Cyclization of the intrinsically disordered α1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function

Abstract

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 α(1S) 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.

FIGURE 1.

Expression and characterization of the cyclic II-III loop. A, the pNW1120 skDHPR II-III loop expression vector with the His₆ tag (gray box) and nine-residue linker (white box). The location of Intein N and Intein C in the vector is shown by the red arrows (modified from Ref. 17). B, Coomassie Blue-stained 12% BisTris-Cl polyacrylamide gels containing Invitrogen BenchMark^TM prestained protein ladders (Invitrogen) in the left lanes. The linear skDHPR II-III loop and the cyclic skDHPR II-III loop are in the right lanes. The dotted line passes through the 20-kDa marker. C, Coomassie Blue-stained 12% BisTris-Cl polyacrylamide gels of the linear (left gel) and cyclized (right gel) skDHPR II-III loop after a 4-h digestion with carboxypeptidase-Y. The first lane in the left gel shows the 20-kDa marker from the protein ladder. In each gel, lanes labeled U show the undigested protein, and lanes labeled 1–4 show aliquots taken after 1–4-h periods of digestion. The dotted lines indicate the position of the undigested protein.

FIGURE 2.

Spectroscopic comparison of the linear and cyclic II-III loops. A, CD spectra for the cyclized and linear skDHPR II-III loop. The linear loop has a negative peak (filled arrow) at 198 nm, and the cyclized loop has two negative peaks (open arrows) at 208 and 222 nm. B, ¹⁵N HSQC NMR spectra for the cyclized loop (green contours) overlaid on the spectrum for the linear II-III loop (purple contours) spectra showing the distribution of amino acid residues. Residues not affected by cyclization in the C region are labeled, whereas some residues adjacent to the linker (Lys⁶⁸⁵ (K685) and Met⁶⁸⁶ (M686)) are also labeled (underscored).

FIGURE 3.

Spectral shifts in assigned residues occurring as a result of cyclization of the II-III loop. The A–D regions are shown at the top of A, with line thickness reflecting α-helical strength. A, the top graph shows the ¹HN chemical shift perturbations, and the lower graph shows ¹⁵NH chemical shift perturbations for the II-III loop residues indicated on the lower axis. The divisions of the loop into A, B, C, and D regions are indicated along the central bar. The magnitude of change was calculated by comparing the ¹H and ¹⁵N HSQC spectra of the cyclized II-III loop and the linear II-III loop. It is notable that the residues most strongly affected by the cyclization are clustered in the A, B, and D regions of the II-III loop. B, SSP scores for the two II-III loop constructs. The purple bars denote the linear whereas the green bars represent the intein II-III loop. Positive values denote α-helical structure propensity, and negative values represent β-strand or extended structure propensity. A SSP score of 1 or −1 corresponds to a fully formed α- or β-structure, respectively.

FIGURE 4.

Transverse relaxation rates (R2) of the circular and the linear loop as a function of residue number. A–D regions are shown at the top, with line thickness reflecting α-helical strength. The black points denote the linear R2 values, whereas the blue points represent the intein II-III loop. The shaded regions cover consecutive residues that undergo significant increase for the cyclized loop.

FIGURE 5.

The relative fluorescence values of SPRY2 (F₃₄₀) plotted as a function of the concentration (μm) of the linear loop (triangles) and cyclic (squares) skDHPR II-III loop. The fluorescence values were corrected for dilution effects and the fluorescence contribution arising from the quenchers. The data were analyzed as described previously (11), using a non-linear regression fit to the relative fluorescence intensity at 340 nm (F₃₄₀) as a function of quencher concentration (μm). The curve fitting was performed using the GraphPad Prism software (GraphPad Software), from which the K_d measurements were derived. The inset at the top shows the cyclic loop data and curve using an expanded y axis.

FIGURE 6.

Effects of the linear (A) and cyclic (B) II-III loops and the linker peptide (C) on single RyR1 channel activity. In each panel, the recordings show 3-s segments of representative channel activity recorded at −40 mV under control conditions before the addition of the construct and then after the addition of 10, 50, 100, and 200 nm concentrations of each construct, as indicated on the left. The final record in each panel was obtained after perfusion of the cis chamber to remove the protein from the solution.

FIGURE 7.

Average relative single channel parameters measured with each concentration of the linear loop (filled bins), cyclic loop (hatched bins), and the linker peptide (cross-hatched bins). Data are shown for relative open probability (A), mean open time (B), and mean closed time (C). Each bar indicates the average ± S.E. (error bars) of 4–8 measurements. The asterisk indicates a significant difference from the control data, the filled symbol indicates a significant difference between the linear and cyclic loops at the particular concentration. Data are presented as the log₁₀ of the relative values in order to better display values that were less than control.

FIGURE 8.

Effects of the linear and cyclic loop constructs on the distribution of open and closed times. Exponential open and closed time constants were determined. Open and closed times were collected into logged bins, and the square root of the relative frequency of events (P½) was plotted against the logarithm of the open (open circles) or closed times (filled circles) in ms (23). Examples are shown for data from individual channels under control conditions (A and D) and after exposure to 50 nm linear and cyclic loops (B and E) and then 200 nm linear and cyclic loops (C and F). The numbers of events generally varied between 500 and 1000. The broken lines in each graph show the fit of multiple exponential functions to the data. The arrows indicate individual open time constants (τ_o1, τ_o2, and τ_o3) and individual closed time constants (τ_c1, τ_c2, and τ_c3). The variables τ_o3 and τ_c3 (see description under “Results”) are shown in red and blue, respectively. A–C, exposure to linear loop. D–F, exposure to cyclic loop.

FIGURE 9.

Effects of the linear and cyclic loops on the average open (A and B) and closed (C and D) time constants. The probability of events falling into each time constant is plotted against the time constant in ms (open time or closed time). Time constants are shown for control data (filled circles), 10 nm loop (open circles), 50 nm loop (open squares), 100 nm loop (open diamonds), and 200 nm loop (crosses). The red circles indicate regions of major difference between the effects of the linear and the cyclic loops, which are primarily in τ_o3 and τ_c3. Data from four bilayers were analyzed for the linear loop and seven bilayers for the cyclic loop. Each data point is the average of 8–16 measurements (at +40 and −40 mV) for the linear and cyclic loops, respectively. Error bars indicate S.E.