Presentation is loading. Please wait.

Presentation is loading. Please wait.

Structural predictions of HCN/CNG ion channels: Insights on channels’ gating Candidate:Supervisors: Alejandro GiorgettiProf. Paolo Carloni Prof. Vincent.

Similar presentations


Presentation on theme: "Structural predictions of HCN/CNG ion channels: Insights on channels’ gating Candidate:Supervisors: Alejandro GiorgettiProf. Paolo Carloni Prof. Vincent."— Presentation transcript:

1 Structural predictions of HCN/CNG ion channels: Insights on channels’ gating Candidate:Supervisors: Alejandro GiorgettiProf. Paolo Carloni Prof. Vincent Torre

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 HCN channels 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) S1S2S3S4S5 S N-Terminal CNBD P-helix-Loop C-Linker mV Cytoplasm Extracellular Heart and brain pacemaking regulation Sea urchin sperm (spHCN)Mammalian heart and brain: HCN1-4

5 Cones Rods CNG channels PhotoreceptorsOlfactory 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 Participate in sensory perception and signalling throughout the nervous system

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

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

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

10 Known Structures (templates) Template(s) selection Coordinate Mapping Structure Evaluation 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

11 Known Structures (templates) Template(s) selection Structure Evaluation 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, 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.

12 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 Structure Evaluation

13 chargediameter length MTSET: Å 10 Å MTSES: Å 10 Å MTSEA: Å10 Å Cd 2+ coordinates to two or more cysteins CONVENTIONRange (Å) Maximum Allowed distance(Å)[1][1] -79 Cd – – 56 - Cd - 5 – [1][1] Maximum allowed distance considering the thermal fluctuations of the protein (Careaga and Falke, 1992). CuP favours disulphide bond formation Rothberg and Yellen, 2002 Rulisek and Havlas,2000 Accessibilities experiments: MTS reagents Experimental Data  Distance Restraints (Cysteine scanning mutagenesis) Extracted from pdb

14 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

15 Lys433 Validation controls: C428 blocked upon CuP exposure C428 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)

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

17 S1S2S3S4S5 S N-Terminal P-helix-Loop C-Linker Cytoplasm S4S5 S CNG channels CNBD

18 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-Helix C-Linker

19 Closed Open V Å13.4 Å G Å13.5 Å S Å14.0 Å CNG channels: S6-Helix/C-linker Modelling d(Opposite Cα) ≈ 11 Å N402 A406 Q409 A414 Q417 F375 S6-Helix C-Linker

20 CNG channels: P-Helix-Loop Modelling F380PotentiationBlock--D(F380Cα- C314Cα) < 8 Å F380C-L356CNo Effect --D(F380Cα- L356Cα) ≈ 6 Å T360BlockNo EffectMTSES Poten MTSES Poten D(Cα- Cα) ≈ 11 Å (Open) D(Cα- Cα) > 14 Å (Closed) S5-helix P-helixS6-helix Template: KcsA at 2.00 Å resolution for S6 region Overall Identity: KcsA-SpIh: 18 %

21 CNG channels: P-Helix-Loop Models E363T355 L358 T360 d(Cα-Cα)≈11 Å Open TMA + E71 E363 T355 L358 Closed T360 d(Cα-Cα)≈14 Å Upper View F380 L356 S6-Helix P-Helix Open P-Helix Closed L356 F380 I361 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 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

25 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. Acknowledgements

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.


Download ppt "Structural predictions of HCN/CNG ion channels: Insights on channels’ gating Candidate:Supervisors: Alejandro GiorgettiProf. Paolo Carloni Prof. Vincent."

Similar presentations


Ads by Google