Structural basis for Ca2+ activation of the heteromeric PKD1L3/PKD2L1 channel

Abstract

The heteromeric complex between PKD1L3, a member of the polycystic kidney disease (PKD) protein family, and PKD2L1, also known as TRPP2 or TRPP3, has been a prototype for mechanistic characterization of heterotetrametric TRP-like channels. Here we show that a truncated PKD1L3/PKD2L1 complex with the C-terminal TRP-fold fragment of PKD1L3 retains both Ca²⁺ and acid-induced channel activities. Cryo-EM structures of this core heterocomplex with or without supplemented Ca²⁺ were determined at resolutions of 3.1 Å and 3.4 Å, respectively. The heterotetramer, with a pseudo-symmetric TRP architecture of 1:3 stoichiometry, has an asymmetric selectivity filter (SF) guarded by Lys2069 from PKD1L3 and Asp523 from the three PKD2L1 subunits. Ca²⁺-entrance to the SF vestibule is accompanied by a swing motion of Lys2069 on PKD1L3. The S6 of PKD1L3 is pushed inward by the S4-S5 linker of the nearby PKD2L1 (PKD2L1-III), resulting in an elongated intracellular gate which seals the pore domain. Comparison of the apo and Ca²⁺-loaded complexes unveils an unprecedented Ca²⁺ binding site in the extracellular cleft of the voltage-sensing domain (VSD) of PKD2L1-III, but not the other three VSDs. Structure-guided mutagenic studies support this unconventional site to be responsible for Ca²⁺-induced channel activation through an allosteric mechanism.

Fig. 1. The PKD1L3-CTD/PKD2L1 heterocomplex retains Ca²⁺ and acid-induced channel activity.

a Topology diagram of PKD1L3 and PKD2L1. PKD1L3, and PKD2L1 proteins assemble to a heterocomplex with a stoichiometric ratio of 1:3. The constructs used for structural determination in this study, mouse PKD1L3-CTD (residues 1632–2151) and PKD2L1 (residues 64–629), are indicated by the dashed box. CTL C-type lectin domain, REJ sperm receptor for egg jelly, PLAT Polycystin-1, Lipoxygenase, α-toxin domain, PMD polycystin-mucolipin domain, PD pore domain, VSD voltage-sensing domain. b The Ca²⁺ and acid-induced channel activity of the PKD1L3-CTD/PKD2L1 heterocomplex is similar to that of the full-length (FL) channel. The Ca²⁺ and acid-induced currents were recorded from Xenopus oocytes expressing either full-length PKD1L3 or PKD1L3-CTD with PKD2L1. Shown here are representative traces for gap-free recording at −80 mV. c Scatter plots and bar graphs of the Ca²⁺-induced currents at −80 mV recorded from oocytes expressing the indicated proteins. The number of oocytes is shown below each bar. Data are presented as mean ± SD in the bar graph. Currents in each group are compared with that of PKD1L3-FL/2L1-injected group with two-sided Student’s t test. n.s.: not significant; ****P < 0.0001. d The side (left) and intracellular (right) views of the cryo-EM structure of the PKD1L3-CTD/PKD2L1 complex. For simplicity, we will call it PKD1L3/2L1. The glycosyl moieties are shown as sticks. The black and red arrows highlight the different structures of the S4–S5 linkers in PKD1L3 and the three PKD2L1 subunits, respectively. e Structural comparison of PKD1L3/2L1 with and without added Ca²⁺. The two structures will be referred to as “Ca²⁺-loaded” and “apo”. All structural figures were prepared in UCSF chimera if not otherwise indicated.

Fig. 2. A closed pore with an elongated intracellular gate.

a Difference in the architecture of PD between PKD1L3/2L1 and PKD1/2 (PDB code: 6A70). PKD1L3 has a conventional PH1-SF-PH2 segment that is missing in PKD1. Inset: Comparison of the PD segments of PKD1 (yellow) and PKD1L3 (blue). The S6 segment of PKD1 bends in the middle, resulting in a S6a half helix that aligns with a typical pore helix PH1. The sequence connecting S6a and S5 is invisible in PKD1. b Conformational shifts between the S6 tetrahelical bundles of PKD1L3/2L1 and PKD1/2. The PD of apo PKD1L3/2L1 is superimposed with that of PKD1/2. A secondary structural element transition in the middle of S6 from an α helical turn in PKD2L1 to a π helix in PKD2 results in an iris-like rotation of the S6 tetrahelical bundle. The conformational shifts of the corresponding segments from PKD1L3/2L1 to PKD1/2 are indicated by red arrows. c The PD is sealed by an elongated intracellular gate. The permeation path of the apo heterotetramer, calculated by HOLE, is illustrated by gray dots. The pore radii of PKD1L3/2L1 (red), homotetrameric PKD2L1 (blue, PDB code: 5Z1W), and homotetrameric PKD2 (green, PDB code: 5T4D) are compared (right). The intracellular gate of PKD1L3/2L1 is extraordinarily elongated and will be illustrated as two layers. Right two panels: Composition of the two layers of the intracellular gate. Shown here are extracellular views. The densities are contoured at 5 σ. d PD comparison between PKD1L3/2L1 and PKD2L1 homotetramer. Longer S6 segments of PKD2L1 were resolved in PKD1L3/2L1 than those in the PKD2L1 homotetramer. Note that the PKD2L1 subunits exhibit nearly identical conformations in the two channels. e Structural deviations of the S4–S5 linkers of the four subunits in PKD1L3/2L1. Left: Whereas the linkers between S4 and S5 in PKD2L1, similar to those in other VGIC proteins, forms a short helix, that in PKD1L3 is a loop. Even among the three PKD2L1 subunits, the S4–S5 linker in VSD_III is distinct from the other two. The C-terminal short helix (dark green) of the S4–S5_III bends toward the PD, pushing S6 of PKD1L3 inward. Right: The S4–S5 helix of PKD2L1-III interacts with the S5 and S6 helices of PKD1L3 through specific hydrogen bonds (upper panel) and extensive van der Waals contacts (lower panel). The hydrogen bonds are indicated by black dashed lines.

Fig. 3. A Lys switch for Ca²⁺ entrance to the “KDDD” selectivity filter (SF).

a An asymmetric SF. Left: The pore domain of the apo and Ca²⁺-loaded complexes exhibits nearly identical overall conformation. Right: The distinct composition of the SF segments in PKD1L3 and PKD2L1 results in an asymmetric SF in the heterotetramer. Shown in the insets are the side views of the SF in the two diagonal subunits of the apo structure. b A Ca²⁺ ion is found in the SF in the Ca²⁺-loaded structure. Left: Ca²⁺ is positioned off the central axis and closer to PKD1L3. Shown here is an extracellular view of the PD. Right: The bound Ca²⁺ ion is mainly coordinated by three carbonyl oxygen groups on the SF loop of PKD1L3. The carboxylate group of Asp523 and two carbonyl oxygens from the two preceding residues in PKD2L3-III may also contribute to water-mediated coordination. The distances are indicated as black dashed lines and labeled in unit Å. c The upper site of the SF is open for Ca²⁺ entry with an outward swing of the long side chain of Lys2069. Left: Distinct conformations of Lys2069 between the apo (pink) and the Ca²⁺-loaded (domain colored) structures. Right: The Ca²⁺ was clearly resolved in the map of Ca²⁺-loaded complex. The densities, shown as gray mesh, are contoured at 4 σ. d An enlarged outer mouth and redistributed surface charges of the SF upon addition of Ca²⁺. Shown here are extracellular views of the electrostatic surface potential of the PD calculated in PyMol.

Fig. 4. An unconventional Ca²⁺ binding site in VSD_III of the heterocomplex may be responsible for Ca²⁺ activation.

a A Ca²⁺ ion is found in the extracellular cleft of VSD_III. Left two panels: An extra density is found in VSD_III only in the presence of added Ca²⁺. The densities are contoured at 4 σ. Right two panels: Different local conformations of the three VSDs of PKD2L1 reveal the molecular basis for the VSD_III-only binding site for Ca²⁺. Arg343 in VSD_III projects toward the PMD domain to interact with Asn313 in PMD_III and Ser1761 in PKD1L3, whereas in VSD_I (silver) and VSD_II (pale cyan) it points to the interior of the VSD and occupies the Ca²⁺-binding site. The down conformation of Arg343 in these two VSDs is likely owing to repulsion by His213 from the neighboring PKD2L1 subunit. b VSD_III deviates to a larger degree between the apo and Ca²⁺-loaded structures than the other three VSDs. Shown here is the heatmap of RMSD between the two structures. c Relatively minor but important conformational changes of VSD_III, S4–S5_III, and PD between apo and Ca²⁺-loaded states implicate the molecular basis for Ca²⁺-induced channel activation. Left: Shifts of the VSD_III segments. Right: Key residues that may mediate channel activation upon Ca²⁺ binding to VSD_III. d Functional validation of the residues in panel a that constitute the Ca²⁺-activation site. e Functional validation of the residues in panel c that may be involved in the conformational coupling for Ca²⁺-induced channel activation. Data are presented as mean ± SD in the bar graph. Currents of other groups are compared with that injected with PKD1L3-CTD/PKD2L1 with two-sided Student’s t test. ***P < 0.001; ****P < 0.0001. No significant or mild changes in acid-induced current were observed in these mutants (Supplementary Fig. 10).

Fig. 5. A working model for Ca²⁺ activation of PKD1L3-CTD/2L1.

a An overall schematic illustration of the conformational changes of the heterochannel upon Ca²⁺ binding to the SF and VSD_III. The Ca²⁺ and Na⁺ ions are shown as red and orange circles, respectively. b A more detailed illustration of the change of channel segments corresponding to the two states shown above. Two diagonal views are shown.