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“Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel” by Ricarda J.C. Hilf & Raimund Dutzler Presented by Ceyda.

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Presentation on theme: "“Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel” by Ricarda J.C. Hilf & Raimund Dutzler Presented by Ceyda."— Presentation transcript:

1 “Structure of a potentially open state of a proton-activated pentameric ligand-gated ion channel” by Ricarda J.C. Hilf & Raimund Dutzler Presented by Ceyda Yaramanoglu

2 W HAT HAS ALREADY BEEN DONE ? X-ray structure of a pentameric ligand-gated ion channel from Erwinia chrysanthemi (ELIC) showed the closed conformation of a pentameric ligand-gated ion channel (pLGIC) It is a Homo-pentameric protein Has an Ion-conducting, but discontinuous pore located on the fivefold axis of symmetry

3 H OW DO YOU KNOW ELIC IS THE CLOSED CONFORMATION ? Bulky hydrophobic residues towards the extracellular side constrict the pore that spans the lipid bilayer These seem to be barriers that hinder ions from diffusing through the pore It is the thermodynamically stable state There is no ligand bound (but it also has not yet been identified)

4 H ILF AND D UTZLER FOUND A POTENTIALLY OPEN - STATE CONFIGURATION ! A protein homologous to ELIC was isolated from the cyanobacterium Gloebacter violaceus (GLIC) GLIC has proton-gated channels, that are activated by a decrease in extracellular pH Crystallizing GLIC at pH 4.0 increased probability to isolate the open form of the channel

5 E FFECT OF P H Extracellular pH A decrease in extracellular pH activates GLIC Ligand = low pH in this case Figure: pH-activation of GLIC. Two-electrode voltage clamp recordings of Xenopus oocytes expressing GLIC in comparison to water-injected oocytes. Currents were recorded at 40 mV. The pH of the solution is indicated.

6 GLIC STRUCTURE a ) Ribbon representation of GLIC as viewed from within the membrane at 3.1Å resolution (extracellular region is above and the grey lines represent the membrane). b) Structure of the pentameric channel viewed from extracellular side

7 W HAT DO ELIC AND GLIC HAVE IN COMMON ? They are both prokaryotic proton-activated pentameric ligand-gated ion channels Form ion channels that are selective for cations over anions Resemble conduction properties of the cation-selective branch including acetylcholine and serotonin receptors Both structures suggest that a tilt of the pore-forming helices opens the channel Extracellular domains have 10 β-strands that compose two tightly interacting beta sheets Transmembrane domain is composed of 4 α-helices Position of α1 is the same, but great variances in α2 and α3

8 S UPERPOSITION OF GLIC & ELIC c) GLIC is shown in green and ELIC in brown. The superposition was generated by a least squares fit of the Cα positions of the respective α1 helices of the five subunits of the pentameric channel.

9 H OW ARE ELIC AND GLIC DIFFERENT ? Transmembrane pore is constricted on its extracellular side No rotation of α2 and α3 No signs of an attached ligand Same region has a funnel-shaped opening, with narrowest part in intracellular entry 9° rotation of α2 and α3 Ligand-binding domain has changed through low pH ELICGLIC

10 S IGNIFICANCE OF ROTATION OF TRANSMEMBRANE ALPHA - HELICES In GLIC, α2 and α3 are rotated by approx. 9°around an axis that intersects with residue Val 267 This axis runs parallel to the lipid membrane This causes Outward movement of the helices away from the pore, in the extracellular region Inward movement of the helices towards the pore in intracellular region

11 C ONFORMATIONS OF THE ALPHA -2 AND - 3 HELICES IN GLIC VS. ELIC Left: Conformation of the α2-α3 helix pair of a single subunit as observed in the structures. Right: Conformation of the α2-α3 helix pair after a least squares fit of the Cα position of the respective helix pair of ELIC and GLIC. GLIC is depicted in shades of green, whereas ELIC is shown in brown.

12 M OVIE ppinfo/nature07461.html

13 S TRUCTURE OF GLIC Extracellular half is dominated by hydrophobic residues Some residues carry bulky side-chains Intracellular half is lined by polar side chains Serine and Threonine residues maintain hydrophilic character in the water-filled channel Entrance and Exit of the channel are framed by rings of acidic residues Contribute to the overall negative electrostatic environment in the pore At pore entry: ring of five glutamate residues

14 S IGNIFICANCE OF 5 GLUTAMATE RESIDUE - RING The negatively charged side chains of these glutamate residues form a ring of interacting ionizable groups Side chains directly interact with the partly desolvated permeant cations Narrowest part of the channel, “narrow selectivity filter” Can directly interact with ions Impede free diffusion of ions Allow selectivity for cations

15 P ORE STRUCTURE OF GLIC a) View of α2 helices of GLIC defining the pore region. Glutamate ring

16 T ESTING THE I ONIZATION ACTIVITY OF THE RING Soaked the GLIC crystals in solutions containing: Rb + Cs + Zn 2+ Locating the electron density of bound ions in intra- and extracellular regions showed that they interacted with the side chains of the 5 glutamate ring

17 I ON INTERACTIONS OF THE 5 GLUTAMATE RING IN GLIC Intracellular part of the pore region in GLIC. Rb + is blue, Cs + is grey, and Zn 2+ is red. Clearly shows interaction of the ions with the residues of the 5 glutamate ring.

18 P ROOF THAT THE GLUTAMATE RING ALLOWS SELECTIVITY They created a mutant that is missing the residue E221A at 3.5 Å Changed conformation of Glu 221 side chain Unaltered backbone, but intracellular constriction of the pore was removed It had decreased cation selectivity Shows the significance of the 5 glutamate residues for ion conduction

19 L ET ’ S COMPARE WITH THE NICOTINIC ACETYLCHOLINE RECEPTOR nAChR Acetylcholine receptors are also cation-selective It’s extracellular part is also funnel shaped, as with GLIC But diameter is closer to ELIC Supposed to be in closed conformation Also has an “intermediate ring of charges” Determines cation selectivity

20 L IGAND BINDING SITE Ligand binding sites of all pLGICs are located at the interface between two subunits and are covered by the beta strands β9 and β10. GLIC’s binding site is different than with ELIC Structure around GLIC β9 is weakly conserved and 7 residues shorter than in ELIC ELIC has a mobile, poorly defined loop connecting β9 and β10, but in GLIC the loop is well defined with residues involved in ionization The conformation of GLIC’s binding site closely resembles that of acetylcholine-binding protein (AChBP) in the presence of its substrate analogue carbamylcholine Another proof that GLIC is in active conformation

21 E XTRACELLULAR DOMAIN OF GLIC VS. ELIC This is the view of the ligand binding domains in GLIC (green) and ELIC (brown). The inner beta sheets in GLIC are tilted by 5° towards the membrane plane. This change affects the regions connecting β1/β2 and β6/β7 strands with the α2/α3 loop.

22 C ONSERVED STRUCTURES CRITICAL FOR CHANNEL OPENING Arg 191 at the end of β10 forms two salt bridges: With Asp31 in the β1/β2 turn With Aso 121 in the β6/β7 loop Mutations of these salt bridges in the open conformation of nAChR and other pLGIC’s have shown that this drastically decreases the open probability of the channel THUS: These loop regions are very important for the transduction of conformational signals from the ligand-binding site to the pore domain

23 S ALT BRIDGES IN GLIC Cα representation of the interface region between the extracellular domain and the pore in GLIC (green) and ELIC (brown) viewed from the extracellular side. Ion interactions/ salt bridges are shown in dotted lines. Arg 191 (R191) forms salt bridges with Asp31 (D31) and Asp 121 (D121) in GLIC (green).

24 M ORE EVIDENCE.. We see differences in radii of ELIC and GLIC Due to funnel shape of GLIC ELIC has lower Electrostatic Potential Proposes that this is the closed position (closer to equilibrium) GLIC has a higher Electrostatic Potential Proposes that it is the open position

25 C OMPARISON OF RADIUS AND ELCETROSTATIC POTENTIAL OF ELIC AND GLIC Pore radius (r) in the transmembrane region of GLIC (green) is larger than in ELIC (brown). Electrostatic potential (φ) is more negative in GLIC than in ELIC. (This was calculated from a numerical solution of the linearized Poisson-Boltzmann equation at 0 and 150nM monovalent salt concentration.) Top picture shows the orientation of GLIC. (Note the funnel shape)

26 C ONCLUSIONS Comparing ELIC and GLIC gave structural insight into the mechanisms underlying pore opening But they are still ambiguous The change in conformation most likely occurs via a tilt of the helices α2 and α3 NOT via a rotation of the pore-lining helices around their helix axis Hilf and Dutzler provided the first structural view at high resolution into how pLGIC may open and selectively conduct ions

27 R EFERENCES ations/ligand- gated_ion_channel_Nature_2009_Dutzler.pdf ref/nature07461-s1.pdf

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