Structural comparisons of the high and low pH PcTx1-bound states with the desensitized state (PDB code: 3HGC) show that the upper palm β-strands and knuckle domains define a structurally conserved scaffold (Supplementary Fig. 6
), reminiscent of the scaffold of P2X receptors39
. Relative to the desensitized state, the lower palm domain and the wrist of the high pH Δ13-PcTx1 complex rotate by as much as ~13° around an axis positioned below the scaffold (). This rotation shifts subunit-subunit interfaces compared to those of the desensitized state structure, separating the thumb and palm domains of adjacent subunits by 2–3 Å and displacing the finger domains of the high and low pH complexes ().
Conformational changes in the extracellular domain
The consequences of toxin binding are manifested in the flexing of a blanket of β-sheets encapsulating the negatively charged central vestibule40
, a cavity composed of the lower palm domains, poised between the toxin binding site and the wrist ( and Supplementary Fig. 7
). With the Cα atom of Val 75 as a landmark, the distances between adjacent subunits are ~11 Å and ~12 Å in the low and high pH structures, respectively; upon formation of the desensitized state, the distance diminishes to ~7 Å. Chemical modification of the E79C mutant of ASIC341
, a residue equivalent to Glu 80 in cASIC1a and predicted to face the interior of the central vestibule, slows the rate and extent of channel desensitization, consistent with the notion that the central vestibule contracts upon transition from the PcTx1-bound states to the desensitized state. Small molecules that activate23
or potentiate the steady state current of ASIC322, 42
, respectively, bind within the central vestibule23
, or at the subunit interface near Ala 82, and stabilize the central vestibule in an expanded conformation, recalling the ATP-dependent expansion of the extracellular vestibule of P2X receptors39
A striking conformational change in the palm domain, common to both PcTx1 complexes and located within the β1-β2 linker, is a ~180o
flip of the Thr 84-Arg 85 peptide bond (Supplementary Fig. 8
), inducing a shift of β1 away from the equivalent β-sheet of the adjacent subunit. In addition, Ala 413, Leu 414, and Asn 415 in the β11-β12 linker in the high pH structure adopt conformations in which the side chains of Leu 414 and Asn 415 have effectively swapped positions ( and Supplementary Fig. 9
). Indeed, the Cα atoms of residues Ala 82 and Ala 413 are farther apart in the high pH state (8.1 Å), in comparison to the low pH state (6.4 Å), consistent with the notion that a disulfide can form between the equivalent residues in shark ASIC1b, stabilizing the channel in the desensitized state25
(Supplementary Fig. 10
). Studies at sites equivalent to Leu 86, a residue that interacts with Leu414 in the high pH Δ13-PcTx1 state, and Asn 415 reinforce the conclusion that the β1-β2 and β11-β12 linkers are crucial to gating42–44
The structures of the high and low pH Δ13-PcTx1 complexes suggest molecular mechanisms underlying the pH dependent conformations of the β1-β2 and β11-β12 linkers. We speculate that at high pH, the carboxyl group of Glu 80 is ionized, favoring an expanded conformation of the central cavity (Supplementary Fig. 7
). As the pH drops, acidic residues in the vestibule bind protons, allowing Glu 80 and adjacent residues to adopt a ‘contracted’ central vestibule conformation. In fact, the distance between Cα of Glu 80 and Glu 412, residues that flank both linkers, diminishes from 14.1 Å to 11.3 Å in the high and low pH states, respectively. Neutralization of Glu 80 by mutation to Ala results in a channel that desensitizes ~40-fold more rapidly than the parent construct and that does not yield measureable steady state current upon application of PcTx1 at pH 7.25 (Supplementary Fig. 11
), thus underscoring the role that titratable residues play in pH dependent conformational changes of the central vestibule.
A highly conserved non polar residue in the β11-β12 linker, Leu 414, is also important to the conformational transitions of the central vestibule. In the high pH Δ13-PcTx1complex, Leu 414 interacts with Leu 86 of the β1-β2 linker. Upon transition to the low pH Δ13-PcTx1 complex and to the desensitized state, however, Leu 414 forms multiple hydrophobic contacts involving Leu 281, Ile 306, and Val 368 of the adjacent subunit. In addition, Asn 415 hydrogen bonds with Tyr 416 and the backbone nitrogen of Ala 83 in the β1-β2 linker (Supplementary Fig. 9c, d
). Together, these rearrangements highlight the importance of the β1-β2 and β11-β12 linkers and nearby subunit interfaces to conformational transitions of the central vestibule.