TY - JOUR
T1 - Instability in a coiled-coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase
AU - Weichsel, Andrzej
AU - Kievenaar, Jessica A.
AU - Curry, Roslyn
AU - Croft, Jacob T.
AU - Montfort, William R.
N1 - Publisher Copyright:
© 2019 The Protein Society
PY - 2019/10/1
Y1 - 2019/10/1
N2 - How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC-1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N-terminus in sGC activates cyclase activity near the C-terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small-molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled-coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad-repeating pattern for coiled-coil motifs, but also invariant positions that disfavor coiled-coil stability. Full-length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled-coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.
AB - How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC-1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N-terminus in sGC activates cyclase activity near the C-terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small-molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled-coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad-repeating pattern for coiled-coil motifs, but also invariant positions that disfavor coiled-coil stability. Full-length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled-coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.
KW - GC-1
KW - YC-1
KW - cGMP
KW - guanylate cyclase
KW - leucine zipper
KW - nitric oxide
KW - stimulator compound
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U2 - 10.1002/pro.3707
DO - 10.1002/pro.3707
M3 - Article
C2 - 31411784
AN - SCOPUS:85071279586
SN - 0961-8368
VL - 28
SP - 1830
EP - 1839
JO - Protein Science
JF - Protein Science
IS - 10
ER -