Research ArticleSTRUCTURAL BIOLOGY

Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM

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Science Advances  29 Jul 2020:
Vol. 6, no. 31, eabb2964
DOI: 10.1126/sciadv.abb2964
  • Fig. 1 Functional properties of WT and mutant RyR1 and RyR2.

    (A and B) Representative traces of [Ca2+]cyto (top) and [Ca2+]ER (bottom) signals determined with G-GECO1.1 and R-CEPIA1er, respectively, for RyR1 WT (black) and RyR1 R164C (green) and for RyR2 WT (black) and RyR2 R176Q (red). Note that cells expressing RyR2, unlike cells expressing RyR1, show spontaneous Ca2+ oscillations. (C and D) Average [Ca2+]ER for RyR1 WT (empty bar) and RyR1 R164C (green bar) and average threshold and nadir [Ca2+]ER for RyR2 WT (empty bars) and RyR2 R176Q (red bars) expressed in HEK293 cells. Data are means ± SD (n = 49 to 79). (E) Average Ca2+ oscillation frequency in normal Krebs solution for RyR2 WT (empty bar) and RyR2 R176Q (red bar). Data are means ± SD (n = 115 to 121). (F and G) Ca2+-dependent [3H]ryanodine binding of RyR1 R164C (green) and RyR2 R176Q (red) with respect to their respective WT (black) counterparts. The mutants show a higher probability of channel opening and lower probability of inactivation at high [Ca2+] as compared to WT. (H and I) Calculated CICR activity of RyR1 R164C (green) and RyR2 R176Q (red) at resting Ca2+ (pCa 7) with respect to their WT counterparts. Data are means ± SD (n = 3). The mutants display higher sensitivity (~15- and ~2-fold) to activation by Ca2+ as compared to WT. *P < 0.05 versus WT, ***P < 0.001 versus WT.

  • Fig. 2 The flexion angle indicates partial unfurling of the CytA in RyR1 R164C.

    (A and B) The NTDA of RyR1 R164C (yellow) undergoes a 6° rocking with respect to WT closed RyR1 (green). This is not far from the 8° rocking observed during channel opening (closed and open WT RyR1 in green and orange, respectively). (C) The plot of flexion angle of multiple RyR structures including ours indicates that the closed and open structures have a positive and negative flexion angle, respectively, for both isoforms. PDB codes are provided in the plot. The presence of FKBP, which stabilizes RyR flexion angle, is indicated. The CytA of RyR1 R164C adopts a conformation that was so far only seen in the presence of activators Ca2+ with ATP and caffeine [PDB: 5T9V (23)] or absence of FKBP [(22), PDB: 4UWA (61)]. (D) The flexion angle (thick blue line) measures the downward movement of the CytA with respect to the ER membrane and correlates with opening and priming. For RyR1 R164C (left), RyR2 R176Q (middle), and RyR2 WT (right), the values were −0.5°, +0.3°, and 0°, respectively, indicating that mutant RyR1 had a conformation between closed and open states, while mutant and WT RyR2 were closed.

  • Fig. 3 The NTDA+/CD interface plays a role in channel hyperactivation.

    (A) Sequence alignment of rabbit RyR1 and mouse RyR2 (UniProt: P11716 and E9Q401, respectively) shows that the NTDA:β8-β9 loop is highly conserved between the two isoforms. The arrow indicates the equivalent Arg residue that is mutated to Cys (C) in RyR1 and to Gln (Q) in RyR2. (B) Inset images show a zoomed-in view of the NTDA+/CD interface, with the NTDA+:β8-β9 loop shown in black. A distinct “hook”-like density of R3984 and R3938 is observed in RyR1 R164C (green) and RyR2 R176Q (salmon), respectively. The R3984-G160 interaction acts as a “molecular latch” in RyR1 R164C and helps to stabilize a new relative position of NTDA+ and CD and appears to hinder the return to the typical closed-state configuration of this domain pair. (C) Overview showing both NTDA+/CD and CD/CTD interfaces in RyR1 R164C (green) and RyR2 R176Q (salmon). (D) Inset images with emphasis on the Ca2+ binding site indicate alteration of the site observed in RyR1 R164C (green) resulting from a decrease in the T5001-E3893 distance to 3.4 Å (from 4.9 Å in closed RyR1), potentially inducing a better fit of the Ca2+ ion. In contrast, this is not observed in the RyR2 counterpart. (E) Simple schematic explaining the long-range effect that the NTDA mutation (*) has on HD2 (magenta), CD (green), and CTD (tan), resulting in downward rocking of the distal HD2 [a trait associated with the open-state conformation (22) and shrinking of the Ca2+ binding site (#), which facilitates activation (31)]. These changes could alter the energy landscape and explain the increased probability of opening.

  • Fig. 4 Local effects of the mutation.

    (A and B) The NTDA:β8-β9 loop is held together by an intricate network of putative H-bonds (dashed blue lines) in both isoforms. In RyR1 R164C, R3984 from the CD interacts with the backbone O atom of G160 in the NTDA:β8-β9 loop and props the NTDA+ domain upward, thereby stabilizing an altered NTDA+/CD conformation of the channel. This interaction is not observed in the RyR2 R176Q structure. (C and D) The density of R169 reaches across to the Q176 in mutant RyR2, while that of R157 does not reach across to the shorter C164 in mutated RyR1, indicating that the size of the mutated residue might play an important role. (E and F) The R176-R169 interaction in WT RyR2 and the equivalent R164-R157 interaction in WT RyR1 appear to preserve and stabilize the shape of the NTDA:β8-β9 loop. (G) The mutation R164C in RyR1 caused separation of NTDA+/NTDB interface. (H) The mutation stabilized a new relative orientation of the NTDA+/CD interface (atomic model of closed WT RyR1) (PDB: 5TB0 in translucent gray). Solid black lines illustrate dilation of the NTDA:β8-β9 loop.

  • Fig. 5 Validation of cryo-EM–derived models using [3H]Ry binding and Ca2+ imaging of reciprocal mutations.

    Mutation to larger Gln (Q) as compared to smaller Cys (C) from Arg (R) resulted in a [3H]Ry binding profile and live Ca2+ handling more similar to WT RyR, likely due to better preservation of the putative H-bond network. [3H]Ry binding values from Fig. 1 included for easier comparison. (A and B) Ca2+-dependent [3H]Ry binding of RyR1 R164C (green) and RyR1 R164Q (blue) and of RyR2 R176Q (red) and RyR2 R176C (blue) with respect to WT counterparts (black). (C and D) Calculated CICR activity of RyR1 R164C (green bar) and RyR1 R164Q (blue bar) and of RyR2 R176Q (red bar) and RyR2 R176C (blue bar) at resting Ca2+ (pCa 7) with respect to WT counterparts (empty bar). Data are means ± SD (n = 3). (E and F) Representative traces of [Ca2+]cyto (upper) and [Ca2+]ER (lower) signals determined with G-GECO1.1 and R-CEPIA1er, respectively, for RyR1 WT (black) and RyR1 R164Q (blue) and for RyR2 WT (black) and RyR1 R176C (blue). Note that cells expressing RyR2, unlike cells expressing RyR1, show spontaneous Ca2+ oscillations. (G and H) Average [Ca2+]ER for RyR1 WT (empty bar), RyR1 R164C (green bar), and RyR1 R164Q (blue bar) and average threshold and nadir [Ca2+]ER for RyR2 WT (empty bar), RyR2 R176Q (red bar), and RyR2 R176C (blue bar) expressed in HEK293 cells. Data are means ± SD (n = 49 to 79). (I) Average Ca2+ oscillation frequency for RyR2 WT (empty bar), RyR2 R176Q (red bar), and RyR2 R176C (blue bar). Data are means ± SD (n = 67 to 121). ***P < 0.001 versus WT, *P < 0.05 versus WT, ###P < 0.001 versus original disease mutant, and #P < 0.05 versus original disease mutant.

  • Fig. 6 RMSD analysis reveals the mechanism of the long-range allosteric effect.

    RMSD between mutant and closed WT RyR1 was subtracted from RMSD between mutant and open WT RyR1 such that greater similarity to the closed conformation results in a positive value. (A) Pairwise 1- and 2-domain comparisons indicate that, in most cases, domains are more akin to the closed conformation except for the NTDA+/CD interface of RyR1 R164C (inset), which bore greater similarity to open RyR1 (PDB: 5T9V). The two domains change in relative orientation with respect to each other near the site of the mutation, while individually they were more similar to the closed state. (B) Schematic representation of domain movements observed in RyR1 during channel opening (top), visualized for one monomer. The size and direction of the arrows indicate magnitude of movement observed. In RyR1 R164C closed (bottom), domain translocations, mainly confined to the large CytA, are similar to the opening motion near the site of the mutation (red asterisk). Other domains are differentially affected, changing the configuration of the closed channel. These motions may also affect the DHPR, which is physically connected to RyR1’s CytA.

Supplementary Materials

  • Supplementary Materials

    Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM

    Kavita A. Iyer, Yifan Hu, Ashok R. Nayak, Nagomi Kurebayashi, Takashi Murayama, Montserrat Samsó

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