Structural predictions of HCN/CNG ion channels: Insights on channels’ gating

1. Structural predictions of HCN/CNG ion channels:Insights on channels’ gating

2. 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.

3. 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

4. Extracellular P-helix-Loop + + + + + + + + + + + + + -50 mV + S6 S1 S2 S3 S4 S5 + + - - - - - - - - - - - - - - Cytoplasm C-Linker N-Terminal CNBD 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)

5. CNG channels Participate in sensory perception and signalling throughout the nervous system Other tissues (aorta, kidney, testis,..) Photoreceptors Olfactory receptors • 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 Cones Rods

6. 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.

7. Known Structures (templates) Comparative Modeling Template(s) selection Target sequence Idea:Proteins evolving from a common ancestor maintained similar core 3D structures. Sequence Alignment Structure Evaluation Coordinate Mapping Final Structural Models

8. Protein Data Bank PDB Database of templates Sequence Similarity Structure quality (resolution, experimental method) Experimental conditions (ligands and cofactors) Comparative Modeling Known Structures (templates) Template(s) selection Target sequence Sequence Alignment Structure Evaluation Coordinate Mapping Final Structural Models

9. Comparative Modeling Known Structures (templates) Template(s) selection Target sequence • KcsA • MthK (open) • KirBac1.1 • KvAp Sequence Alignment Structure Evaluation Coordinate Mapping • mHCN2 C-Linker Final Structural Models

10. Comparative Modeling Known Structures (templates) Template(s) selection Target sequence • Used program: ClustalW • Alignment improvement: • Secondary Structure Predictions • Transmembrane Helix Predictions (PHD program) • Experimental information on regions important for gating and selectivity. Sequence Alignment Structure Evaluation Coordinate Mapping Final Structural Models

11. Satisfaction of Spatial Restraints: MODELLER Known Structures (templates) • 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. Template(s) selection Target sequence Sequence Alignment Structure Evaluation Coordinate Mapping Final Structural Models Comparative protein modeling by satisfaction of spatial restraints. A. Šali and T.L. Blundell. J. Mol. Biol. 234, 779-815

12. Comparative Modeling Known Structures (templates) Template(s) selection 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- Sequence Alignment Structure Evaluation Coordinate Mapping Final Structural Models

13. Experimental Data  Distance Restraints(Cysteine scanning mutagenesis) Cd2+ coordinates to two or more cysteins Reversible Extracted from pdb Accessibilities experiments: MTS reagents Irreversible charge diameter length MTSET: + 5.8 Å 10 Å MTSES: - 4.8 Å 10 Å MTSEA: + 4.8 Å 10 Å Rothberg and Yellen, 2002 Rulisek and Havlas,2000 CuP favours disulphide bond formation Extracted from pdb [1] Maximum allowed distance considering the thermal fluctuations of the protein (Careaga and Falke, 1992).

14. 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%)

15. 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)

16. MthK Open L95 G461 E96 A108 T464 E92 N465 A111 T112 KcsA Q468 V115 Close Template Target: spHCN HCN channels: Gating Model • T464C: irreversible Cd2+ block • N465C: reversible Cd2+block • Q468C: reversible Cd2+ block d(T464Cα - T464Cα) ≈ 11 Å

17. CNG channels + + S6 S4 S5 + + P-helix-Loop + + + + + + + + + + + + + + S6 S1 S2 S3 S4 S5 + + - - - - - - - - - - - - - - Cytoplasm C-Linker N-Terminal CNBD S6-Helix Closed-Open P-Helix Accessibilities C-Linker Closed-Open Filter Accessibilities

18. State independent reversible Cd2+ blockage CNG channels: S6-Helix/C-linker Modelling S6-Helix C-Linker • 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

19. F375 S6-Helix N402 C-Linker A406 Q409 A414 Q417 CNG channels: S6-Helix/C-linker Modelling d(Opposite Cα) ≈ 11 Å

20. CNG channels: P-Helix-Loop Modelling S5-helix P-helix S6-helix • Template: KcsA at 2.00 Å resolution for S6 region • Overall Identity: KcsA-SpIh: 18 %

21. F380 L356 Upper View F380 S6-Helix L356 P-Helix Open P-Helix Closed CNG channels: P-Helix-Loop Models T355 E363 Closed E71 E71 L358 T360 d(Cα-Cα)≈14 Å I361 T355 E363 Open TMA+ L358 T360 d(Cα-Cα)≈11 Å S6 rotation  F380/L456  P-Helix  T360  I361  Pore occlusion

22. CNG channels: Final Models

23. 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

24. HCN vs CNG: Selectivity and Gating • 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.

25. 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.

26. 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.