The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase
Jonathan A. Winger, Emily R. Derbyshire, Meindert H. Lamers, Michael A. Marletta and John Kuriyan Abstract / Figures from the paper / Coordinates / Supplementary Information
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Figure 1. Structural alignment of selected guanylate and adenylate cyclase catalytic domains. Secondary structure annotation and numbering correspond to the guanylate cyclase homolog CYG12 from C. reinhardtii. Sequences are grouped as follows: A, atypical soluble guanylate cyclases; B, membrane-bound guanylate cyclases; C, NO-sensing soluble guanylate cyclases; D, putative bacterial guanylate cyclases; E, mammalian and bacterial adenylate cyclases. Functional residues are indicated by symbols: metal binding (*); ribose binding (Δ); guanine/adenine binding (◆); triphosphate binding (#). Accession numbers are as follows: Chlamydomonas reinhardtii CYG12 (GenBank XP_001700847), Caenorhabditis elegans GCY35 (GenBank O02298), Rattus norvegicus sGCβ2 (GenBank BAB68564), Drosophila melanogaster GYC-88E (GenBank Q8INF0), Homo sapiens RetGC1 (GenBank Q02846), R. norvegicus GCA (GenBank P18910), C. elegans GCY7 (GenBank AAQ62451), Strongylocentrotus purpuratus mGC (GenBank P16065), R. norvegicus sGCβ1 (GenBank BAC55087), zias latipes sGCβ1 (GenBank BAA76691), duca sextaCβ1 (GenBank AAC61264), melanogasterβ1 (GenBank), norvegicus sGCα1 (GenBank AAB17953), O. latipes sGCα1 (GenBank BAA76690), M. sexta sGCα1 (GenBank AAC61263), D. melanogaster sGCα1 (GenBank AAF56917), Anabaena sp. PCC7120 all1118 (GenBank NP_485161), Nostoc punctiforme PCC73102 NpR1313 (GenBank YP_001864972), N. punctiforme PCC73102 NpR0352 (GenBank ACC79135), Trichodesmium erythraeum IMS101 Tery_4585 (GenBank ABG53561), T. erythraeum IMS101 Tery_3412 (GenBank ABG52512), Synechocystis sp. PCC6803 sll0646 (GenBank BAA16969), Canis familiaris ACV_C1 (GenBank 1CJU_A), R. norvegicus ACII_C2 (GenBank 1CJU_B), Mycobacterium tuberculosis Rv1264 (GenBank 1Y11_A), M. tuberculosis Rv1900c (GenBank 1YBU_C), Spirulina platensis CyaC (GenBank 1WC1_C). Initial alignments were carried out using the program ClustalX [67]. Sequences were adjusted manually with comparison to results from a structural homology search using the DALI server [45]. Figure 1 was prepared using the program ESPRIPT [68]. Regions containing residues of > 70% equivalence (red letters) are boxed with a thin blue line, and absolutely conserved residues are highlighted in red.
Figure 2.
Structural features of the guanylate cyclase domain.
A) Structural representation of a guanylate cyclase domain
monomer. Elements of secondary structure are labeled
according to the nomenclature depicted in Figure 1.
B) The guanylate cyclase catalytic domain. Monomer
A is colored green, and monomer B is multi-colored,
ranging from blue at the N-terminus to red at the
C-terminus. Two of eight phosphate ions are shown and
are depicted as stick figures: phosphorus, orange; oxygen, red.
Figure 3.
Comparison between guanylate and adenylate cyclase active
sites. Monomer B of the guanylate cyclase catalytic domain
was superimposed onto the C2 domain of mammalian adenylate
cyclase (PDB ID: 1CJU) [49]. Residues and structural
elements involved in catalysis and nucleotide recognition
are shown. A) Comparison of guanylate and adenylate cyclase
catalytic residues. B) Comparison of guanylate and
adenylate cyclase base recognition residues. Side chains
and structural elements from the guanylate cyclase and
adenylate cyclase catalytic domains are colored green and
grey, respectively. The nucleotide 2',3'-dideoxyadenosine
triphosphate (ddATP) and the two Mg2+ ions are from 1CJU.
Non-carbon atoms are colored as follows: phosphorus, orange;
oxygen, red; nitrogen, blue; sulfur, yellow; arsenic, violet; magnesium, white.
Figure 4.
Proposed guanylate cyclase activation mechanism. Comparison
of helix α1 in the guanylate cyclase structure with helix α1
of the active mammalian adenylate cyclase structure indicates
that the guanylate cyclase structure is in an inactive
state. Monomer B of the guanylate cyclase structure was
superimposed onto the C2 domain of the active adenylate
cyclase structure (PDB ID: 1CJU) [49] and monomer A was
superimposed onto the C1 domain of the same adenylate
cyclase structure. The C2 domain is omitted for clarity.
The C1 domain of the active adenylate cyclase structure
is colored blue, and the guanylate cyclase structure is
colored green. The nucleotide 2',3'-dideoxyadenosine
triphosphate (ddATP) and Mg2+ ions from 1CJU are shown as a
stick figure and spheres: phosphorus, orange; oxygen, red; nitrogen, blue; magnesium, white.
Figure 5.
Communication between active sites. Plot of guanylate
cyclase activity at increasing concentrations of substrate
GTP. Guanylate cyclase (5 μg) was incubated for 2 min at
24 °C with the indicated concentrations of GTP in the
presence of 4 mM MnCl2 and cGMP was measured. Data were
fit to the equation (Vmax(S)n)/((S0.5)n+Sn)), where Vmax is the maximum activity,
S is the concentration of GTP, S0.5 is the substrate
concentration at which half-maximal velocity is reached,
and n is the Hill coefficient. From the fit, Vmax = 2795 ± 117
nmoles cGMP/min/mg, S0.5 = 269 ± 26 μM, and n = 1.49 ± 0.16. A
Hill coefficient greater than 1 indicates the presence of interacting active sites.
Figure 6.
A potential binding site for a regulatory control element.
A) Structure of the mammalian adenylate cyclase catalytic
domain bound to the activator Gsα (PDB ID 1CJU) [49]. The
switch II helix of Gsα binds in a groove on the C2 domain
between the α1–α2 and α3-β4a loops, priming the catalytic
domain for nucleotide binding. A surface representation of
the adenylate cyclase catalytic domain is shown, and Gsα is
shown as a ribbon cartoon. The C1 domain is colored blue,
the C2 domain is colored orange, and Gsα is colored teal.
B) Surface representation of the guanylate cyclase catalytic
domain in the same orientation as the adenylate cyclase
domain in A. A groove similar to that used by adenylate
cyclase to bind to Gsα is located between the α1–α2 and
α3-β4a loops, and may serve as a site for interaction of
control elements with the guanylate cyclase catalytic domain.
Monomer A is colored teal, and monomer B is colored green.
Additional file 2.
Activation mechanism of mammalian adenylate cyclase.
Helix α1 in the C1 domain of mammalian adenylate cyclase
undergoes a significant conformational change going
from an inactive structure to an active structure.
A hypothetical inactive structure of the mammalian
adenylate cyclase catalytic domain was generated as
previously described [35]: the C2 domain structure from
1AZS was superimposed on chain A from the structure of
a C2 domain homodimer (PDB ID: 1AB8) [41], and the C1
domain structure from 1AZS was superimposed on chain
B from 1AB8. The nucleotide- and Gsα-bound active C1/C2
dimer structure 1CJU [49] was then superimposed onto
the C2 domain of the inactive model. The inactive
model is colored white, and the active structure is
colored blue. Gsα and the C2 domain of 1CJU are omitted
for clarity. The nucleotide 2',3'-dideoxyadenosine
triphosphate (ddATP) and Mg2+ ions from 1CJU are shown
as a stick figure and spheres: phosphorus, orange; oxygen, red; nitrogen, blue; magnesium, white.