Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K⁺ channel

1) This well-argued paper could perhaps be improved somewhat if the very long run-on paragraphs are broken up to allow the reader to follow the discussion better.

We have revised the manuscript, especially the Introduction and Discussion, to improve readability. Numerous paragraphs have been shortened and tightened.

2) The figures are notable for their clarity, but the addition of a few more labels could provide guides for the non-specialist reader to follow more readily. For example, in Figure 1, identify the structural module colored blue. In Figure 2, point out where the channel subunit is, and where the beta subunit is. We don't mean to overemphasize this point, but the authors may consider readers who are not overly familiar with the architecture of the voltage-gated channels.

Labels have been added as requested to Figure 1, as well as a shaded area to demarcate the membrane, two additional toxins added to Figure 1B (as requested below), coloration to distinguish components in Figure 3 (we believe the reviewer was referring to Figure 3 in the comment about Figure 2 above), we have changed the orientation in Figure 4A to show secondary structure elements in toxin density (as requested below), and we have added ion site labels S1–S4 on Figure 6C.

3) The Introduction should point out that the structures of the scorpion toxins (mention the important ones) are very similar, and they are expected (known?) to bind the channels in similar ways. This point is key to the arguments in the Discussion, but is taken for granted. In particular, the concluding part of the paper says that: “The CTX family of K⁺ channel toxins has a remarkable diversity in sequence and binding affinities to specific channel subtypes.” Given this, might the reader not assume that the binding modes might be different, and that some toxins might require conformational changes and others might not?

We have addressed this point by adding to the Introduction the sentences below (in quotes) and we now show three different toxin structures in Figure 1b:

“The positive charge was later identified as Lys27, a residue that is conserved in all members of the CTX‐like toxin family (Figure 2A) (Park and Miller, 1992; Goldstein and Miller, 1993). Studies with other members of the CTX toxin family, most extensively Agitoxin2 (AgTx2), supported the conclusion that they function in a manner similar to CTX (Garcia et al., 1994; Krezel et al., 1995; Hidalgo and MacKinnon, 1995; Ranganathan et al., 1996). Most notably, conservation of toxin shape and the functionally important lysine suggested they all bind with a similar orientation on a K⁺ channel and inhibit through a common mechanism whereby a lysine amino group functions as a K⁺ ion mimic to block the pore (Miller, 1995) (Figure 1b).”

4) A crystallographer might find it helpful to see a top-down view along the four-fold axis of the actual (pre-modeling) electron density map, and the refined map after modeling. This would help understand the statement that initially “certain secondary structural features were discernible…”

We have addressed this point by showing a different orientation of toxin omit map density in Figure 4A. This makes the point better than the suggested view down the four-fold axis.

5) When discussing the Kaliotoxin NMR “structure”, the authors might state whether or not the contacts inferred from the NMR data are consistent with the crystal structure.

This is a very important point that we did not address explicitly, but in response to this comment we have made modifications (below). Basically, the solid state NMR data did not contain distance restraints between toxin and channel, and therefore the origin of the “structure” is entirely unclear. The paper simply shows some chemical shift changes in both the toxin and channel upon binding, which very possibly do not have their origin in structural changes. Thus, we have removed the reference to Lange et al. from the last paragraph of the Discussion (and kept the references to Takeuchi et al. and Yu et al., which refer to NMR models based on distance restraints). Regarding the conformational changes proposed in the Kaliotoxin NMR “structure” (Lange et al.) we have added the following to the Discussion:

“This, we note, is in contrast to the solid state NMR ‘structure’ of a KcsA mutant with Kaliotoxin, where such rearrangements in the channel were proposed (Lange et al., 2006). In this NMR study chemical shift changes induced by toxin were interpreted as resulting from dihedral angle changes (i.e., structural); however, such chemical shift changes could have other origins (i.e., electrostatic). Moreover, no channel–toxin distance restraints were included in the determination of this solid state NMR ‘structure’ (Lange et al., 2006). In the crystal structure presented here…”

6) Could the authors comment on why active CTX requires its N-terminus to be blocked with pyroglutamate, if any hints of this emerge from the structure?

In order to say something conclusively about this, we would need more mutagenesis results with same channel-toxin pairs, which we do not have (i.e., mutations on the N-terminus have been done with toxin-channel pairs that are different than our structure and therefore we would have to speculate on the basis of homology modeling). We’d rather not. However, for purposes of intellectual exchange we offer the following thoughts here should the reviewers want to think about it for future work:

The most subtle experiment has been done in the Goldstein, Pheasant, and Miller paper in Neuron (1994) where the pyroGlu was mutated to a proline resulting in a near-isosteric but charge inequivalent substitution. The result was an 8-fold increase in Koff and a 570-fold change in K_d. Now if one looks at the region of the channel where the pyroGlu approaches the channel and compares an alignment between Kv1.2 and Shaker, here is what we see –

Kv1.2 …D E R D S Q F P…

Shaker …G S E N S F F K…

The underlined residue, which is Q in the structure and is Phe in Shaker. In one conformation of the Phe side chain, it could possibly get into disfavourable interactions with a charged terminus. That would explain the results with Shaker.

(This implies that with the same pyroGlu to proline mutation and with paddle chimera the effect of the pyroGlu to proline mutation would be less–information that we don't know about.)

The other piece of information is with BK from the Park, Hausdorff, and Miller paper in PNAS (1991) where the uncyclized material shows an apparent K_d of 110 nM as compared to 10 nM for the native toxin (Anderson, MacKinnon, Smith and Miller, J. Gen. Physiol., 1988). However, here there may be dual effects from charge and sterics as well. Comparison of the same region for Kv1.2 and BK looks like the following –

Kv1.2 …D E R D S Q F P…

BK …S G D P L D F D…

However, these two regions are much harder to align between Shaker and BK and it is harder to rationalize smaller changes in affinity based on this. Thus, we have chosen to not to try to interpret these results.