PDBsum entry 3bpz

Transport protein

PDB id: 3bpz

Contents

Protein chains

194 a.a. *

Ligands

CMP ×4

Waters ×1044

* Residue conservation analysis

PDB id:

3bpz

Name:

Transport protein

Title:

Hcn2-i 443-460 e502k in the presence of camp

Structure:

Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2. Chain: a, b, c, d. Fragment: ligand biding domain (residues 443-640). Synonym: brain cyclic nucleotide-gated channel 2, bcng-2, hyperpolarization-activated cation channel 1, hac-1. Engineered: yes. Mutation: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Gene: hcn2, bcng2, hac1. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.65Å R-factor: 0.191 R-free: 0.216

Authors:

K.B.Craven,N.B.Olivier,W.N.Zagotta

Key ref:

K.B.Craven et al. (2008). C-terminal movement during gating in cyclic nucleotide-modulated channels. J Biol Chem, 283, 14728-14738. PubMed id: 18367452 DOI: 10.1074/jbc.M710463200

Date:

19-Dec-07 Release date: 25-Mar-08

Protein chains

O88703  (HCN2_MOUSE) -  Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 from Mus musculus

Seq: 863 a.a.

Struc: 194 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M710463200

J Biol Chem 283:14728-14738 (2008)

PubMed id: 18367452

C-terminal movement during gating in cyclic nucleotide-modulated channels.

K.B.Craven, N.B.Olivier, W.N.Zagotta.

ABSTRACT

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens. Here we tested this hypothesis by dynamically manipulating the SB triad in functioning CNGA1 channels. Reversing the charge at positions Arg-431 and Glu-462, two of the SB triad residues, by either mutation or application of charged reagents increased the favorability of channel opening. To determine how a charge reversal mutation in the SB triad structurally affects the channel, we solved the crystal structure of the HCN2 C-terminal region with the equivalent E462R mutation. The backbone structure of this mutant was very similar to that of wild type, but the SB triad was rearranged such that both salt bridges did not always form simultaneously, suggesting a mechanism for the increased ease of opening of the mutant channels. To prevent movement in the SB triad, we tethered two components of the SB triad region together with cysteine-reactive cross-linkers. Preventing normal movement of the SB triad region with short cross-linkers inhibited channel opening, whereas longer cross-linkers did not. These results support our hypothesis that the SB triad forms in the closed channel and indicate that this region expands as the channel opens.

Selected figure(s)

Figure 6.
Crystal structure of the C-terminal region of HCN2-E502K channels. A, E502K tetramer is shown: box indicates area of enlargement (left). Enlargements show two SB triad interfaces between subunits A (red) and C (gold) (middle); subunits B (blue) and D (green) (right). Mesh represents an F[o]-F[c] simulated annealing omit map of SB triad residues contoured to 1.8 σ. Red spheres represent water molecules. B, overlay of E502K structure (blue) and wild-type HCN2-I structure (green) in each configuration. The SB triad residues are shown in stick format. The structures were overlaid using the program LSQMAN (r.m.s.d. = 0.23 Å). C, schemes indicate the SB triad residues: lines indicate which salt bridges form in each configuration, and closest distances between residues are indicated for each salt bridge.

Figure 7.
Chemical structures of MTS reagents. MTS-Butyl, MTS-1-MTS, MTS-3-MTS, MTS-6-MTS are shown. Length (Å) of cross-linker or side-chain indicated (from points of disulfide attachment).

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 14728-14738) copyright 2008.

Figures were selected by an automated process.