Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

<div><p>Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the K_v1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.</p></div

Molecular determinants of ligand recognition in the SthK-C_term in complex with cAMP or cGMP.

<p><b>a-b)</b> Comparison of the amino acids in the ligand binding site involved in recognition of cAMP in SthK (left) and HCN2 (right). Amino acids of the β-roll are shown in blue sticks, residues of the C-helix in green sticks. The C-helix is shown in cartoon representation. The cAMP molecule is shown in yellow. Dashed lines represent hydrogen bonds or salt bridges. <b>c-d)</b> Comparison of the cGMP bound SthK-C_term with the cGMP bound structure of HCN2 illustrates that the cGMP molecule binds in the anti-conformation, whereas previously published structures showed cGMP bound in its syn-conformation. Locked in the anti-conformation, the phosphoribose part of cGMP is able to establish the same interaction with SthK-CNBD as cAMP. However, the anti-conformation appears to inhibit interactions between the purine ring from cGMP and the C-helix from the SthK-CNBD. Most likely, the absence of these interactions explains why binding of cGMP to SthK does not provide the required energy for the opening conformational changes.</p

Molecular architecture of the SthK C-terminal domain.

<p><b>a)</b> Superposition of the SthK-C_term tetramer onto the open gate structure of the Kv1.2 channel. The CNBD is shown in blue, the C-linker in green and the transmembrane domain in yellow. Only two transmembrane subunits are shown for clarity. <b>b)</b> Cartoon representation of a single SthK-C_term monomer. The C-linker is shown in red, the β-roll in blue and the C-helix in green. The cAMP molecule is shown in sphere representation. Blue atoms are nitrogen, white atoms are carbon and red atoms are oxygen. <b>c)</b> Top down cartoon view of the SthK-C_term tetramer along the fourfold symmetry axis. Each of the four subunits is shown in a different color. <b>d)</b> Superposition of the C-linker of SthK (yellow) onto HCN2 (white). <b>e)</b> Superposition of the CNBD of SthK (yellow) onto HCN2 (white)</p

Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels - Table 1

<p>Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels - Table 1 </p

Negative stain electron microscopy.

<p>Representative areas of micrographs with negatively stained purified <i>Alpo</i>1-wt, <i>Alpo</i>1-eGFP, <i>Alpo</i>4-wt, <i>Alpo</i>4-eGFP, <i>Alpo</i>6-wt and <i>Alpo</i>6-eGFP. The insets in <i>Alpo</i>4-wt show class averages corresponding to top and side views of the protein. The edge length of the insets is 220 Å.</p

FSEC-based detergent screen [34].

<p>FSEC profiles of <i>Alpo</i>X-eGFP solubilized by the indicated detergents. The fluorescent signals were approximately equated with each other. * indicates a suitable detergent for the solubilization of the particular protein. A detergent was considered suitable if FSEC resulted in a chromatogram characterized by a high, symmetric peak around 13.5 mL and a lower peak around the void volume (7 mL).</p

Ion selectivity of <i>Alpo</i>5-wt and <i>Alpo</i>6-wt.

<p>Current-voltage curves of <i>Alpo</i>5-wt and <i>Alpo</i>6-wt activated by 10 mM GABA recorded in the presence of extracellular chloride (green) or gluconate ions (red).</p

Large-scale purification of <i>Alpo</i>1, <i>Alpo</i>4 and <i>Alpo</i>6.

<p>SEC-profiles of purified <i>Alpo</i> CLR homologues and SDS-PAGE analyses of the corresponding oligomeric peak fractions. Left: Purification of Alpo1-eGFP and <i>Alpo</i>1-wt in DDM. Middle: Purification of <i>Alpo</i>4-eGFP and <i>Alpo</i>4-wt in LMNG-CHAPS. Right: Purification of <i>Alpo</i>6-eGFP and <i>Alpo</i>6-wt in the presence of glycine and LMNG.</p

Subunit topology and construct design.

<p>β-strands are indicated in dark green, α-helices in red and eGFP in mint green. The signal sequence is colored blue, the His₈-tag yellow. EC: extracellular, IC: intracellular. (A) Cartoon representation of a single pLGIC subunit, seen parallel to the membrane plane. The arrow indicates the position of eGFP in the <i>Alpo</i>X-eGFP constructs. (B) Construct design of <i>Alpo</i>X-wt and <i>Alpo</i>X-eGFP.</p

List of 33 chemical compounds and their concentration used for ligand screening by TEVC.

<p>List of 33 chemical compounds and their concentration used for ligand screening by TEVC.</p