Archaeal Rhodopsins

1

• Archaeal Rhodopsins
• Four heptahelical retinal proteins identified in
  haloarchaea.
• - Bacteriorhodopsin (ion pump).
• - Halorhodopsin (chloride pump).
• - Sensory rhodopsin I (positive negative
  phototaxis).
• - Sensory rhodopsin II (negative phototaxis).

2

• What is expressed when?
• When oxygen and respiratory substrates abundant
• - Sensory rhodopsin II is expressed.
• Absorbs near the solar maximum (500 nm).
• Initiates a negative photo-taxis signal.
• When respiratory substrates and oxygen are in
  short supply
• - Bacteriorhodopsin and halorhodopsin expressed.
• Energy transduction proceeds via
  photosynthesis.
• - Sensory rhodopsin I is expressed to optimise
  light conditions.
• Initiates a positive response _at_ 570 nm.
• Also has a negative response _at_ short
  wavelength.
• The signaling state absorbs a second photon _at_
  400 nm.

3

• Phototaxis and adaption
• Bacteria typically swim in a given direction for
  5 to 50 sec.
• - Spontaneously tumble and change swimming
  direction.
• If improved conditions are found
• - Tumbling is suppressed.
• - Swim further in this direction.
• If disfavourable conditions are found
• - Tumbling is increased.
• - Looks for a new direction to swim.
• In parallel a feed-back mechanism operates which
  reinstates the normal tumbling frequency.
• - Called adaption.
• Combined effect is that bacteria swim up a
  gradient towards favourable conditions.

4

• Signal propagation
• Structural changes in SRII conveyed to HtrII.
• A conformational change in HtrII activates the
  coupling protein (CheW) a histidine kinase
  (CheA).
• CheA phosphorylates CheY.
• - Phosphorylated CheY a switch factor on the
  Flagellar motor.
• - Tumbling of bacteria suppressed or increased.
• Competitive Adaption' controlled by a
  methylesterase (CheB) a
• methyltransferase (CheR) (reestablish an
  equilibrium).
• A negative phototaxis response initiated by
  SRII.
• A slightly more complex dual response initiated
  by SRI.

5

• Structure of sensory rhodopsin II
• Sensory rhodopsin II from Natronbacterium
  pharaonis more stable than from Halobacterium
  salinarium.
• - Lipidic cubic phase crystals diffract to 2.1 Å
  .
• Fold extremely similar to bacteriorhdopsin.
• - Only helices A and B have any significant
  changes relative to bacteriorhodopsin and
  halorhodopsin.

6

• Spectral tuning of sensory rhodopsin II
• Sensory rhodopsin has a maximum absorption at
  500 nm.
• - Compare with bacteriorhodopsin (570 nm).
• Spectral tuning tailored to suit function.
• - Negative phototaxis response at solar maximum.
• Waters similar but Arg72 oriented towards
  extracellular side.
• - Alters the Schiff base, counter-ion
  interactions.
• A number of polar residues within the binding
  pocket alter the polarity of the retinal
  environment.
• - Thr204 (Ala in bR) Val108 (Met in bR) Gly130
  (Ser in bR).
• - Retinal less curved in sensory rhodopsin II.

7

• K-state of sensory rhodopsin II
• As with bR, illuminate at low temperature (100
  K) and determine the difference Fourier map.
• - Negative density seen on Wat402.
• - More difference density peaks seen along the
  retinal.
• - Retinal less constrained within its pocket.
• - Also observed using FTIR spectroscopy.

8

• Why make the binding pocket less constrained
• Sensory rhodopsin II is a sensory receptor
• Requires long-lived signaling states.
• Bacteriorhodopsin requires high turnover (short
  photocycle).
• The signaling state of sensory rhodopsin II is
  the M-state (with deprotonated retinal).
• Movements of Lys205's carbonyl oxygen (Lys216 in
  bR) is suppressed in sensory rhodopsin II.
• Does not create sites for waters to order.
• Asp96 is absent in sensory rhodopsin II.
• Extends the lifetime of the deprotonated state.

9

• Crystal structure of complex to 2 Å
• Co-crystallised sensory rhodopsin II and a two
  transmembrane helices segment (residues 1 to 114)
  of the transducer.
• - Packs as homo-dimer of hetro-dimers.
• Inter-helical contacts primarily hydrophobic.
• - No H-bonds between TM1 TM2 (only van der
  Waals contacts) of the transducer dimer.
• - TM2 of the transducer packs closely up to
  helices F G of sensory rhodopsin II.
• - Closer to helix G (4.06 Å average van der Waals
  contacts) than helix F (4.22 Å average van der
  Waals contacts).

10

• H-bonds to TM2 of HtrII
• Y199 of sensory rhodopsin II identified as
  forming a H-bond to N74 of the transducer.
• F-G loop (extracellular side) has three H-bonds
  T189 G43 S62.
• Proposed to provide Anchor points''.
• T199 very close to Pro175 (Pro185 in
  bacteriorhodopsin), which provides a hinge''
  about which helix F flexes.
• Residues 82 to 114 not visible.
• - Cannot see if a charged patch'' interacts
  with a negative patch'' on HtrII in this
  region.

11

• Signaling state of sensory rhodopsin II
• A positively charged patch unique to sensory
  rhodopsin II identified
• from the ground state structure.
• Proposed to interact with negatively charged
  residues in the transducer.
• Not visible in the complex structure.
• A model for the signaling state can be built by
  analogy with the bR-triple mutant structural
  model of the M-intermediate.
• - Show the largest movements overlap with the
  charged patch.

SRII resting state
SRII signalling state model
12

• Functional Mechanism?
• Light activation of Sensory rhodopsin II
  isomerises the retinal.
• - Retinal pushes up upon a conserved Trp171.
• - Helix F swings out.
• - Movement is hinged near a conserved Pro175.
• Outwards tilting of cytoplasmic half of helix
  proposed to
• - Collide tangentially with TM2 \ induce its
  rotation.
• - TM2 anchored to helix G axis of rotation
  between two helices.
• - Rotation may unwind the cytoplasmic helices of
  HtrII.
• - May be the trigger which shifts the equilibrium
  from inactive to active state, recognised by the
  CheA.

13

• Summary
• Four rhodopsins found in halophilic archaeal
  bacteria.
• Display a conserved structural mechanism of
  functionality.
• Bacteriorhodopsin is the best understood.
• - Water molecules play a key role.
• - Steric clashes with the retinal play a key
  role.
• Sensory rhodopsin II (as with bacteriorhodopsin)
  becoming a prototype
• system of study.
• Homologues identified in eubacteria and other
  prokaryotes.

14

• Structural mechanism of the the Ca2-ATPase
• Ca2 activates muscle contraction when enters
  the cytoplasm.
• To be effective Ca2 must be pumped out'' by
  something.
• Ca2-ATPase performs this task.
• - Constitutes 90 of protein within the
  membrane.
• P-type ATPase (include Na/K-ATPase
  H/K-ATPase).
• So-called since an aspartate residue is
  autophosphorylated.
• Two Ca2 transported per ATP consumed ( two or
  three H counter transported)

8 Å Electron microscopy map of the Ca2-ATPase
15

• Ca2 ATPase crystal packing
• Crystals grown by dialysis in a mixture'' of
  purified protein phospholipid
  (phosphatidylcholine).
• Type I crystal packing.
• Diffract to 2.6 Å.
• Distance between adjacent layers is 146 Å
• Could estimate membrane boundary from
  distribution of water molecules.

16

• Protein Arrangement
• One transmembrane domain
• - Ten a-helices contain two Ca2 binding sites.
• P-domain.
• - Contains the phosphorylation residue (Asp351)
• N-domain.
• - Contains the nucleotide (ATP) binding domain.
• A-domain.
• - Act as an anchor for domain N.

17

• Ca2 Binding Site
• Two Ca2 binding sites identified
• Site I between M5 M6
• Coordinated by six oxygens side chains of a Thr,
  Asn, Asp, Glu of M6, a water a Glu of M8.
• Site II formed almost on'' helix M4.
• - Coordinated by six oxygens three backbone
  carbonyl oxygens of M4 a Glu of M4 an Asn,
  Asp of M6.
• Helix M4 is unwound'' at this position (M6
  also disrupted).
• - Realises efficient coordination geometry rows
  of oxygen atoms guide Ca2 to the binding site
  remove solvating waters.

18

• ATP Binding Site
• Identified by soaking TNP-AMP into crystals.
• - Located in domain N.
• - More than 25 Å from the phosphorylation site
  (Asp351).
• Conformational changes must occur!

ATP site
D351
19

• Structural Changes
• Crystals grown in presence of thapsigargin (a
  potent inhibitor) absence of Ca2
• - Known to lock the protein in a tight
  conformation.
• Data collected to 3 Å resolution.
• Molecular replacement (by moving domains
  blockwise within a low-resolution EM structure)
  used to solve the structure.

With Ca2
Without Ca2
20

• What changes occur?
• N domain inclines nearly 90o with respect to the
  membrane.
• - Equates to a 50 Å movement of the top part of
  N.
• P domain inclines about 30o relative to the
  membrane.
• - N domain inclines a further 50o relative to the
  P-domain.
• - P N domains virtually unchanged (rmsd 0.63
  0.75 Å).
• A domain rotates 110o horizontally.
• - A few hinge-residues _at_ P-A domain interface
  change a lot.

21

• Changes within the membrane
• Ca2 binding site between M4, M5 M6.
• Changed conformation does not have Ca2 bound.
• Helices M1 to M6 tilt about 30o
• Helices M7 to M10 almost unchanged.
• The middle section of M5 straightens out.
• - Strongly affects L67 loop.
• - A key loop in the P-A domain interface
  probably important in transmitting signal of
  phosphorylation to Ca2 binding.

Purple with Ca2 Green without Ca2
22

• How does it pump Ca2?
• Muscles activated by Ca2 concentration going
  up.
• Ca2 enters the cytoplasmic channel binds
  inducing a rearrangement of the P N domains ?
  E1Ca2-state.
• ATP binds between N P domains D351 is
  phosphorylated, closing cytoplasmic channel ?
  E1PCa2-state.
• A slow conformational change occurs, opens the
  luminal channel disrupts Ca2 binding ?
  E2P-state 2 Ca2.
• Water enters, hydrolyses the phosphoenzyme
  reopens the cytoplasmic channel ? E2-state.