Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking

Bradley G. Fritz, Sue A Roberts, Aqeel Ahmed, Linda Breci, Wenzhou Li, Andrzej Weichsel, Jacqueline L. Brailey, Vicki H. Wysocki, Florence Tama, William "Bill" Montfort

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

Original languageEnglish (US)
Pages (from-to)1568-1582
Number of pages15
JournalBiochemistry
Volume52
Issue number9
DOIs
StatePublished - Mar 5 2013

Fingerprint

Molecular Models
Guanylate Cyclase
X ray scattering
X-Rays
Carbon Monoxide
Heme
Manduca
Histidine
Proteins
Tobacco
Guanosine Triphosphate
Soluble Guanylyl Cyclase
Mass spectrometry
Muscle
Catalyst activity
Nitric Oxide
Chemical activation
Ligands
Molecules
Muscle Relaxation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking. / Fritz, Bradley G.; Roberts, Sue A; Ahmed, Aqeel; Breci, Linda; Li, Wenzhou; Weichsel, Andrzej; Brailey, Jacqueline L.; Wysocki, Vicki H.; Tama, Florence; Montfort, William "Bill".

In: Biochemistry, Vol. 52, No. 9, 05.03.2013, p. 1568-1582.

Research output: Contribution to journalArticle

Fritz, Bradley G. ; Roberts, Sue A ; Ahmed, Aqeel ; Breci, Linda ; Li, Wenzhou ; Weichsel, Andrzej ; Brailey, Jacqueline L. ; Wysocki, Vicki H. ; Tama, Florence ; Montfort, William "Bill". / Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking. In: Biochemistry. 2013 ; Vol. 52, No. 9. pp. 1568-1582.
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AU - Fritz, Bradley G.

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AU - Ahmed, Aqeel

AU - Breci, Linda

AU - Li, Wenzhou

AU - Weichsel, Andrzej

AU - Brailey, Jacqueline L.

AU - Wysocki, Vicki H.

AU - Tama, Florence

AU - Montfort, William "Bill"

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AB - Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

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