Structural predictions of HCN/CNG ion channels:
Insights on channels’ gating
Candidate: Supervisors:
Alejandro Giorgetti Prof. Paolo Carloni
Prof. Vincent Torre
Ion channelsMembrane 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
Hyperpolarization-activated and Cyclic
nucleotide- modulated
HCN
Cyclic nucleotide- gated ion channels
CNG
HCN channels
Activated by membrane hyperpolarizationModulated by interaction with cyclic nucleotidesTetramericSimilar topology to voltage-gated K+ channelsCation selective: K+ > Na+ .Problem: No Crystal structure available (pore)
S1 S2 S3 S4 S5 S6+ +
++
N-Terminal
CNBD
P-helix-Loop
C-Linker
+ + + + + + + + + + + +
- - - - - - - - - - - - - -
-50 mV
Cytoplasm
Extracellular
Heart and brain pacemaking regulation
Sea urchin sperm (spHCN) Mammalian heart and brain: HCN1-4
CNG channels in Rods
Cones Rods
CNG channels
Photoreceptors Olfactory receptors Other tissues(aorta, kidney, testis,..)
Gated by interaction with cyclic nucleotidesTetramericCation selective:Na+ ~ K+ > Li+ > Rb+ > Cs+.Similar topology to voltage-gated K channels
Problem: No Crystal structure available More than 70 experimental restraints
Participate in sensory perception and signalling throughout the nervous system
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.
Template(s) selection
Sequence Alignment
Coordinate Mapping
Stru
cture E
valuation
Final Structural Models
Comparative ModelingKnown
Structures (templates)
Target sequence
Idea: Proteins evolving from a common ancestor maintained similar core 3D structures.
Template(s) selection
Sequence Alignment
Coordinate Mapping
Stru
cture E
valuation
Final Structural Models
Comparative ModelingKnown
Structures (templates)
Protein Data Bank PDBDatabase of templates
Sequence Similarity
Structure quality (resolution, experimental method)
Experimental conditions (ligands and cofactors)
Target sequence
Known Structures (templates)
Sequence Alignment
Coordinate Mapping
Stru
cture E
valuation
Final Structural Models
Target sequence
Comparative Modeling
Template(s) selection
KcsA MthK (open) KirBac1.1 KvAp
mHCN2 C-Linker
Known Structures (templates)
Template(s) selection
Coordinate Mapping
Stru
cture E
valuation
Final Structural Models
Target sequence
Used program: ClustalW
Alignment improvement:
Secondary Structure Predictions Transmembrane Helix
Predictions (PHD program) Experimental information on
regions important for gating and selectivity.
Comparative Modeling
Sequence Alignment
Known Structures (templates)
Template(s) selection S
tructu
re Evalu
ation
Final Structural Models
Target sequence
Satisfaction of Spatial Restraints: MODELLER
Sequence Alignment
Coordinate Mapping
Comparative protein modeling by satisfaction of spatial restraints. A. Šali and T.L. Blundell. J. Mol. Biol. 234, 779-815
Homology derived: Obtained from the sequence alignment.
Stereochemical: Obtained from the amino acid sequence of target (CHARMM parameter set - MacKerell et al., 1998 ).
Van der Waals and Coulomb energy terms: from CHARMM force field
‘External’: Include distances restraints in the generation of the model.
Known Structures (templates)
Template(s) selection
Sequence Alignment
Coordinate Mapping
Final Structural Models
Target sequence
Errors in template selection or alignment result in bad models
Iterative cycles of alignment, modeling and evaluation
Validation: experiments?
Iterative cycles of modeling-experiments-modeling-
Comparative Modeling
Stru
cture E
valuation
charge diameter length
MTSET: + 5.8 Å 10 Å
MTSES: - 4.8 Å 10 Å
MTSEA: + 4.8 Å 10 Å
Cd2+ coordinates to two or more cysteins
0%
5%
10%
15%
20%
25%
30%
35%
Fre
qu
en
cie
s
3 4 5 6 7 8
d(Ca@Cys-Ca@Cys)
CONVENTION Range (Å)Maximum Allowed
distance(Å)[1]
Cα@Cys- Cα@Cys 3.6 -7 9
Cd – Cα@Cys 3 – 5 6
Cα@Cys - Cd -Cα@Cys
5 – 9.2 11
[1] Maximum allowed distance considering the thermal fluctuations of the protein(Careaga and Falke, 1992).
0%
5%
10%
15%
20%
25%
Fre
qu
enci
es
3 4 5 6 7 8 9 10
d(Ca@Cys-Cd(II)-Ca@Cys)
CuP favours disulphide bond formation
Rothberg and Yellen, 2002Rulisek and Havlas,2000
Accessibilities experiments:
MTS reagents
Experimental Data Distance Restraints(Cysteine scanning mutagenesis)
Extracted from pdb
Extracted from pdb
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%)
HCN channels: modelling
Activation Gate
S5-Helix
S6-Helix
Lys433
Validation controls:C428 blocked upon CuP exposureC428 blocked upon Cd2+ exposure
C428S recovers wt function
Rotameric Studies of K433 and R405
CNG channels: P-Helix-Loop Models
# Hydrogen-bonds in the filter:
KcsA ~ 26
HCN (more than 180 structures) ~ 21±1
Rigidity/flexibility connected to selectivity properties? (Laio and Torre, 1999)
Close
Open
Target: spHCN
G461
T464N465
Q468
MthK
KcsA
Template
E96
E92
L95
A108
A111T112
V115
T464C: irreversible Cd2+ block
N465C: reversible Cd2+ block
Q468C: reversible Cd2+ block
HCN channels: Gating Model
d(T464Cα - T464Cα) ≈ 11 Å
S1 S2 S3 S4 S5 S6+ +
++
N-Terminal
P-helix-Loop
C-Linker
+ + + + + + + + + + + +
- - - - - - - - - - - - - -Cytoplasm
S4 S5 S6+ +
++
CNG channels
CNBD
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
State independent reversible Cd2+ blockage
S6-
Hel
ixC
-Lin
ker
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: S6-Helix/C-linker Modelling
d(Opposite Cα) ≈ 11 Å
N402
A406
Q409
A414
Q417
F375
S6-
Hel
ixC
-Lin
ker
CNG channels: P-Helix-Loop Modelling
F380 Potentiation Block - - D(F380Cα- C314Cα) < 8 Å
F380C-L356C No Effect No Effect - - D(F380Cα- L356Cα) ≈ 6 Å
T360 Block No Effect MTSESPoten
MTSES Poten
D(Cα- Cα) ≈ 11 Å (Open)
D(Cα- Cα) > 14 Å (Closed)
S5-helix P-helix S6-helix
Template: KcsA at 2.00 Å resolution for S6 region
Overall Identity: KcsA-SpIh: 18 %
CNG channels: P-Helix-Loop Models
E363T355
L358T360
d(Cα-Cα)≈11 Å
Open
TMA+
E71 E71E363
T355
L358
Closed
T360d(Cα-Cα)≈14 Å
UpperView
F380L356
S6-Helix
P-Helix OpenP-Helix
Closed
L356
F380
I361
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
Exhibit slightly different gating mechanisms: in CNG channels the conformational change is transmitted to the P-helix-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 Cyclic-nucleotide gated channels.
HCN vs CNG: Selectivity and Gating
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: EugenioOf course, this thesis is dedicated to Ro and Santi.
Acknowledgements
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.