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This article is part of the supplement: 5th International Conference on cGMP: Generators, Effectors and Therapeutic Implications

Open Access Poster presentation

Biochemical and structural characterization of C-terminal constructs of bovine soluble guanylate cyclase

Franziska Seeger* and Elsa D Garcin

Author Affiliations

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

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BMC Pharmacology 2011, 11(Suppl 1):P67  doi:10.1186/1471-2210-11-S1-P67

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2210/11/S1/P67


Published:1 August 2011

© 2011 Seeger and Garcin; licensee 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.

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. Each of the two homologous subunits (α and β) contains three domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), a central dimerization HNOX associated (HNOXA) and coiled-coil (CC) domain, and a C-terminal catalytic domain (GC).

The enzyme is basally active, but NO binding to the heme group in the β subunit’s regulatory domain enhances sGC catalytic output several hundred fold. The exact mechanism by which the regulatory domain relays the NO activation signal to the catalytic domain remains elusive. Furthermore, it has been proposed that the HNOX regulatory domain inhibits the activity of the catalytic GC domain [1]. Winger et al. showed that the GC heterodimer by itself exhibits catalytic activity in the presence of Mg2+ and Mn2+[1]. On the contrary, Wedel et al. propose that additional amino acids are required for dimerization, folding, and catalytic activity [2].

We aim to test the hypothesis that additional domains are necessary for full activity of the catalytic domain by combining mutagenesis, activity assays, fluorescence spectroscopy, Small-Angle X-ray Scattering (SAXS), and protein crystallography.

Results

We have chosen a divide-and-conquer approach to study sGC catalysis. Here, we report the recombinant expression and purification of bovine C-terminal constructs “αβGC” and “αβCC-GC” in E. coli (Figure 1). Preliminary activity measurements for these constructs in the presence of Mg2+ show that αβCC-GC exhibits higher level of catalytic activity than αβGC (4-fold). This suggests that additional domains are necessary for full catalytic activity. To our knowledge this is the first report that attributes catalytic activity to the αβCC-GC heterodimer in the presence of Mg2+.

thumbnailFigure 1. Full-length sGC and C-terminal truncated constructs αβGC and αβCC-GC.

Conclusion

The C-terminal sGC constructs αβGC and αβCC-GC, both exhibit catalytic activity in the presence of Mg2+. Higher levels of activity of αβCC-GC as compared to αβGC hint at structural differences between the two constructs that will be characterized using protein X-ray crystallography and small-angle X-ray scattering.

Acknowledgements

Work supported by American Heart Association Scientist Development Grant (E. Garcin) and NIH CBI Training Grant NIH T32 GM066706-06.

References

  1. 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. PubMed Abstract | Publisher Full Text OpenURL

  2. Wedel B, Koesling D: Functional domains of soluble guanylyl cyclase.

    Biol Chem 1995, 270:24871-24875. Publisher Full Text OpenURL