Assessment of the Shaker K channel 3D structure by homology modeling

In our model, the geometrical and electrostatic properties of the VSD are derived from the 3D structure of the channel under study (cf. below). Although this structural information is presently available for the Kv1.2 and Kv1.2/Kv2.1 channel chimera, most of the functional data (i.e., gating and ionic current measurements) have been obtained from the Shaker K channel (cf. Introduction). Although these two K channels have a high degree of homology and are thus expected to have a very similar 3D structure (12), differences in charged residues in relevant positions of the VSD may well change the details of their gating behavior. We thus proceeded to generate a 3D structure of the Shaker K channel by homology modeling, using the SWISS-MODEL environment (31, 32, 33) and the Kv1.2/Kv2.1 chimera as a template.

Homology modeling, also known as comparative protein structure modeling, is a computational approach to build 3D structural models for proteins using experimental structures of related protein family members as templates. It generates the structural coordinates of the model based on the mapping between the target residues and the corresponding amino acids of the structural template. Regions of the protein for which no template information is available (i.e., insertions and deletions in loop regions) are built from libraries of backbone fragments or by de novo reconstruction by constrained dynamics. Local suboptimal geometry of the models obtained (i.e., distorted bonds, angles, and too close atomic contacts) are finally regularized by limited MD. The Shaker channel primary sequence (accession {"type":"entrez-protein","attrs":{"text":"P08510.3","term_id":"92090610","term_text":"P08510.3"}}P08510.3) was first aligned to the Kv1.2/Kv2.1 chimera using the PDBviewer software v4.10 (see Fig. S1 F), and then the two aligned sequences, together with the 3D coordinates of the Kv1.2/Kv2.1 chimera, were sent to the SWISS-MODEL web site to find the 3D structure for the Shaker K channels. As shown in Fig. S1 A, the 3D structure model for the Shaker channel appears very similar to that experimentally found for the Kv1.2/Kv2.1 channel chimera, as expected from the very high similarity of the two protein sequences.

Notwithstanding, several residues predicted to reside close to the S4 segment appear to be differently charged in the VSDs of the two channels (see Fig. S1 A, where the negative and positive residues of the two structures are colored in red and yellow, respectively, and the gating charges on the S4 segment in magenta). This is especially evident in their external vestibules, with the Shaker VSD charge density profile displaying a strongly negative charge density peak not present in the chimera channel, originating from several charged residues localized in the loops connecting the helices. Diverse dynamics of the S4 segment in the two channels, due to the marked difference in the electrostatic voltage profile along their gating pores and vestibules, can thus be envisaged. This peculiar feature is expected to stabilize the S4 segment in its activated position, in accordance with a Q-V relationship shift to more hyperpolarized voltages found for the Shaker K channel as compared to the Kv1.2 channel, which has a fixed charge profile virtually identical to the Kv1.2/Kv2.1 chimera (8, 34).

The use of a homology model of the Shaker K channel is quite justified by the high similarity of its primary structure with that of the Kv1.2/Kv2.1 chimera channel (cf. Fig. S1). The reader should, however, be aware that the resulting structural features of the Shaker channel are hypothetical, and subtle differences with the real channel (whose crystal structure is not presently available) may occur.