Structural predictions of HCN/CNG ion
channels:
Insights on channels’ gating
Candidate:
Supervisors:
Alejandro Giorgetti
Prof. Paolo Carloni
Prof. Vincent Torre
Ion channels
Membrane proteins that allow ions to cross the hydrophobic
barrier of the core membrane, guarantying to the cell a
controlled exchange of ionized particles.
Ion permeation is crucial for a variety of biological functions
such as nervous signal transmission and osmotic regulation
(Hille, 2001).
Many diseases are also associated to defects in ionic channels
function, the majority of them arising from mutations in the
genes encoding the channel proteins.
A lot of effort is still necessary to connect these mutations to the
structural and functional changes causing the disorder.
Difficulties on getting high resolution 3D structures, may be
resolved by exploiting structure-based strategies in order to
predict structures and to design specific inhibitors targeting
pharmacologically relevant channels.
Cyclic Nucleotide Gated Ion Channels
Illustrate nicely the evolutionary innovation
of new protein functions
by combining functional domains
from several unrelated proteins
Hille, 2001
Hyperpolarizationactivated and
Cyclic nucleotidemodulated
HCN
Cyclic nucleotidegated ion channels
CNG
HCN channels
Heart and brain pacemaking regulation
Sea urchin sperm (spHCN)
Mammalian heart and brain: HCN1-4
Activated by membrane hyperpolarization
Modulated by interaction with cyclic nucleotides
Tetrameric
Similar topology to voltage-gated K+ channels
Cation selective: K+ > Na+ .
Problem: No Crystal structure available (pore)
Extracellular
++++
+++
P-helix-Loop
++
++
S1
S2
S3
S4
+
----
Cytoplasm
---
---
S5
+++
-50 mV
S6
+
---C-Linker
N-Terminal
CNBD
CNG channels
Participate in sensory perception and signalling
throughout the nervous system
Photoreceptors
Cones
Rods
CNG channels in Rods
Olfactory receptors
Other tissues
(aorta, kidney, testis,..)
Gated by interaction with cyclic nucleotides
Tetrameric
Cation selective:Na+ ~ K+ > Li+ > Rb+ > Cs+.
Similar topology to voltage-gated K channels
Problem: No Crystal structure available
More than 70 experimental restraints
Project Aims
Use of different approaches for model building of two ion
channels, extensively studied in Prof. V. Torre’s lab.:
HCN channels: Construction of a large family of models
in order to extract conclusions regarding the
rigidity/flexibility properties of the filter and gating
mechanism, within the low amount of experiments.
CNG channels: Using a large number of constraints we
will try to present a rather well-defined structure of the
open and closed states in order to provide a rational to the
gating mechanism.
Comparative Modeling
Target
sequence
Known
Structures
(templates)
Template(s)
selection
Coordinate
Mapping
Final Structural
Models
Structure Evaluation
Idea: Proteins evolving from a
Sequence
common ancestor maintained
Alignment
similar core 3D structures.
Protein Data Bank PDB
Database of templates
Template(s)
selection
Target
sequence
Sequence Similarity
Structure quality (resolution,
experimental method)
Sequence
Alignment
Coordinate
Mapping
Experimental conditions
(ligands and cofactors)
Final Structural
Models
Structure Evaluation
Comparative Modeling
Known
Structures
(templates)
Comparative Modeling
Template(s)
selection
Sequence
Alignment
Coordinate
Mapping
mHCN2 C-Linker
Final Structural
Models
Structure Evaluation
KcsA
MthK (open)
KirBac1.1
KvAp
Target
sequence
Known
Structures
(templates)
Comparative Modeling
Target
sequence
Known
Structures
(templates)
Template(s)
selection
Alignment improvement:
Secondary Structure Predictions
Transmembrane Helix
Predictions (PHD program)
Experimental information on
regions important for gating and
selectivity.
Sequence
Alignment
Coordinate
Mapping
Final Structural
Models
Structure Evaluation
Used program: ClustalW
Satisfaction of Spatial Restraints:
Known Obtained from the
Homology derived:
Structures
MODELLER
sequence alignment.
(templates)
Sequence
Alignment
Coordinate
Mapping
Structure Evaluation
Stereochemical: Obtained from the
amino acid sequence of target (CHARMM
parameter set - MacKerell et al., 1998 ).
Template(s)
Target
Van der Waals
and Coulomb energy
selection
sequence
terms: from CHARMM force field
‘External’: Include distances restraints in
the generation of the model.
Final Structural
Models
Comparative protein modeling by satisfaction of spatial restraints.
A. Šali and T.L. Blundell. J. Mol. Biol. 234, 779-815
Comparative Modeling
Target
sequence
Iterative cycles of alignment,
modeling and evaluation
Template(s)
selection
Sequence
Alignment
Coordinate
Mapping
Validation: experiments?
Iterative cycles of modelingexperiments-modeling-
Final Structural
Models
Structure Evaluation
Errors in template selection or
alignment result in bad models
Known
Structures
(templates)
Experimental Data  Distance Restraints
(Cysteine scanning mutagenesis)
Cd2+ coordinates to two or more cysteins
25%
Extracted from pdb
Frequencies
20%
15%
Accessibilities experiments:
10%
MTS reagents
5%
0%
3
4
5
6
7
8
9
charge
10
d(Ca@Cys-Cd(II)-Ca@Cys)
Rothberg and Yellen, 2002
Rulisek and Havlas,2000
CuP favours disulphide bond formation
diameter length
MTSET:
+
5.8 Å
10 Å
MTSES:
-
4.8 Å
10 Å
MTSEA:
+
4.8 Å
10 Å
Range (Å)
Maximum Allowed
distance(Å)[1]
35%
Extracted from pdb
30%
CONVENTION
Frequencies
25%
20%
Cα@Cys- Cα@Cys
3.6 -7
9
15%
Cd – Cα@Cys
3–5
6
10%
Cα@Cys - Cd Cα@Cys
5 – 9.2
11
5%
0%
3
4
5
6
d(Ca@Cys-Ca@Cys)
7
8
[1] Maximum allowed distance considering the thermal fluctuations of the protein
(Careaga and Falke, 1992).
HCN channels: modelling
S5-Helix
S6-Helix
Activation Gate
Template: KcsA at 2.00 Å resolution and KirBac1.1 for
Closed configuration.
Template: MthK for open configuration.
Overall Identity: KcsA-SpIh: 18 %. (P-helix-loop: 33%)
CNG channels:
P-Helix-Loop Models
Lys433
Validation controls:
C428 blocked upon CuP exposure
C428 blocked upon Cd2+ exposure
C428S recovers wt function
Rotameric Studies of K433 and R405
# Hydrogen-bonds in the filter:
KcsA ~ 26
HCN (more than 180 structures) ~ 21±1
Rigidity/flexibility connected to selectivity
properties? (Laio and Torre, 1999)
HCN channels:
Gating Model
T464C: irreversible
Cd2+ block
N465C: reversible Cd2+
block
MthK
L95
E96
A108
Q468C: reversible Cd2+
block
E92
A111
T112
KcsA
Open
V115
G461
d(T464Cα - T464Cα) ≈ 11 Å
Template
T464
N465
Q468
Target: spHCN
Close
CNG channels
++++
+++
P-helix-Loop
++
++
S1
S2
S3
S4
+
----
Cytoplasm
---
S5
+++
S6
+
---
---C-Linker
N-Terminal
CNBD
State independent
reversible Cd2+ blockage
C-Linker
S6-Helix
CNG channels:
S6-Helix/C-linker Modelling
Template: KcsA at 2.00 Å resolution for S6 region
Template: MthK for open configuration
Template for the C-Linker N-term: mHCN2 (> 30 %)
Overall Identity: KcsA-SpIh: 18 %
State dependent
Cd2+ blockage
CNG channels:
S6-Helix/C-linker Modelling
C-Linker
S6-Helix
F375
N402
A406
Q409
A414
Q417
d(Opposite Cα) ≈ 11 Å
Closed
Open
V391
12.0 Å
13.4 Å
G395
12.7 Å
13.5 Å
S399
12.0 Å
14.0 Å
CNG channels:
P-Helix-Loop Modelling
S5-helix
P-helix
S6-helix
F380
Potentiation
Block
-
-
D(F380Cα- C314Cα) < 8 Å
F380C-L356C
No Effect
No Effect
-
-
D(F380Cα- L356Cα) ≈ 6 Å
T360
Block
No Effect
MTSES
Poten
MTSES
Poten
D(Cα- Cα) ≈ 11 Å (Open)
Template: KcsA at 2.00 Å resolution for S6 region
Overall Identity: KcsA-SpIh: 18 %
D(Cα- Cα) > 14 Å (Closed)
CNG channels:
P-Helix-Loop Models
T355
E363
Closed
E71
E71
F380
L356
L358
T360
d(Cα-Cα)≈14 Å
I361
Upper
View
F380
T355
S6-Helix
L356
P-Helix
Closed
P-Helix
Open
E363
Open
TMA+
T360
d(Cα-Cα)≈11 Å
L358
S6 rotation  F380/L456  P-Helix  T360  I361  Pore occlusion
CNG channels:
Final
Models
Summary
HCN: Final structural models in agreement with
experimental results.
Proposed gating mechanisms for HCN and CNG channels.
CNG: Models used for designing experiments.
Models were able to predict coupling mechanism between
S6 and P-helix: L356 and F380.
Proposed interaction between S5 and S6: C314 and F380C
HCN vs CNG: Selectivity and Gating
Exhibit slightly different gating mechanisms: in CNG
channels the conformational change is transmitted to the Phelix-loop region, whilst HCN does not allows a
conformational change to be transmitted to the filter
region.
Differences in gating might be the cause of differences in
rigidity/flexibility of the channel pore and so, directly
related with the highly divergent selectivity properties of
both channels (Laio A. and Torre, 1999).
HCN channels exhibit intermediate properties between
pure voltage-gated K+ channels and pure Cyclicnucleotide gated channels.
Acknowledgements
Anil, Monica, Paolo and Pavel: the ‘experimentalists’ that did the dirty
job.
SISSA and GSK for financial support all these years, and also for very
useful discussions.
Paolo and Vincent, who showed me how to work in this fascinating
field, in which collaboration between theoreticians and
experimentalists is fundamental.
The ‘Zii’ Michele, Katrin, Lorenzo, Ciras, Ruben and Valentina, Pedro,
Andrea, Alessandra and Angelo, because they made us feel like home,
and principally, because in these years they were our ‘local family’.
All the great people from SBP sector: Simone, Claudio, Marco
(Berrera and Punta), Pietro, Matteo, Kamil, Andrea, Giacomo,
Francoise and Juraj. Among them, I wish to say ‘gracias’ to Sergio,
Claudia and Alejandro.
People from Menini’s and Torre’s groups for giving me the ‘window’
Also ‘gracias’ to our ‘Argentinean’ group: Marco, Dani and Marcelo;
Agustin, Caro and Marcelo, and last but not least: Eugenio
Of course, this thesis is dedicated to Ro and Santi.
A last word: used methodology
Because of the constantly improving bioinformatics
techniques and of the rapidly increasing number of
high-resolution protein structures, the combined
experimental/computational approach will play an
increasingly important role in membrane structure
predictions in the next future.