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  • Oral presentation
  • Open Access

Soluble guanylate cyclase crystal clear: 1stcrystal structure of the wild-type human heterodimeric sGC catalytic domains and implications for activity

BMC Pharmacology and Toxicology201314 (Suppl 1) :O14

https://doi.org/10.1186/2050-6511-14-S1-O14

  • Published:

Keywords

  • Nitric Oxide
  • Catalytic Domain
  • Acute Heart Failure
  • Regulatory Domain
  • Dime Interface

Background

Soluble guanylate cyclase (sGC) is the key enzyme in the NO-sGC-cGMP signaling cascade crucial in regulating the cardiovascular system. Low output of this system causes hypertension and acute heart failure, which are the leading causes of death globally.

Mammalian sGC is a heterodimer of two homologous subunits (α and β), which contain four domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), an HNOX associated (HNOXA) domain and a coiled-coil (CC) domain important for dimerization, and a

C-terminal catalytic domain (GC) (Figure 1).
Figure 1
Figure 1

Full-length sGC and C-terminal constructs αβGC and αβCC-GC used in this study

The enzyme is basally active, but NO binding to the heme group in the β subunit regulatory domain enhances sGC catalytic output several hundred fold. The molecular mechanism by which the regulatory domain relays the activation signal to the catalytic domain remains elusive. Several studies have highlighted the crucial role of the HNOXA and CC domains for sGC dimerization necessary for catalytic activity [19]. Others have shown that the C-terminal GC domains, alone, form catalytically active heterodimers that are inhibited in the presence of the βHNOX regulatory domain [10]. Clearly, more information is needed to elucidate the requirements for sGC activity and activation.

We have established a bacterial overexpression system for truncated constructs of sGC containing the catalytic domains. These constructs can be probed for activity and structurally characterized for conformational changes that may occur during activation.

Results and discussion

Here, we report the first crystal structure for the wild type human heterodimeric αβGC catalytic domain to 1.9 Å resolution (Figure 2). Comparison of the heterodimeric αβGC to homodimeric ββGC allows us to identify distinct interactions at the GC dimer interface that can be used to modulate the heterodimer/homodimer equilibrium crucial for activity.
Figure 2
Figure 2

1.9 Å crystal structure of human heterodimeric wild-type catalytic domain of sGC

Structural comparisons with adenylate cyclase in its inactive vs. activated state suggest distinct conformational changes that may occur during sGC activation.

Our structural characterization of αβGC combined with activity assay measurements on αβGC, αβCC-GC, and full-length sGC allow us to propose a molecular mechanism for sGC activation. These findings will provide a basis for understanding the mode of action of current and the rational design of novel sGC agonists for application in cardiovascular diseases.

Declarations

Acknowledgements

Work supported by American Heart Association Scientist Development Grant (E. Garcin), American Heart Association Mid-Atlantic Pre-doctoral fellowship (F. Seeger).

Authors’ Affiliations

(1)
Department of Chemistry Biochemistry, University of Maryland Baltimore County, Baltimore, MD 21250, USA

References

  1. Harteneck C, Koesling D, Söling A, Schultz G, Böhme E: Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits. FEBS Lett. 1990, 272: 221-223. 10.1016/0014-5793(90)80489-6.View ArticlePubMedGoogle Scholar
  2. Hoenicka M, Becker EM, Apeler H, Sirichoke T, Schröder H, Gerzer R, Stasch JP: Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide. J Mol Med. 1999, 77: 14-23. 10.1007/s001090050292.View ArticlePubMedGoogle Scholar
  3. Rothkegel C, Schmidt PM, Atkins DJ, Hoffmann LS, Schmidt HH, Schröder H, Stasch JP: Dimerization Region of Soluble Guanylate Cyclase Characterized by Bimolecular Fluorescence Complementation in Vivo. Mol Pharmacol. 2007, 72: 1181-1190. 10.1124/mol.107.036368.View ArticlePubMedGoogle Scholar
  4. Shiga T, Suzuki N: Amphipathic α-helix Mediates the Heterodimerization of Soluble guanylyl cyclase. Zoological Sci. 2005, 22: 735-742. 10.2108/zsj.22.735.View ArticleGoogle Scholar
  5. Wagner C, Russwurm M, Jäge R, Friebe A, Koesling D: Dimerization of nitric oxide-sensitive guanylyl cyclase requires the alpha 1 N terminus. J Biol Chem. 2005, 280: 17687-17693.View ArticlePubMedGoogle Scholar
  6. Wilson EM, Chinkers M: Identification of sequences mediating guanylyl cyclase dimerization. Biochemistry. 1995, 34: 4696-4701. 10.1021/bi00014a025.View ArticlePubMedGoogle Scholar
  7. Zabel U, Häusle C, Weeger M, Schmidt HHHW: Homodimerization of soluble guanylyl cyclase subunits. J Biol Chem. 1999, 274: 18149-18152. 10.1074/jbc.274.26.18149.View ArticlePubMedGoogle Scholar
  8. Zhao Y, Marletta MA: Localization of the heme binding region in soluble guanylate cyclase. Biochemistry. 1997, 36: 15959-15964. 10.1021/bi971825x.View ArticlePubMedGoogle Scholar
  9. Zhou Z, et al: Structural and Functional Characterization of the Dimerization Region of Soluble Guanylyl Cyclase. Journal of Biological Chemistry. 2004, 279: 24935-24943. 10.1074/jbc.M402105200.View ArticlePubMedGoogle Scholar
  10. Winger JA, Marletta MA: Expression and characterization of the catalytic domains of soluble guanylate cyclase: interaction with the heme domain. Biochemistry. 2005, 44: 4083-4090. 10.1021/bi047601d.View ArticlePubMedGoogle Scholar

Copyright

© Seeger and Garcin; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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