Signaling by the Escherichia coli Aspartate Chemoreceptor Tar with a Single Cytoplasmic Domain per D

1
Signaling by the Escherichia coli Aspartate
Chemoreceptor Tar with a Single Cytoplasmic
Domain per Dimer

• Tatsuno, I., Homma, M., Oosawa, K., Kawagishi, I.
• Science. 274. 423-425 (1996)

Attractant Signaling by an Aspartate
Chemoreceptor Dimer with a Single Cytoplasmic
Domain.
Gardina., P. J. and Manson, M. D. Science. 274.
425-426 (1996)
2
Hughson and Hazelbauer paper

• Designed to understand mechanism of movement of
  chemoreceptors during transmembrane signaling.
• Assays were performed using intact cells.

3
Methodology
1. Clone Trg and its derivatives on an IPTG
inducible plasmid. 2. Cells were deleted for
other chemoreceptors, as well as for cheB and R
They also had a plasmid making RBP. 3.
Derivatives had Cysteins engineered along the
transmembrane helices (30 in TM1 24 in TM2
13 double) 4. S-S crosslinks induced by treating
cells with the oxidizing agent
Cu-phenanthroline 5. Samples analyzed by
SDS-PAGE and western blots with Trg antibodies.
Measure the effects of ligand occupancy on the
rates of oxidative cross-linking between
cysteines introduced into the Trg chemoreceptor
4
Effect of ligand occupancy is confined to
intrasubunit rather than intersubunit cross-links.
5
If TM1 does not move, TM2 must. Data consistent
with a downward motion
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Conclusions
Effects observed were subtle Data consistent
with intra rather than intersubunit
movement Axial sliding of one helix relative to
the other most likely Data not consistent with
other kinds of movement eg. Radial/Sheer
(helices move together or apart in different
planes) or Rotational movements
7
Signaling by the Escherichia coli Aspartate
Chemoreceptor Tar with a Single Cytoplasmic
Domain per Dimer

• Tatsuno, I., Homma, M., Oosawa, K., Kawagishi, I.
• Science. 274. 423-425 (1996)

Attractant Signaling by an Aspartate
Chemoreceptor Dimer with a Single Cytoplasmic
Domain.
Gardina., P. J. and Manson, M. D. Science. 274.
425-426 (1996)
8
Structure of Tar receptor
N 1 - 6 TM1 7 30 Peri 31-188 TM2 189
212 C 213 - 552

• Full-length receptor
• TM1 and TM2 the first and second trasmembrane
  segments
• K1 and R1 predicted a-helical coilded-coil
  domains that include 4 regulatory methylation
  sites (dots)
• Signaling domain (cytoplasmic domain) is
  conserved.

9

• Nature 365 343 (1993)

10
(No Transcript)
11
Hydrogen-bonding interaction scheme at the
aspartate binding site. The boxed amino acids are
from subunit A, the ovals, from subunit B. Water
is indicated with W, and hydrogen bonds with
dash lines.
Science. 2541345 (1991)
12
Signaling by the Escherichia coli Aspartate
Chemoreceptor Tar with a Single Cytoplasmic
Domain per Dimer

• Tatsuno, I., Homma, M., Oosawa, K., Kawagishi, I.
• Science. 274. 423-425 (1996)

13
Signaling system
14
HypothesisSignaling by Tar may be mediated by
interaction between dimers or conformational
changes within a single cytoplasmic domain.

• Method (In vivo intersubunit suppression)
• 1. Substitute amino acid and delete known domains
  of Tar.
• 2. Coexpress amino acid substituted mutants and
  test chemotatic response

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In vivo intersubunit suppression
Tar-A19K is mutation in TM1 Tar-A198E is mutation
in TM2 Tar-T154P is mutation in sensing domain
The A19K in TM1 is suppressible by A198E
mutatation in TM2
A heterodimer having mutation in TM1, TM2, and
sensing domain is still functional.
The heterodimer is functional.
16
Heterodimer with single cytoplasmic domain is
functional
One of the two cytoplasmic signaling domains in a
dimer is dispensible for generating an attractant
response to aspartate
17
The linker domain may be important
The linker domain may be important for
intersubunit interaction for signaling
18
Gardina., P. J. and Manson, M. D. Science. 274.
425-426 (1996)

• Attractant Signaling by an Aspartate
  Chemoreceptor Dimer with a Single Cytoplasmic
  Domain.

19
Method (In vivo intragenic complementation)
Create mutants in Tar subunits, which have to
form heterodimers to bind aspartate. Tar-R73K
and Tar-T154I on compatible plasmids, pMK113 and
pRBB16. Introduce these plasmids into strains
lacking Tar and Tsr receptors. Test the response
of these mutants to aspartate by forming
characteristic expanding rings in soft agar with
0.1mM aspartate.
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Do heterodimers restore aspartate response?

• Wild type,
• R73K and T154I homodimers, respectively,
• R73K and T154I heterodimers

Coexpression of the mutants proteins, which form
heterodimers with one intact binding site, did
restore aspartate response
21
Test whether a heterodimer containing one
full-length and one cytoplasmically truncated
subunit mediates responses to aspartate

• Prepare Plasmids which contain a UAA nonsense
  codon.
• pMK113-229oc terminates Tar 17 amino acids after
  TM2, so the protein lacks 95 of cytoplasmic
  domain.
• pMK113-258oc terminates 46 amino acids after TM2,
  so the protein lacks 85 of the cytoplasmic
  domain
• Both proteins fail to support any aspartate
  chemotaxis.

22
Does a heterodimer containing one cytoplasmically
truncated subunit mediate responses to aspartate?
Empty circles pRBB16(tar) pMK113-?tar Filled
circles pRBB16-R73K pMK113-258oc Empty
triangles pRBB16-R73K pMK113-T154Ioc Filled
triangles pRBB16-R73K pMK113-?tar
A heterodimer containing only one cytoplasmic
domain can mediate an attractant signal.
The segment of Tar between residues 229 and 258
is necessary to form functional heterodimers.
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Interpretations proposed

• Tar heterodimers with only one cytoplasmic domain
  can maintain aspartate response (Cis-inhibition).
• A signal generated at an aspartate-bound
  heterodimer could counter CheA activity
  maintained by full-length homodimers that cannot
  bind aspartate (Trans-inhibition).

24
Conclusions

• Heterodimers with only one intact cytoplasmic
• domain are functional.
• Linker domain may be important for intersubunit
• interaction for signaling.