Yamuna Karunasekara

Using Deubiquitylating Enzymes as Research Tools

Ubiquitin is synthesized in eukaryotes as a linear fusion with a normal peptide bond either to itself or to one of two ribosomal proteins and, in the latter case, enhances the yield of these ribosomal proteins and/or their incorporation into the ribosome. Such fusions are cleaved rapidly by a variety of deubiquitylating enzymes. Expression of heterologous proteins as linear ubiquitin fusions has been found to significantly increase the yield of unstable or poorly expressed proteins in either bacterial or eukaryotic hosts. If expressed in bacterial cells, the fusion is not cleaved due to the absence of deubiquitylating activity and can be purified intact. We have developed an efficient expression system, utilizing the ubiquitin fusion technique and a robust deubiquitylating enzyme, which allows convenient high yield and easy purification of authentic proteins. An affinity purification tag on both the ubiquitin fusion and the deubiquitylating enzyme allows their easy purification and the easy removal of unwanted components after cleavage, leaving the desired protein as the only soluble product. Ubiquitin is also conjugated to epsilon amino groups in lysine side chains of target proteins to form a so-called isopeptide linkage. Either a single ubiquitin can be conjugated or other lysines within ubiquitin can be acceptors for further conjugation, leading to formation of a branched, isopeptide-linked ubiquitin chain. Removal of these ubiquitin moieties or chains in vitro would be a valuable tool in the ubiquitinologists tool kit to simplify downstream studies on ubiquitylated targets. The robust deubiquitylating enzyme described earlier is also very useful for this task.

A dihydropyridine receptor α1s loop region critical for skeletal muscle contraction is intrinsically unstructured and binds to a SPRY domain of the type 1 ryanodine receptor

The II-III loop of the dihydropyridine receptor (DHPR) alpha(1s) subunit is a modulator of the ryanodine receptor (RyR1) Ca(2+) release channel in vitro and is essential for skeletal muscle contraction in vivo. Despite its importance, the structure of this loop has not been reported. We have investigated its structure using a suite of NMR techniques which revealed that the DHPR II-III loop is an intrinsically unstructured protein (IUP) and as such belongs to a burgeoning structural class of functionally important proteins. The loop does not possess a stable tertiary fold: it is highly flexible, with a strong N-terminal helix followed by nascent helical/turn elements and unstructured segments. Its residual structure is loosely globular with the N and C termini in close proximity. The unstructured nature of the II-III loop may allow it to easily modify its interaction with RyR1 following a surface action potential and thus initiate rapid Ca(2+) release and contraction. The in vitro binding partner for the II-III was investigated. The II-III loop interacts with the second of three structurally distinct SPRY domains in RyR1, whose function is unknown. This interaction occurs through two preformed N-terminal alpha-helical regions and a C-terminal hydrophobic element. The A peptide corresponding to the helical N-terminal region is a common probe of RyR function and binds to the same SPRY domain as the full II-III loop. Thus the second SPRY domain is an in vitro binding site for the II-III loop. The possible in vivo role of this region is discussed.

The β1a Subunit of the Skeletal DHPR Binds to Skeletal RyR1 and Activates the Channel via Its 35-Residue C-Terminal Tail

Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.

The voltage-gated calcium-channel β subunit: more than just an accessory

Voltage-gated Ca2+ channels (VGCCs) are involved in a number of excitatory processes in the cell that regulate muscle contraction, neurotransmitter release, gene regulation, and neuronal migration. They consist of a central pore-forming α1 subunit together with a number of associated auxiliary subunits including a cytoplasmic β subunit. With the aid of X-ray crystallography, it has been found that the β subunits of VGCCs (β2a, β3, and β4) interact strongly with the I–II loop of the pore-forming α1 subunit. Here we discuss the potential interaction sites of β1a with its α1 subunit as well as the skeletal ryanodine receptor. We suggest that not only can β1a interact with the α1 subunit I–II loop, but more subtle interactions may be possible through the II–III loop via the β1a SH3 domain. Such findings could have important implications with respect to EC coupling.

An α-helical C-terminal tail segment of the skeletal L-type Ca2+ channel β1a subunit activates ryanodine receptor type 1 via a hydrophobic surface

Excitation‐contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C‐terminal 35 residues of the β1a subunit adopt a nascent α‐helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide β1aV490‐M524, corresponding to the C‐terminal 35 residues of β1a, reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2‐fold activation of RyR1 by 10 nM wild‐type peptide. An upstream hydrophobic heptad repeat implicated in β1a binding to RyR1 does not contribute to RyR1 activation. Wild‐type β1aA474‐A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2‐ and 2.2 ± 0.3‐fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue β1a C‐terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.—Karunasekara, Y., Rebbeck, R. T., Weaver, L. M., Board, P. G., Dulhunty, A. F., Casarotto, M. G. An α‐helical C‐terminal tail segment of the skeletal L‐type Ca2+ channel β1a subunit activates ryanodine receptor type 1 via a hydrophobic surface. FASEB J. 26, 5049–5059 (2012). www.fasebj.org

Structural and functional characterization of interactions between the dihydropyridine receptor II-III loop and the ryanodine receptor.

1 Excitation–contraction coupling in skeletal muscle is dependent on a physical interaction between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR). 2 A number of peptides derived from the II–III loop region of the DHPR have been shown to be functionally active in stimulating the release of calcium via RyR channels. Their function has been found to correlate with the presence of a basic helical region located at the N‐terminus of the II–III loop. 3 The entire recombinant skeletal DHPR II–III loop is an efficient activator of RyR1 and RyR2. 4 The skeletal DHPR II–III loop is comprised of a series of a‐helices, but its tertiary structure has been determined to be unstructured and flexible. 5 Fluorescence quenching experiments have been used to identify and measure the binding affinity of the II–III loop with fragments of the RyR.

Cyclization of the Intrinsically Disordered α1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α1S subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and β-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.

Molecular recognition of the disordered dihydropyridine receptor II-III loop by a conserved spry domain of the type 1 ryanodine receptor.

1 The dihydropyridine receptor (DHPR) II–III loop is an intrinsically unstructured region made up of α‐helical and β‐turn secondary structure elements with the N and C termini in close spatial proximity. 2 The DHPR II–III loop interacts in vitro with a ryanodine receptor (RyR) 1 SPRY domain through α‐helical segments located in the A and B regions. Mutations within the A and B regions in the DHPR II–III loop alter the binding affinity to the SPRY2 domain. 3 The A and C peptides derived from DHPR II–III loop show negative cooperativity in binding to the SPRY2 domain. 4 The SPRY2 domain of the RyR1 (1085–1208) forms a β‐sheet sandwich structure flanked by variable loop regions. An acidic loop region of SPRY2 (1107–1121) forms part of a negatively charged cleft that is implicated in the binding of the DHPR II–III loop. 5 The mutant E1108A located in the negatively charged loop of SPRY2 reduces the binding affinity to the DHPR II–III loop.

The inhibitory glutathione transferase M2-2 binding site is located in divergent region 3 of the cardiac ryanodine receptor.

The muscle-specific glutathione transferase GSTM2-2 modulates the activity of ryanodine receptor (RyR) calcium release channels: it inhibits the activity of cardiac RyR (RyR2) channels with high affinity and activates skeletal RyR (RyR1) channels with low affinity. The C terminal domain of GSTM2-2 (GSTM2C) alone physically binds to RyR2 and inhibits its activity, but it does not bind to RyR1. We have now used yeast two-hybrid analysis, chemical cross-linking, intrinsic tryptophan fluorescence and Ca(2+) release studies to determine that the binding site for GSTM2C is in divergent region 3 (D3) of RyR2. The D3 region encompasses residues 1855-1890 in RyR2. Specific mutagenesis shows the binding primarily involves electrostatic interactions with residues K1875, K1886, R1887 and K1889, all residues that are present in RyR2, but not in RyR1. The significant sequence differences between the D3 regions of RyR2 and RyR1 explain why GSTM2-2 specifically inhibits RyR2. This specific inhibition of RyR2 could modulate Ca cycling and be useful for the treatment of heart failure. RyR2 inhibition during diastole may improve filling of the SR with Ca(2+) and improve contractility.

Structural and biophysical analyses of the skeletal dihydropyridine receptor β subunit β1a reveal critical roles of domain interactions for stability

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β1a. Removal of the intrinsically disordered N and C termini and the hook region of β1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2–3 °C between the β1a and β2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6–8 °C. Similar to other β isoforms, β1a bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β1a while also providing a conduit for allosteric signaling events.