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.