Structure and dynamics underlying elementary ligand binding events in human pacemaking channels
- University of Wisconsin-Madison, United States
Figures
Figure 1 with 2 supplements
Imaging ligand binding to single molecules in ZMWs.
(A) HCN2 channel subunit transmembrane topology is homologous to canonical voltage-gated potassium channel subunits with the exception of a CNBD following the pore lining S6 helix. (B) Isolated CNBD …
Figure 1—figure supplement 1
Bulk solution fluorescence imaging of fcAMP binding.
(A) Bound probability obtained by measuring fcAMP bulk solution anisotropy plotted as a function of CNBD concentration for wild type and mutant HCN2 CNBDs. (B) Solution FRET between bound fcAMP and …
Figure 1—figure supplement 2
Imaging ZMW arrays.
Brightfield (A) and fluorescence (B) images of an array of ZMW nanoholes with diameters of approximately 200 nm. Bright spots in the fluorescence image reflect the subset of ZMWs that contain a …
Figure 2 with 1 supplement
Singe-molecule FRET imaging of binding dynamics at micromolar concentrations.
(A) Cartoon depicting smFRET during fcAMP binding. Direct excitation of the donor fcAMP results in stimulated emission from the acceptor on the CNBD due to efficient FRET while the donor is bound up …
Figure 2—figure supplement 1
Specific fcAMP binding at single molecules.
(A–C) Single-molecule fluorescence time series for individual acceptor-labeled CNBDs in the absence (A) and presence (B) of 3 µM fcAMP, and during competition between 3 µM fcAMP and 5 mM …
Figure 3 with 4 supplements
Single-molecule cyclic nucleotide binding dynamics at HCN2 CNBDs.
(A) Bound probability from the total fraction of time spent bound for all single molecules as a function of fcAMP concentration fit with the equation Bmax/(1+Kd/[fcAMP]), where Bmax = 0.75 is the …
Figure 3—figure supplement 1
Maximum likelihood estimates of biexponential parameters for single-molecule dwell time distributions.
(A) Histograms of unbound and bound single-molecule dwell time distributions (gray) overlaid with maximum likelihood estimates for monoexponential (blue dashed) and biexponential (red) …
Figure 3—figure supplement 2
Dwell time correlations within single molecules.
Contour plots of two dimensional histograms for (A) the average bound time versus average unbound time per molecule, (B) first order correlation between the dwell times of sequential unbound (event …
Figure 3—figure supplement 3
CNBD tetramer.
Size exclusion chromatography confirms that the GCN4pLI-HCN2(CNBD) tetramer is stable in solution and exhibits no detectable dissociation into monomers. (Inset) SDS-PAGE overloaded with respect to …
Figure 3—figure supplement 4
Binding with and without smFRET.
Comparison of dwell time distributions from monomeric CNBDs either before acceptor bleaching (i.e. from smFRET trace) or after acceptor bleaching (i.e. fcAMP fluorescence alone). See Figure 2B for …
Figure 4 with 3 supplements
Comparison of apo and holo forms of the HCN2 CNBD.
(A) X-ray crystal structures of apo (this work) and (B) holo (PDB 3U10) conformations of the HCN2 CNBD colored according to protein secondary structure. Bound cAMP in the holo structure is shown as …
Figure 4—figure supplement 1
Overview of the X-ray structure.
(A) X-ray crystal structure of the HCN2 CNBD in the absence of ligand as a fusion protein with MBP. CNBD segments rainbow colored from N- (blue) to C-terminus (red). MBP shown in gray. (B) CNBD …
Figure 4—figure supplement 2
Comparison of the X-ray and NMR structures of the apo CNBD.
X-ray (red) and NMR (cyan) structures of the apo CNBD superposed over all Cα atoms. The P-helix in the X-ray structure is highlighted in gold. Although the α-helical termini are similar in both …
Figure 4—figure supplement 3
Molecular packing in the crystal of apo MBP-HCN2.
Shown are six asymmetric unit cells viewed down the b axis. MBP is shown in white and HCN2 in rainbow representation. HCN2 moieties are arranged in a loosely packed layer that connects MBP layers …
Figure 5
Global and local structural features stabilized by cAMP.
(A) RMSDs from apo (this work) and holo (PDB 3U10) crystal structures for the HCN2CNBD (residues 515–632) during 1 µs simulations starting in either the apo or holo structure both with and without …
Figure 6
A structural model of HCN2 CNBD binding dynamics.
A structural model of cAMP (red spheres) binding dynamics at monomeric CNBDs from HCN2 channels. Rate constants (s-1 or M-1s-1) were optimized using HMM modeling of idealized single-molecule fcAMP …
Tables
Table 1
Kinetic model rate constants. Optimized rate constants (s-1 or M-1s-1) for models shown in Figure 3E. U* and B* denote unbound and bound states, respectively.
Model | U1→ B1 | B1→ U1 | B1→B2 | B2→B1 | U1→U2 | U2→U1 | U1→B2 | B2→U1 |
|---|---|---|---|---|---|---|---|---|
1 | 1.3 × 105 | 0.34 | - | - | - | - | - | - |
2 | 1.4 × 105 | 0.91 | 0.52 | 0.31 | - | - | - | - |
3 | 1.3 × 105 | 0.98 | 0.49 | 0.23 | - | - | 0.10 × 105 | 0.04 |
4 | 2.3 × 105 | 0.95 | 0.51 | 0.31 | 0.04 | 0.15 | - | - |
5 | 2.2 × 105 | 1.00 | 0.49 | 0.25 | 0.04 | 0.15 | 0.14 × 105 | 0.03 |
6 | 2.4 × 105 | 1.00 | 0.48 | 0.27 | 0.01 | 0.04 | - | - |
7 | 2.8 × 105 | 1.11 | 0.85 | 0.56 | 0.17 | 0.55 | - | - |
Model | U2→B2 | B2→U2 | U2→U3 | U3→U2 | B2→B3 | B3→B2 |
|---|---|---|---|---|---|---|
6 | 0.24 × 105 | 0.02 | - | - | - | - |
7 | - | - | 0.02 | 0.07 | 0.03 | 0.08 |
Table 2
Crystallographic statistics.
Data collection | |
|---|---|
Space group | P21 |
Unit cell dimensions | |
a, b, c (Å) | 61.5, 42.0, 198.4 |
α, β, γ (°) | 90, 90.9, 90 |
Resolution (Å) | 24.82–2.07 (2.11–2.07) |
Unique reflections | 62519 |
Redundancy* | 3.4 (3.4) |
Average [I/s] * | 7.6 (2.2) |
Completeness (%)* | 99.9 (100) |
Rmerge (%)* | 11.5 (66.7) |
Refinement | |
Number of atoms | |
protein | 7913 |
maltose | 46 |
solvent | 381 |
Rwork (%)* | 18.0 (21.6) |
Rfree (%)* | 22.1 (26.0) |
Twin fraction | 0.122 |
Average B-factors, (Å2) | |
protein (MBP), protein (HCN2) | 22.9, 43.2 |
solvent | 28.9 |
R.M.S. deviations, bond angles (°) | 1.322 |
R.M.S. deviations, bond lengths, (Å) | 0.009 |
Ramachandran plot (%) | |
favored | 98 |
allowed | 2 |
| * Values in parentheses are for the outer resolution shell. | |
Table 3
cAMP-dependent change in H-bonding within PBC.
Apo (this work) | Holo (PDB 3U10) | ||||||
|---|---|---|---|---|---|---|---|
carbonyl | amide | d, Å | pattern | carbonyl | amide | d, Å | pattern |
G581 | C584 | 3.42 | i + 3 | G581 | — | — | — |
E582 | L585 | 3.02 | i + 3 | E582 | L586 | 3.07 | i + 4 |
I583 | L586 | 3.10 | i + 3 | I583 | L586 | 3.10 | i + 3 |
I583 | T587 | 2.71 | i + 4 | I583 | T587 | 2.71 | i + 4 |
C584 | — | — | — | C584 | R588 | 2.91 | i + 4 |
