surface
ligands. Mn2+ is also hexa-coordinate with D457
complementarity (Sc)(19) of 0.63. The buried
surface area is comparable in magnitude to the
buried surface area for the C. reinhardtii cyclase
GCCYG12 (1370 Å2; 3ET6), for the cyclase GCCya2
from Synechocystis (1548.9 Å2; 2W01), and for
the human sGC heterodimer GCHum (1230 Å2;
3UVJ). The shape complementarity statistic, Sc, is
also similar in magnitude to those of other
adenylyl and guanylyl cyclases (AC, 0.59;
GCCYG12, 0.64; GCCya2, 0.67; GCHum, and 0.74).
Part of the dimer interface is formed by an
B
Od1 and D501 Od1 and Od2 of molecule B, O1
and O3 atom of the tartrate molecule, and a single
water molecule as ligands. Tartrate is perfectly
sandwiched between the side chains of the
catalytically important residue R545 of both
monomer A and monomer B. Apart from metal
ion coordination, tartrate also forms hydrogen
bonds with amino acids D457, T462, and D501 of
molecule A, D457, I458, T462, and D501 of
molecule of B, and with several water molecules.
unusual interaction of b-strands b4b and b5 of
monomer A with those of monomer B. In other
GC dimeric structures(9-11), this b-sheet interacts
with strands b2 and b3 to form the dimer interface.
Significantly, monomers A and B of GCRho are
connected by a disulfide bridge involving the
catalytically important residue C566(8,20,21) in
b5 from both monomers (Fig. 5C), reinforcing the
conclusion that this is an inactive form of the
protein.
Dependence on Mn2+
Discovery of the disulfide crosslink in the
GCRho dimer caused us to revisit the AUC
experiments where we could show that no dimer
species was formed in the presence of a reducing
agent (5 mM DTT). Likewise, subsequent crystal
trials with ddGTP and Mn2+ in the presence of
DTT produced only crystals with monomer in the
asymmetric unit and cell dimensions and space
group identical to those observed in the absence of
nucleotide. We, therefore, conducted a series of
After initial refinement, difference Fourier
density appeared at the cavity formed by the
7

Structure and monomer/dimer equilibrium of guanylyl cyclase
experiments to determine what factors were
dimer, it did crystallize from PEG20000 in the
presence of 10 mM potassium hydrogen tartrate
with the same space group and unit cell
dimensions as found with the GCRho monomer.
The structure was solved by molecular
replacement using the GCRho monomer as a search
model. The final structure was determined to a
resolution of 1.4 Å with Rwork/Rfree of 0.190/0.208.
A single molecule was in the asymmetric unit with
overall structure identical to that of the GCRho
important for formation of the disulfide
crosslinked GCRho dimer as assayed by SDS-
PAGE. As is shown in Fig. 6, the single factor
most important was the presence of Mn2+. The
presence of GTP, cGMP, and inorganic
pyrophosphate in the absence of metal ion were
without effect. Mg2+ could substitute for Mn2+ in
this reaction but with reduced efficiency. Notably,
addition of the CC domain dramatically decreased
the amount of dimer formed under all conditions.
It should be noted that disulfide bond
monomer.
The rmsd for Ca atoms from
superposition of the two structures is only 0.1 Å
suggesting that the mutations had minimal effect
on secondary and tertiary structure of the protein.
Amino acids G559 and D560 in the b4-b5 loop
and E610-G615 in the b7-b8 loop were highly
disordered and not modeled in the final structure.
The calculated omit map clearly revealed the two
mutations in the protein (Fig. 7B), and K497 and
D566 were modeled in the structure where their
side chains were found to form a salt bridge.
formation required a lengthy incubation period.
The reactions shown in Fig. 6 included a twenty hr
incubation to mimic the original AUC and SEC
conditions (Fig. 4). No crosslinked dimer band
was observed when the incubation period was
omitted.
Structure of the GCAC domain
Previously, we constructed a mutant of
RhoGC in which the activity of the enzymatic
domain was changed from a guanylyl cyclase to an
Indirect evidence for functional dimerization
Indirect evidence for formation of an
adenylyl cyclase(8).
This required the
introduction of two mutations in the GC domain of
the protein: E497K and C566D. Given that the
reactive C566 is replaced by Asp, it was of interest
to determine a crystal structure for the isolated
“adenylyl cyclase” domain, GCAC, of the mutant.
GCAC displays the same substrate specificity (Fig.
7A) as was observed for the full-length mutant(8),
and was used for crystal trials under a host of
different conditions. While GCAC did not form
crystals under the same conditions as for the GCRho
active dimer was obtained by following the
dependence of enzyme activity on the
concentration of protein in the assays. As is
shown in Fig. 8, guanylyl cyclase activity showed
a distinctly nonlinear dependence on total enzyme
concentration that was well fit by equation 1 in
Experimental Procedures for a monomer-dimer
equilibrium in which only the dimer is active. The
parameters used to fit equation 1 to the data are
presented in Table 2. The inclusion of DTT in the
8

Structure and monomer/dimer equilibrium of guanylyl cyclase
reaction had no effect on activity demonstrating
DISCUSSION
that the disulfide crosslinked dimer observed in
the crystal structure does not form during the short
incubation times used here in the activity assays. It
is evident from the data in Fig. 8 and Table 2 that
the effect of the CC domain on quaternary
structure of the protein is a rather modest 3-4 fold
decrease in KD for formation of the dimer.
The purpose of this study was to provide
an initial purification and characterization of the
isolated guanylyl cyclase domain of RhoGC, with
(GCwCCRho) and without (GCRho) the coiled-coil
domain (CC), in preparation for a more in depth
mechanistic exploration of the full-length protein.
Genes for His6-tagged versions of both proteins
were co-expressed in E. coli with GroEL and
GroES(16) to improve the yield of properly folded
protein in the soluble fraction of cellular extracts.
The proteins were purified by Ni-affinity
chromatography in a yield of about 50 mg/L cell
culture.
To eliminate concern that the C-terminal
His6-tag might interfere with GCRho dimerization,
a new construct was generated with a N-terminal
His6-tag and TEV-proteolysis site inserted
between the His6-tag and remainder of the GCRho
catalytic domain, and the dependence of activity
on enzyme concentration repeated with the TEV
proteolyzed enzyme. The data were again well fit
by equation 1 with an obviously non-linear
dependence of activity on enzyme concentration
giving a KD value of 64 ± 9 µM, well within
experimental error of the KD values for GCRho
reported in Table 2.
Both proteins displayed robust guanylyl
cyclase activity in a reaction that was dependent
on added metal ion, Mg2+ or Mn2+. As has been
observed with other cyclases, GCRho was
significantly more active with Mn2+ than with
Mg2+(9-12,16). Interestingly, including the CC
domain in the GCwCCRho construct eliminated this
difference in activity with the two metal ions.
GCwCCRho displays a kcat (2.5 s-1) that is about 4-
fold less than the GCRho domain with Mn2+ (10 s-1)
but significantly more than GCRho with Mg2+ (0.16
s-1), and perhaps most importantly, about the same
as the apparent kcat for light-stimulated guanylyl
cyclase activity for the full-length RhoGC (2.2 s-
1)(8). The latter comparison suggests that a
function of the remaining domains in RhoGC (i.e.,
N-term and rho) is to suppress constitutive activity
of GCwCCRho, such that the full-length protein
expresses guanylyl cyclase activity only after
Active-state dimer
Since we could not crystallize an active-
state dimer of GCRho, we modeled what the dimer
might look like based upon superposition with the
published structure of the mammalian adenylyl
cyclase VC1IIC2 catalytic domain in complex with
Mn2+ and ddATP (Fig. S4)(14). The active site
residues at the dimer interface align nicely
between the two structures, although side chain
conformations show some differences, as is
expected given that GCRho crystallized as a
monomer.
9

Structure and monomer/dimer equilibrium of guanylyl cyclase
activation with light. This appears to be the case
Trypanosoma brucei(23). Rv1625c forms an
inactive dimer in solution(22) whereas the T.
brucei enzymes display weak subunit affinity and
transient formation of catalytically active dimers
in solution(23).
also for ligand-activated guanylyl cyclases, where
regulatory domains suppress constitutive activity
of the catalytic domains(9-11,16).
While there were clear similarities of
GCRho and GCwCCRho in comparison with other
guanylyl cyclases, a striking difference was
encountered in crystal structures of the GCRho
domain where the protein crystallized under
standard conditions with only a single subunit in
We were able to crystallize a GCRho dimer
in the presence of ddGTP, Mn2+, and added
potassium sodium tartrate. The two subunits of
the dimer are nearly identical in overall fold to the
isolated GCRho monomer and to the individual
subunits found in dimer structures of other
guanylyl cyclases, but the arrangement of the two
subunits is very different from the canonical
wreathlike structure first described for adenylyl
cyclase(13-15) and later found in the guanylyl
cyclases(9-11). The subunits of GCRho dimer are
displaced by 6 Å and rotated by 101° with respect
to the arrangement of subunits in the adenylyl
cyclase dimer(14) such that the conserved active
site residues are dispersed in the protein and are
unlikely to support binding of nucleotide. In
addition, the GCRho dimer contains a disulfide
bond involving C566, an active-site residue known
to be critical for activity of the protein(8). Thus, it
is clear that this dimer cannot be part of the
catalytic cycle of the enzyme.
the asymmetric unit.
This was unexpected
because both GCwCCRho and GCRho display
significant guanylyl cyclase activity. All other
guanylyl cyclases crystallize as dimers with two
monomers in the asymmetric unit(9-11), and the
active sites of adenylyl and guanylyl cyclases are
known to be formed at the interface of the two
protomers in a dimeric complex(17,18). The
unusual crystallization results for GCRho were
supported by SEC and AUC experiments showing
that the protein was 100% monomer in solution.
To be sure, monomers of other guanylyl cyclases
have been observed in solution studies(9,16), but
the monomer/dimer equilibria are more heavily
shifted toward dimer, and, as noted above, the
other guanylyl cyclases all crystallized with a
dimer in the asymmetric unit. There are two
reports in which adenylyl cyclases crystallized as a
monomer: one for a Phe to Arg mutant involving
Unusual dimer structures have been
described for both the Rv1264(24) and
Rv1625(22) adenylyl cyclases from M.
tuberculosis, and in this context the domain
swapped head-to-head dimer observed for WT
Rv1625(22) shares some similarity with the head-
to-head dimer we see here for GCRho. It is unlikely
that the disulfide in GCRho would form under
a
residue in the dimer interface of the
Mycobacterium tuberculosis guanylyl cyclase
Rv1625c catalytic domain(22), and the other for
the catalytic domains of the adenylyl cyclases,
GRESAG4.1
and
GRESAG4.3,
from
10

Structure and monomer/dimer equilibrium of guanylyl cyclase
intracellular reducing conditions, and it seems
Irrespective of whether or not the
observed dimer is involved in GCRho function, it
seems clear that the catalytically active state must
be a dimer, similar in arrangement of subunits to
that of adenylyl cyclase(13,14). We have not been
able to provide evidence for such a dimer by direct
methods such as SEC, AUC, and crystallography,
but we could provide evidence through the non-
linear dependence of guanylyl cyclase activity on
enzyme concentration. It is likely that Mn2+ and
substrate assist in formation of the dimer under
these conditions, but even so, the KD for formation
(20-80 µM) is much higher than for other guanylyl
cyclases (e.g., the KD for the catalytic domain of
the mammalian soluble guanylyl cyclase is 0.45
µM)(16). The extremely high KD suggests that
formation of the active state dimer might be part
of the light-activation mechanism. Future work in
the laboratory will explore the role of domain
dimerization in the light-dependent activation of
full-length RhoGC.
most probable that the disulfide forms subsequent
to dimerization as a consequence of a high local
concentration of the two Cys residues in an
oxidizing environment. Thus, the disulfide could
function as a kinetic trap for the dimer. The dimer
structure does not appear to result from two
monomers that were brought together in a random
and haphazard association as would be expected if
initial formation of a disulfide crosslink were
followed by formation of the dimer. To begin, the
GCRho dimer has a buried surface area and
geometric
surface
complementarity
(Sc)
comparable in magnitude to those of other
guanylyl and adenylyl cyclases. The C2 axis of the
GCRho dimer is rotated by 90º with respect to the
canonical AC dimer such that six of the conserved
active site residues, three identical residues from
each subunit (D457, D501, and R545), come
together to form a ligand binding pocket for
tartrate and two Mn2+ ions. The Asp residues bind
the Mn2+ ions much as do the corresponding
residues in adenylyl cyclase, and the conserved
Arg residues bind to the tartrate carboxylates
much as the corresponding residue in adenylyl
cyclase binds the terminal phosphate in
ddATP(14). Interestingly, the disulfide bond and
Mn2+/tartrate ligand-binding site are located on the
C2 axis, at opposite ends of the GCRho dimer
interface. Nonetheless, the disulfide-linked dimer
is likely a crystallization artifact, and it remains to
be determined experimentally if this particular
oligomeric state has any relevance for regulation
of guanylyl cyclase activity.
EXPERIMENTAL PROCEDURES
Materials
Unless specified otherwise, all materials
were as described previously(8).
Protein expression and purification
Two constructs containing the guanylyl
cyclase domain (GC) of RhoGC were used in
these studies: one composed of the GC domain
alone (“GCRho”; amino acids 443-627), and the
11

Structure and monomer/dimer equilibrium of guanylyl cyclase
other which included the coiled-coil domain (CC)
The frozen cell pellets were thawed on ice
and resuspended to a final volume of 50 mL with
lysis buffer (25 mM HEPES, pH 7.0, 100 mM
NaCl, and 20 mM imidazole) containing 1 mM
PMSF. The cell suspension was sonicated on ice
with a Misonix Sonicator 3000 at 70W power with
a cycle of 20 s on and 20 s off for a total of 4 min.
Sonicated cells were centrifuged in a Beckman
Coulter JA-20 rotor at 16,000 rpm for 30 min at 4
°C to pellet cell debris, and the supernatant
fraction passed through a 0.22 µm filter before
loading onto a preequilibrated 5 mL prepacked
HiTrapFF Ni-Sepharose column (GE Healthcare
Life Sciences) at 1 mL/min. The column was
washed with 10 column volumes of lysis buffer
followed by 20 volumes of lysis buffer containing
40 mM imidazole, and the protein eluted with an
80 mL linear gradient of 40 to 500 mM imidazole
in lysis buffer. Protein was monitored by
absorbance at 280 nm, and fractions containing
GCRho (or GCwCCRho) were identified by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and pooled before concentration
with an exchange of buffer to remove imidazole
(10 kDa molecular mass cutoff Amicon Ultra
Centrifuge Filter from EMD Millipore). Purified
GCRho and GCwCCRho were frozen in small
concentrated aliquots at -80 °C until needed.
as well (“GCwCCRho”; amino acids 397-627).
Both were cloned into a pET15b vector for
expression in E. coli BL21(DE3) cells and
included a C-terminal 6x His-tag (His6) for
purification on a Ni-affinity column. Both genes
also included a Val codon after the start
methionine to improve bacterial expression. The
BL21(DE3) cell line harbored an additional pGro7
plasmid (Takara Bio Inc.) containing the genes for
the GroEL and GroES molecular chaperone
proteins under the control of the AraB promoter.
Competent cells were transformed with either
pET15b-GCRho or pET15b-GCwCCRho according
to standard procedures and incubated overnight at
37 °C on LB-agar plates containing 100 µg/mL
ampicillin and 20 µg/mL chloramphenicol. Single
colonies were used to inoculate small growth
cultures (10 mL LB containing 100 µg/mL
ampicillin and 20 µg/mL chloramphenicol) which
were grown for 14-18 hr with shaking at 220 rpm
and 37 °C. One mL of the overnight culture was
used to inoculate a liter of LB containing both
antibiotics. 500 mg of L-(+)-arabinose (Acros
Organics) was added upon inoculation to begin
induction of GroEL/ES. Cells were grown at 37 °C
until an OD600 0.4-0.7 was reached, at which point
expression of the protein was induced by addition
of IPTG to a final concentration of 250 µM. Cells
were then incubated with shaking at 20 °C for 20
hr and harvested by centrifugation at 4000 rpm in
a Beckman Coulter JLA-8.1000 rotor for 15 min at
4 °C. Cell pellets were stored at -80 °C until
needed.
Size exclusion chromatography (SEC) on
an Äkta FPLC system (Amersham Biosciences,
Uppsala, Sweden) equipped with a Superdex-200
10/300 GL gel filtration column (GE Healthcare
Life Sciences) was used to determine oligomeric
state of the proteins. 300 uL of 200 uM purified
12

Structure and monomer/dimer equilibrium of guanylyl cyclase
GCRho (or GCwCCRho) was loaded onto the
protein(25). All centrifuge steps were carried out
using ProteomeLab Optima XL-A
Superdex column which had been pre-equilibrated
with 25 mM HEPES, pH 7.0, and 100 mM NaCl at
4 °C, and the column run at a constant rate of 0.5
mL/min. Protein was monitored by absorbance at
280 nm, and elution volume was compared to a
standard curve using soluble protein standards to
estimate the molecular mass of the peak fraction.
A separate GCRho construct, GCNHisTEV,
a
Ultracentrifuge (Beckman Coulter). In all cases
395 µL of protein was loaded into double sector
center pieces balanced by 400 µL of reference
solution containing all components of the sample
solution except the protein. All cell housings were
loaded into a four-hole An-60 Ti rotor and spun at
35,000 rpm at 22 °C for 15 hr. Absorbance at 280
nm was measured to track protein concentration
during sedimentation. Data were processed and
analyzed using SEDFIT software (NIH, Version
14.4fb). Data were fit using a continuous c(s)
distribution model based on theory for a soluble
and globular (spherical) protein with self-
association. Parameters used to fit the raw
absorbance data to a confidence level of 95%
included assumptions for average protein partial
specific volume (0.73) as well as for buffer density
(1.00 g/mL) and buffer viscosity (0.01002 units)
being identical to pure water as no viscous agents
such as glycerol were used to stabilize the protein
in solution.
was designed with
a
TEV-protease site
(ENLYFQG) inserted immediately after a N-
terminal His6-tag and followed by a Met and the
remainder of GCRho sequence to determine if the
affinity tag interfered with dimerization of the
protein in solution. The protein was expressed and
purified as presented above. The purified protein
was proteolyzed with a 30:1 ratio of GCNHisTEV to
TEV-protease for 9 hr at 4 °C in 25 mM HEPES
buffer, pH 7.0, containing 100 mM NaCl, 1 mM
DTT, and 0.5 mM EDTA in a total volume of 1
mL. The cleaved protein was separated from
undigested GCNHisTEV and TEV-protease by Ni-
affinity chromatography, as confirmed by SDS-
PAGE and anti-His6 western blot analysis. Both
TEV-cleaved and -uncleaved GCNHisTEV were
shown to be monomeric in solution by SEC on a
Superdex-200 equilibrated in 25 mM HEPES, pH
7.0, 100 mM NaCl running at 0.5 mL/min at 4 °C.
Enzymatic activity assays
Guanylyl cyclase activity was measured
using reversed phase high-performance liquid
chromatography (RP-HPLC) to follow formation
of cGMP and disappearance of GTP. 100 µL
reactions were prepared in 50 mM Tris buffer, pH
7.6, containing 50 mM NaCl and 0.5 mM EDTA.
Reactions contained GTP and metal ion (MgCl2 or
MnCl2) at the concentrations indicated in the
figures and were initiated by addition of enzyme.
Analytical ultracentrifugation
Sedimentation velocity experiments were
designed to identify factors (e.g., salt, enzyme
concentration, divalent cation, etc.) that could
impact the oligomeric state of isolated GCRho
13

Structure and monomer/dimer equilibrium of guanylyl cyclase
20 µL aliquots were quenched at specified time
following equation for a reaction in which the only
active species is a dimer of the enzyme in a
monomer/dimer equilibrium:
points (30 s - 5 hr) by combining with an equal
volume of 1 N HCl. Precipitated protein was
removed with 0.22 µm Spin-X centrifugal filters
(Corning Costar) by centrifugation at 5,000 rpm
for 5 min in a tabletop centrifuge. The samples
were then neutralized with 20 µL of 1 M
potassium phosphate (pH 8) and applied to a 250 x
2.1 mm ACE 5 C18-AR reversed phase 5 μm
column connected to an Agilent 1260 Infinity
HPLC system with a G136D 1260 multi-
wavelength detector. Nucleotides were separated
by isocratic elution with a 100 mM potassium
phosphate buffer, pH 6.2, at a flow rate of 0.4
mL/min and monitored by absorbance at 254 nm.
Peaks were integrated with OpenLab CDS
ChemStation software and compared to peaks
from standards for GTP and cGMP of known
concentration.
4ꢅ + ꢆꢇ
−
ꢆꢇꢈ + 8ꢅ×ꢆꢇ
8
ꢀ = ꢁꢂꢃꢄ
×
ꢉꢊ 1
where v = reaction rate (μM/s), kcat = turnover
number (s-1), E = total enzyme concentration
(μM), and KD is the equilibrium dissociation
constant for dimerization of the enzyme, defined
ꢐ
as ꢆꢇ = [ꢋꢌꢍꢌꢋꢎꢏ] with units of μM. KD and kcat
[ꢑꢒꢋꢎꢏ]
are parameters determined through the fitting
process. These reactions contained 10 mM GTP
and 20 mM metal ion (Mn2+ or Mg2+), which was
saturating for all enzyme concentrations tested.
SDS-PAGE analysis of disulfide crosslinks
Samples of GCRho and GCwCCRho were
Assays for adenylyl cyclase activity were
incubated for 20 hr under various conditions, as
indicated in Fig. 6, to test for the formation of
intermolecular disulfide bonds. Each sample
contained 1 µM GCRho or GCwCCRho in 25 mM
HEPES, pH 7.0, and 100 mM NaCl. Metal ion
(Mn2+ or Mg2+), GTP substrate, and cGMP and
inorganic pyrophosphate products were included
as indicated in the figure. Each sample was
diluted 1:2 with gel load buffer, with or without 5
mM DTT, and then loaded onto a 10% Mini-
PROTEAN TGX polyacrylamide gel (Bio-Rad)
for analysis. Protein bands were visualized with
Coomassie Brilliant Blue stain.
performed identically except that the reaction
contained ATP instead of GTP, and the HPLC
column running buffer was 100 mM potassium
phosphate, pH 6.2, containing 10% (v/v)
methanol.
The concentration of stock solutions was
determined
spectrophotometrically
using
extinction coefficients of 13,700 M-1cm-1 at 252
nm for GTP, 12,320 M-1cm-1 at 260 nm for cGMP,
15,400 M-1cm-1 at 259 nm for ATP, and 15,000 M-
1cm-1 at 260 nm for cAMP.
The dependence of activity on enzyme
concentration was evaluated through a non-linear
least squares fit (MATLAB) of the rate data to the
14

Structure and monomer/dimer equilibrium of guanylyl cyclase
Crystallization
Å resolution for the GCRho dimer. Data sets were
Crystallization trials were performed by
integrated using iMosflm version 7.2(26) and
scaled using SCALA version 3.3(27) from the
CCP4 software suite version 7.0(28,29).
Diffraction data were processed in space group
sitting drop vapor diffusion at room temperature
using Hampton (Hampton Research, CA) and Jena
Bioscience (Jena Bioscience, Jena, Germany)
sparse matrix crystallization screens. Drops were
set with a Phoenix robot (Art Robbins Instruments,
CA) by mixing protein (GCRho, GCwCCRho or
GCAC; 20 mg/mL) in 25 mM HEPES, pH 7.0,
containing 100 mM NaCl with crystallization
mother liquor in a 1:1 ratio. Crystals of GCRho
appeared after about 3 weeks in 25% w/v
PEG3350, 100 mM BIS-TRIS, pH 5.5, and 200
mM NaCl while GCAC crystals formed in 15% w/v
PEG20000 and 10 mM potassium hydrogen
tartrate. These crystals all contained a single
subunit in the asymmetric unit. In some trials, the
protein solution also contained 10 mM MgCl2 (or
MnCl2) and 1 mM ddGTP. In this case (MnCl2),
crystals appeared in 20% (w/v) PEG3350 and 200
mM potassium sodium tartrate and contained a
disulfide crosslinked dimer in the asymmetric unit.
1
P22121 for the GCRho and GCAC monomers and
C2221 for the GCRho dimer. Complete data
collection statistics are listed in Table 1.
The GCRho monomer structure was solved
by molecular replacement using PHASER version
2.6(30) with the structure of the catalytic domain
of homodimeric human soluble GC (PDB entry
2WZ1) molecule A as a search model. The GCAC
monomer and GCRho dimer structures were solved
using the final refined model of the GCRho
monomer as
a search model. Rigid body
refinement followed by positional and B-factor
refinement was carried out using phenix.refine(31)
from the PHENIX software suite version 1.11(32).
Simulated annealing was included in earlier
refinements to minimize the initial model bias.
Manual model building was done using COOT
version 0.8(33). Water molecules were included in
the final refinement after satisfying the criteria of
3σ Fo – Fc and 1σ 2Fo – Fc. Several iterative cycles
of refinement were carried out before final
submission of data. Data collection and final
refinement statistics are given in Table 1. Data sets
for the GCRho monomer (PDB entry 6AO9), GCAC
(PDB entry 6AOA), and GCRho dimer (PDB entry
6AOB) have been submitted to the Protein Data
Bank. All crystal structure figures in this paper
were prepared using PyMol version 1.8
(Schrödinger LLC, Portland, OR).
Data collection, processing, and refinement
Crystals were soaked in reservoir solution
containing 15% glycerol as cryoprotectant before
flash freezing in liquid nitrogen. Diffraction data
were collected at 100 K with beam line 8.2.1 at the
Advanced Light Source (Lawrence Berkeley
National Laboratory, Berkeley, CA) using an
ADSC Q315R CCD detector (Area Detector
Systems Corporation, CA). The best crystals
diffracted up to 1.1 and 1.4 Å resolution for the
GCRho and GCAC monomers, respectively, and 1.7
15

Structure and monomer/dimer equilibrium of guanylyl cyclase
ACKNOWLEDGEMENTS
AUTHOR CONTRIBUTIONS
This research used resources of the
D.D.O. conceived and coordinated the
study. R.P.K. and B.R.M. were responsible for all
aspects of the experimental program. J.F. was
involved in the initial development of the
expression and purification system for the
guanylyl cyclase domain. M.M.T. was involved in
the development of the HPLC method for
enzymatic assays and in the construction of the
GCAC mutant. D.H.Z. performed assays for
dependence of activity on enzyme concentration
(Fig. 8). M.O.L. developed and performed
disulfide crosslinking assays (Fig. 6). D.D.O.,
R.P.K., and B.R.M. wrote the paper. All authors
discussed and commented on the manuscript.
Advanced Light Source, which is a DOE Office of
Science User Facility under contract no. DE-
AC02-05CH11231. We are grateful to the staff at
the Advanced Light Source-Berkeley Center for
Structural Biology for their assistance in X-ray
data collection.
We thank the Adar family and friends for
continued support throughout this work and
support from the Brandeis University Provost’s
research fund. We would also like to thank Prof.
Timothy Street and Jackson Halpin for sharing
their HPLC and providing technical advice. We
thank Prof. Bruce Foxman for helpful discussions
on crystallography.
CONFLICT OF INTEREST
The authors declare no competing
financial interest.
16

Structure and monomer/dimer equilibrium of guanylyl cyclase
REFERENCES
1.
2.
Avelar, G., Schumacher, R., Zaini, P., Leonard, G., Richards, T., and Gomes, S. (2014) A rhodopsin-
guanylyl cyclase gene fusion functions in visual perception in a fungus. Curr. Biol. 24, 1234-1240
Ernst, O. P., Lodowski, D. T., Elstner, M., Hegemann, P., Brown, L. S., and Kandori, H. (2014)
Microbial and animal rhodopsins: structures, functions, and molecular mechanisms. Chem. Rev.
114, 126-163
3.
4.
Spudich, J. L., Yang, C. S., Jung, K. H., and Spudich, E. N. (2000) Retinylidene proteins: Structures
and functions from archaea to humans. Annu. Rev. Cell Dev. Biol. 16, 365-392
Gao, S., Nagpal, J., Schneider, M. W., Kozjak-Pavlovic, V., Nagel, G., and Gottschalk, A. (2015)
Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-
cyclase opsin CyclOp. Nat. commun. 6, 8046
5.
6.
Scheib, U., Stehfest, K., Gee, C., Korschen, H., Fudim, R., Oertner, T., and Hegemann, P. (2015)
The rhodopsin-guanylyl cyclase of the aquatic fingus Blastocladiella emersonii enables fast
optical control of cGMP signaling. Sci Signal. 8, RS8
Lamarche, L. B., Kumar, R. P., Trieu, M. M., Devine, E. L., Cohen-Abeles, L.E., Theobald, D. L., and
Oprian,
D.
D.
(2017)
Purification
and
characterization
of
RhoPDE,
a
retinylidene/phosphodiesterase fusion protein and potential optogenetic tool from the
choanoflagellate Salpingoeca rosetta. Biochemistry 56, 5812-5822
7.
8.
Yoshida, K., Tsunoda, S. P., Brown, L. S., and Kandori, H. (2017) A unique choanoflagellate
enzyme rhodopsin with cyclic nucleotide phosphodiesterase activity. J. Biol. Chem. 292, 7531-
7541
Trieu, M. M., Devine, E. L., Lamarche, L. B., Ammerman, A. E., Greco, J. A., Birge, R. R., Theobald,
D. L., and Oprian, D. D. (2017) Expression, purification, and spectral tuning of RhoGC, a
retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic fungus
Blastocladiella emersonii. J. Biol. Chem. 292, 10379-10389
9.
Rauch, A., Leipelt, M., Russwurm, M., and Steegborn, C. (2008) Crystal structure of the guanylyl
cyclase Cya2. Proc. Natl. Acad. Sci. U. S. A. 105, 15720-15725
10.
11.
12.
13.
Winger, J. A., Derbyshire, E. R., Lamers, M. H., Marletta, M. A., and Kuriyan, J. (2008) The crystal
structure of the catalytic domain of a eukaryotic guanylate cyclase. BMC Struct. Biol. 8, 42
Allerston, C. K., von Delft, F., and Gileadi, O. (2013) Crystal structures of the catalytic domain of
human soluble guanylate cyclase. PLoS one 8, e57644
Hurley, J. H. (1999) Structure, mechanism, and regulation of mammalian adenylyl cyclase. J. Biol.
Chem. 274, 7599-7602
Tesmer, J. J., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Crystal structure of the
catalytic domains of adenylyl cyclase in a complex with Gsalpha.GTPgammaS. Science 278, 1907-
1916
14.
15.
16.
Tesmer, J. J., Sunahara, R. K., Johnson, R. A., Gosselin, G., Gilman, A. G., and Sprang, S. R. (1999)
Two-metal-Ion catalysis in adenylyl cyclase. Science 285, 756-760
Zhang, G., Liu, Y., Ruoho, A. E., and Hurley, J. H. (1997) Structure of the adenylyl cyclase catalytic
core. Nature 386, 247-253
Winger, J. A., and Marletta, M. A. (2005) Expression and characterization of the catalytic
domains of soluble guanylate cyclase: interaction with the heme domain. Biochemistry 44, 4083-
4090
17.
Lucas, K. A., Pitari, G. M., Kazerounian, S., Ruiz-Stewart, I., Park, J., Schulz, S., Chepenik, K. P., and
Waldman, S. A. (2000) Guanylyl cyclases and signaling by cyclic GMP. Pharmacol. Rev. 52, 375-
414
17

Structure and monomer/dimer equilibrium of guanylyl cyclase
18.
19.
20.
Sinha, S. C., and Sprang, S. R. (2006) Structures, mechanism, regulation and evolution of class III
nucleotidyl cyclases. Rev. Physiol. Biochem. Pharmacol. 157, 105-140
Lawrence, M. C., and Colman, P. M. (1993) Shape complementarity at protein/protein
interfaces. J. Mol. Biol. 234, 946-950
Sunahara, R. K., Beuve, A., Tesmer, J. J., Sprang, S. R., Garbers, D. L., and Gilman, A. G. (1998)
Exchange of substrate and inhibitor specificities between adenylyl and guanylyl cyclases. J. Biol.
Chem. 273, 16332-16338
21.
Tucker, C. L., Hurley, J. H., Miller, T. R., and Hurley, J. B. (1998) Two amino acid substitutions
convert a guanylyl cyclase, RetGC-1, into an adenylyl cyclase. Proc. Natl. Acad. Sci. U. S. A. 95,
5993-5997
22.
23.
24.
25.
26.
Barathy, D., Mattoo, R., Visweswariah, S., and Suguna, K. (2014) New structural forms of a
mycobacterial adenylyl cyclase Rv1625c. IUCrJ. 1, 338-348
Bieger, B., and Essen, L. O. (2001) Structural analysis of adenylate cyclases from Trypanosoma
brucei in their monomeric state. EMBO J. 20, 433-445
Tews, I., Findeisen, F., Sinning, I., Schultz, A., Schultz, J. E., and Linder, J. U. (2005) The structure
of a pH-sensing mycobacterial adenylyl cyclase holoenzyme. Science 308, 1020-1023
Schuck, P. (2000) Size-distribution analysis of macromolecules by sedimentation velocity
ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606-1619
Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R., and Leslie, A. G. (2011) iMOSFLM: a
new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol.
Crystallogr. 67, 271-281
27.
28.
29.
Evans, P. (2006) Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62,
72-82
Potterton, E., Briggs, P., Turkenburg, M., and Dodson, E. (2003) A graphical user interface to the
CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131-1137
Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M.,
Krissinel, E. B., Leslie, A. G., McCoy, A., McNicholas, S. J., Murshudov, G. N., Pannu, N. S.,
Potterton, E. A., Powell, H. R., Read, R. J., Vagin, A., and Wilson, K. S. (2011) Overview of the
CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235-242
McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J.
(2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658-674
Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov,
M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H., and Adams, P. D. (2012) Towards automated
crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68,
352-367
30.
31.
32.
Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung,
L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R.
J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a
comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D
Biol. Crystallogr. 66, 213-221
33.
34.
Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot.
Acta Crystallogr. D Biol. Crystallogr. 66, 486-501
Omasits, U., Ahrens, C. H., Muller, S., and Wollscheid, B. (2014) Protter: interactive protein
feature visualization and integration with experimental proteomic data. Bioinformatics 30, 884-
886
18

Structure and monomer/dimer equilibrium of guanylyl cyclase
FOOTNOTES
1. The equivalent positions for this non-standard setting of P21212 are (x, y, z); (x, -y, -z); (-x, ½ + y, ½ -
z); (-x, ½ - y, ½ + z).
Accession codes: The atomic coordinates and structure factors have been deposited in the Protein Data
Bank. RCSB PDB entry 6AO9, 6AOA, and 6AOB for the GCRho-monomer, GCAC, and GCRho- dimer,
respectively.
Abbreviations: RhoGC, rhodopsin-guanylate cyclase; GCRho, isolated RhoGC guanylyl cyclase domain;
CC, RhoGC coiled-coil domain; GCwCCRho, GCRho with the CC domain; GCAC, GCRho double mutant
with adenylyl cyclase activity; GCCya2, catalytic domain of the guanylyl cyclase Cya2 from Synechocystis
PCC6803; GCCYG12, catalytic domain of the guanylyl cyclase CYG12 from Chlamydomonas reinhardtii;
GCHum, catalytic domain of the human soluble guanylyl cyclase sGC; GTP, guanosine 5’-triphosphate,
ddGTP, 2’,3’-dideoxyguanosine 5’-triphosphate; cGMP, cyclic 3’,5’-guanosine monophosphate.
TABLES AND FIGURES
Table 1. Crystallographic data collection and refinement statistics.
Table 2. KD and kcat values for the fits of equation 1 to the data of Fig. 8.
Figure 1. Model for the transmembrane topology and orientation of RhoGC. Domains are: N-term,
N-terminal domain; rho, microbial type I rhodopsin domain; CC, coiled-coil domain; and GC, guanylyl
cyclase domain. Transmembrane helices were predicted and drawn by Protter
(wlab.ethz.ch/protter/start)(34). Cyan, N-term; green, rho; purple, CC; yellow, GC; blue square, K384,
presumed site of covalent attachment to the chromophore; and red circles, E497 and C566, residues
controlling specificity for the GTP substrate.
Figure 2. Purification and initial characterization of the isolated GCwCCRho and GCRho domains. A,
Purification of GCwCCRho and GCRho. Left panel, Coomassie-stained SDS-PAGE gel showing fractions
for purification of the isolated GCwCCRho domain from transformed E. coli BL21(DE3)-pGro7 cells (co-
expressing GroEL/ES) using Ni-affinity chromatography. Right panel, Coomassie-stained SDS-PAGE gel
showing fractions from purification of GCRho. The last lane contains purified GCwCCRho for size
19

Structure and monomer/dimer equilibrium of guanylyl cyclase
comparison. B, Initial rate data for guanylyl cyclase activity from GCRho and GCwCCRho in the presence
of MnCl2 (left panel) or MgCl2 (right panel). Reactions were performed as described in Experimental
Procedures and contained 10 µM enzyme, 5 mM GTP, and 10 mM divalent cation. C, Äkta FPLC profiles
for size exclusion chromatography of GCRho (solid line) and GCwCCRho (dashed line) on a Superdex-200
10/300 GL column in 25 mM HEPES buffer, pH 7.0, containing 100 mM NaCl. Both proteins were
loaded onto the column at a concentration of 200 µM. Molecular mass standards: 1. blue dextran, 2000
kDa, 8.5 mL (void volume); 2. aldolase, 158 kDa, 13.15 mL; 3. albumin, 67 kDa, 14.57 mL; 4.
ovalbumin, 43 kDa, 15.41 mL; 5. chymotrypsinogen-A, 25 kDa, 16.8 mL; and 6. ribonuclease-A, 14 kDa,
18 mL. Both GCRho and GCwCCRho elute as monomers under these conditions.
Figure 3. Overall structure of the GCRho monomer. Secondary structure elements are labeled according
to conventional nomenclature and are color coded: red, α-helix; yellow, β-strand; and green, loop.
Figure 4. Formation of GCRho dimers. A, Normalized c(s) distribution plot for AUC of GCRho in the
absence (black) and presence (red) of 1 mM MnCl2 and 60 µM ddGTP (ddGTP equimolar with GCRho).
B, SEC profiles for GCRho in the absence (solid line) and presence (dotted line) of MnCl2 and ddGTP.
Solid line, data for GCRho in the absence of MnCl2 and ddGTP is from Fig. 2C. Dotted line, data from
protein that was subjected to the same conditions as for the AUC sample in A containing MnCl2 and
ddGTP. Superdex-200 column was run in 25 mM HEPES, pH 7.0, containing 100 mM NaCl at 4 °C with
a flow rate of 0.5 mL/min.
Figure 5. Structure of the GCRho homodimer. A, Overall structure of the GCRho homodimer. Cartoon
representation of molecule A and B are colored green and gold, respectively. Selected secondary
structure elements are labeled according to convention. Tartrate is modeled as sticks and shown in yellow.
Manganese ions are shown as green spheres. Cysteine residues are shown as sticks and colored according
to the respective main chain coloring. B, Structural superposition of the GC homodimer with the ‘active
conformation’ of the human AC heterodimer (PDB entry 1CJU). Cartoon representations are color-coded
as follows: GCRho molecule A, green; GCRho molecule B, gold; and human AC molecules A and B, blue.
N- and C-terminal amino acids are marked and color-coded according to the coloring of the respective
molecule. C, Disulfide crosslink at the dimer interface. FO - FC omit map is contoured at 3 σ cut off.
Parts of the protein backbone have been omitted for clarity. D, Tartrate binding site at the dimer
interface. FO - FC omit map is contoured at 4 σ cut off. Metal ion coordination and hydrogen bonding are
indicated with dotted lines. Parts of the protein backbone and additional solvent molecules have been
omitted for clarity.
20

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 6. Analysis of disulfide crosslinking by SDS-PAGE. Protein samples (GCRho or GCwCCRho
)
were incubated for twenty hr under the conditions indicated in the figure before being loaded onto 10%
polyacrylamide gels so as to simulate the AUC conditions in Fig. 4A. All samples were prepared in 25
mM HEPES, pH 7.0, 100 mM NaCl using either 1 µM GCRho or GCwCCRho. Top panels, GCRho; bottom
panels, GCwCCRho; left panels, without DTT; and right panels, with 5 mM DTT. Other conditions are as
indicated in the figure: Mn, 2 mM MnCl2; Hi Mn, 20 mM MnCl2; Mg, 10 mM MgCl2; cGMP, 1 mM
cGMP; GTP, 1 mM GTP; and PPi, 1 mM pyrophosphate.
Figure 7. GCAC activity data and omit map. A, Initial rate data for catalytic activity of GCAC when
provided ATP (black circles) or GTP (red circles) as a substrate. The reactions contained 5 mM substrate
(GTP or ATP) and 10 mM MnCl2. B, The FO – FC omit map contoured at 3σ cut off shows electron
density for the two mutated active site residues K497 and D566 in GCAC. Secondary structure elements
are colored green and labeled according to convention. Amino acid side chains are shown as sticks.
Hydrogen bonding is indicated by the dotted line. Part of the protein backbone has been removed for
clarity.
Figure 8. Guanylyl cyclase activity as a function of enzyme concentration. A, Rate of cGMP
formation as determined by the HPLC assay is plotted as a function of total enzyme concentration
showing a non-linear dependence that is well fit by equation 1 in Experimental Procedures relating initial
rate to a monomer-dimer equilibrium model in which only the dimer is active. The construct and
condition for each set of data are as indicated in the figure. B, Data for GCRho with MgCl2 is re-plotted
from panel A on a more sensitive scale to show clearly the non-linear dependence on enzyme
concentration. All reactions contained 10 mM GTP and 20 mM metal ion (Mn2+ or Mg2+). The dotted
curves are fits of equation 1 to the data using the parameters of KD and kcat listed in Table 2. The enzyme
concentration is total enzyme added to the reaction, expressed in terms of the µM concentration of
monomer. An expanded view of the data for GCwCCRho-Mn is shown in Figure S3A. A control
experiment showing that the non-linear behavior is not a result of non-specific protein interactions at low
concentrations is shown in Figure S3B.
21

Structure and monomer/dimer equilibrium of guanylyl cyclase
Table 1
GCRho-monomer GCRho-dimer GCAC-monomer
PDB ID
6AO9
6AOB
6AOA
Data collection statistics
Space group
P22121
C2221
P22121
Resolution range (Å)
Highest resolution shell (Å) 1.19 - 1.13
Unit cell parameters (Å)
20 - 1.13
20 - 1.70
1.79 - 1.70
a = 91.8
b = 95.9,
c = 100.1;
617510
20 - 1.40
1.48 - 1.40
a = 34.4,
b = 65.4,
c = 88.4;
280921
a = 34.5,
b = 65.6,
c = 90.6;
980795
Total reflections
Unique reflections
77710
48364
40049
Completeness %a
99.9 (99.7)
7.7 (100)
15.2 (2.2)
12.6 (10.2)
99.3 (97.7)
10.6 (130)
16.1 (2.0)
12.8 (12.3)
99.8 (100)
11.1 (42.3)
8.7 (2.9)
7.0 (7.0)
a
Rmerge
%
I/σ (I)a
Redundancy
Refinement statistics
Resolution range (Å)
No. of reflections used
20 - 1.13
77532
18.8
19.5
1384
0
20 - 1.70
48333
16.4
19.6
2842
10
18 - 1.40
39937
19.0
20.8
1388
0
Rwork
Rfree
%
%
Protein atoms
Ligand atoms
Metal atoms
0
2
0
Water molecules
338
507
320
r.m.s.d. in bond lengths (Å) 0.005
0.006
0.8
0.005
0.8
r.m.s.d. in bond angles (°)
0.8
a Highest-resolution shell values are given in parenthesis
22

Structure and monomer/dimer equilibrium of guanylyl cyclase
Table 2
kcat (s-1)
KD (μM)
Construct & Condition
GCRho – MnCl2
GCRho – MnCl2 & DTT
GCRho – MgCl2
78 ± 12
83 ± 30
76 ± 25
23 ± 15
10 ± 1
9.6 ± 1.6
0.16 ± 0.02
2.5 ± 0.5
GCwCCRho – MnCl2
23

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 1
24

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 2
25

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 3
26

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 4
27

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 5
28

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 6
29

Structure and monomer/dimer equilibrium of guanylyl cyclase
Figure 7
30