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SEMINAR
Decrypting the Excitation Contraction coupling machinery in skeletal muscle
Abstract
Excitation-contraction (EC) coupling in skeletal muscle requires a physical coupling between the voltage-gated calcium channel (Cav1.1) in the surface membrane and the skeletal ryanodine receptor (RyR1) Ca2+ release channel in the membrane of the sarcoplasmic reticulum Ca2+ store1. Although the exact molecular mechanism relating to EC coupling remains unresolved, both the α1s and β1a subunits of Cav1.1 are implicit in this process. In this study we aim to define the structural and interactive framework involving key regions of the Cav1.1 α1s and β1a subunits.
We have determined the structure of the β1a core using X-ray crystallography and similar to other solved β isoforms, β1a consists of an SH3/guanylate kinase (GK) domains separated by a hook region. Unsurprisingly, the GK domain was found to interact strongly with a α1s I-II loop peptide, AID (Kd ~5 nM) but an unanticipated finding was an interaction between the α1s II-III loop and the β1a SH3 domain (~3 μM). The interaction site of the II-III loop was localised to the C region which contains multiple polyproline elements which are synonymous with SH3 domain recognition. Notably, this interaction does not occur for the cardiac β subunit (β2a) and we have mapped a region of the β subunit SH3 RT loop as the reason for this binding variability. It was also noted that in the presence of the AID peptide, no binding of the II-III loop to β1a was detected. This is a significant observation indicating a degree of crosstalk between the I-II and II-III loop interaction sites.
To date, no myopathy-related polymorphisms in β1a have been discovered however, a mutation, V156P with a strong malignant hyperthermia phenotype has recently been confirmed in a male patient. Fittingly, this novel mutation is located in the SH3 domain of β1a in a region that directly impacts the hydrophobic packing of the α1 helix to the core of the molecule. It is quite likely that disruption of this interaction would destabilise the conformation of β1a and impact interactions with other partner proteins.
References
(1) Dulhunty, A. F.; Haarmann, C. S.; Green, D.; Laver, D. R.; Board, P. G.; Casarotto, M. G. Progress in biophysics and molecular biology 2002, 79, 45.
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BIOGRAPHY
1991 PhD (University of Melbourne - Department of Pharmaceutical Sciences)
1991-1996 Welcome Trust Postdoctoral Research Associate Leicester University, UK
1996-2001 Research Fellow (Level B) in Biological NMR (JCSMR)
2004-2012 Fellow and Head of the Biomolecular Structure Laboratory (JCSMR)
2012-present Associate Professor Biomolecular Interactions Laboratory (JCSMR)
My field of research involves the structural and biophysical studies of molecules involved in health and disease. My research program consists of a wide range of medically relevant projects that include: (1) Structural and functional studies of Ca2+ ion channel proteins, the dihydropyridine and ryanodine receptors (DHPR & RYR). The 15 kD intracellular II-III loop region of the DHPR is known to be implicated in signaling to RyR1 and I have been responsible for elucidating the first high resolution structure study of this region as well as its interaction with RyR1. This structure represents one of the most comprehensive structural investigations of a large, intrinsically unstructured protein to date and its importance extends beyond the immediate discipline of EC coupling to the general field of intrinsically disordered proteins and their molecular recognition (JBC, 2011).
(2) I have also played a leading role in several additional projects, including the seminal work involving the cell translocation properties of glutathione transferases (GSTs). Although GSTs play a well known role as detoxifying enzymes, my group has characterized the novel capacity of proteins with the GST fold to efficiently enter cells. This work has laid the platform for the development of a GST-based technology as a potential means of delivering biological cargoes into cells as evidenced by patents (PCT AU2006/001387) and publications (BBA Biomembranes 2009 & PLoS ONE 2011). (3) Virions or virus ion channels are small proteins capable of forming multimeric ion channels and they are expressed in a number of viruses including influenza A (M2), HIV-1 (Vpu) and Hepatitis-C (P7). I have developed strategies to measure the affinity of a number of antiviral drugs to these ion channels and in the case of the M2 ion channel, have used this technology to elucidate the mode of drug binding. This work was published in PNAS (2010) and a follow-up translational study appeared in Antiviral Research in 2012.
Selected Publications
1. Rosenberg, M. R., and M. G. Casarotto. 2010. Coexistence of two adamantane binding sites in the influenza A M2 ion channel. Proceedings of the National Academy of Sciences of the United States of America 107:13866-13871.
This is an important piece of work that resolves a lingering controversy as to where the drug-binding site resides on the influenza A M2 ion channel. It also addresses the issue of drug resistance and explains why adamatane drugs are no longer used.
2. Zhao X, Jie Y, Rosenberg MR, Wan J, Zeng S, Cui W, Xiao Y, Li Z, Tu Z, *Casarotto MG, *Hu W. (2012) Design and synthesis of pinanamine derivatives as anti-influenza A M2 ion channel inhibitors. Antiviral Res. 96:91-9. * Joint senior authors
As a direct result of the PNAS paper (above), several imidazole and guanazole derivatives of pinanamine were developed to inhibit influenza A M2. This translational study provides a new insight into the structural nature of drugs required to inhibit WT A/M2 and its mutants.
3. Tae, H. S., Y. Cui, Y. Karunasekara, P. G. Board, A. F. Dulhunty, and M. G. Casarotto. 2011. Cyclization of the intrinsically disordered alpha1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function. The Journal of biological chemistry 286:22589-22599.
This paper examines the structural and functional implications of cyclizing a disordered protein (DHPR II-III loop). Using solution state NMR we were able to show demonstrate that structural binding elements were altered upon cyclization despite being distant from the linker site. These structural and dynamic changes are correlated with in vitro function of RyR1.
4. Karunasekara Y, Rebbeck RT, Weaver LM, Board PG, Dulhunty AF, Casarotto MG. 2012 An α-helical C-terminal tail segment of the skeletal L-type Ca2+ channel β1a subunit activates RyR1 via a hydrophobic surface. FASEB J. 26:5049-59.
In this paper we address the mechanism of interaction between the DHPR and RyR1 in skeletal muscle EC coupling. We had shown that DHPR β1a subunit binds to and activates RyR1 and in this publication we define β1a residues that critically interact with RyR1 and the structure of the binding domain.
5. Morris, M. J., D. Liu, L. M. Weaver, P. G. Board, and M. G. Casarotto. 2011. A structural basis for cellular uptake of GST-fold proteins. PloS one 6:e17864.
This paper details that the C-terminal domain of GST proteins is responsible for cell entry and destabilising this domain leads to an increase in cell translocation efficiency. |
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