Diapositiva 1

1
NTP BINDING PROPERTIES OF THE BLUE-LIGHT
SENSITIVE YTVA PROTEIN FROM BACILLUS SUBTILIS
Aba Losi
Università degli studi di Parma, Italy
Max Planck Institut Für Bioanorganische Chemie,
MH-Germany
Wolfgang Gärtner
Partly supported by DFG-FOR526, Blue-Light
Photoreceptors
2
Blue-light is an ubiquitous stimulus for living
organisms, and has probably represented one of
the driving evolutionary force, given its unique
capability to penetrate deeply into sea water.
The attenuation coefficient is extremely low in
clear sea water, today as well as in the Archean
era (3.92.5 Ga ago)
Cockell, C. S. (2000) Ultraviolet radiation and
the photobiology of earth's early oceans. Origins
Life Evol. Biosphere 30, 467-99
http//www.whoi.edu/oceanus/viewArticle.do?id2472
3
5I
FAD, R
4I
3I
2I
1I
8a
9
9a
10a
8
1
2
4
4a
5a
7
FMN, R PO3H2
6
7a
riboflavin, R H
The three flavin chromophores that can be
incorporated into known photosensors. Riboflavin
consists of a 7,8-dimethyl-isoalloxazine ring
linked to D-ribitol. FMN (Flavin mononucleotide)
and FAD (Flavin adenin di-nucleotide) are
riboflavin derivatives. Biosynthesis occurs in
Archaea, Bacteria, plants and fungi via one of
the most ancient known metabolic pathways.(see
Markus Fischer and Adelbert Bacher Nat . Prod.
Rep., 2005, 22, 324 350)
4
The LOV paradigm formation of a FMN-Cys
photoadduct
LOV447
LOV390
plant phototropin
Plant phot are light-activated kinases carrying
two LOV domains that bind a flavin mononucleotide
(FMN).
LOV domain (Light, Oxygen, Voltage) a PAS fold
Salomon et. al, Biochemistry, 39, (2000) 9401.
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Bacterial LOV proteins retain phot-like
photochemistry 17 of sequenced bacteria have LOV
proteins
1B. subtilis YtvA
STAS Sulphate Transporters AntiSigma-factor
antagonists). Conserved architecture other
Firmicutes. The STAS domain has a poorly
characterized function in YtvA
2Caulobacter crescentus
Demonstrated photochemistry
3Pseudomonas putida
4Pseudomonas syringae
1. A. Losi, E. Polverini, B. Quest and W. Gärtner
Biophys. J., 2002, 82, 2627-2634 2. A. Losi,
Photochem. Photobiol. Sci., 2004, 3, 566-574.3.
U. Krauss, A. Losi, W. Gärtner, K.-E. Jaeger and
T. Eggert,, Phys. Chem. Chem. Phys., 2005, 7,
2229-2236.4. Losi Gärtner, unpublished
6
Multitude of full-length protein domain
organizations found in the anoxygenic
phototrophs, cyanobacterial and archael LOV
domain containing proteins.
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Phylogenetic-LOV tree
LOV2 - Eukarya
Exclusively Gram Negative Proteobacteria
LOV1 - Eukarya
Archaea
Firmicutes
Cyanobacteria
ZTL - Eukarya
Pseudomonads
Two independent origins of LOV domain evolution
Once among the phototrophs (Erythrobacter) with
Lateral Gene Transfer as one evolutionary
mechanism and 2nd among the Archea which gave
rise to Firmicutes and evtl. Cyanobacterial and
Pseudomonas short LOVs (Krauss et.al, submitted)
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Conservation of the full-length protein domain
organization in the Firmicutes, Pseudomonads,
eukaryotic phot- and ZTL-LOV domain containing
proteins.
Main
questions 1. Physiological role of LOV proteins
in microrganisms 2. Molecular mechanism of
light-to-signal transduction
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Physiological studies on the role of YtvA. 1.

• B.subtilis wildtype produces brown
  agar-diffusible pigment(s) whereas B. subtilis
  ?ytvA remains unpigmented
• Pigmentation is a sign for sporulation in
  Bacillus (melanin-like pigment) (Gärtner and
  coworkers, unpublished)

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2.T. A. Gaidenko, T. J. Kim, A. L. Weigel, M. S.
Brody and C. W. Price, The Blue-Light Receptor
YtvA Acts in the Environmental Stress Signaling
Pathway of Bacillus subtilis, J. Bacteriol.,
2006, 188, 6387-6395.
Enviromental stress
1A

• YtvA upregulates ?B in the absence of stress and
  C62 is required.
• 2. Forms large complexes with its paralogs
• (STAS proteins)
• 3. YtvA transcription is upregulated by the
• Spx global regulator of disulfide stress.

(All experiments were performed in light)
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3. M. Avila-Perez, K. J. Hellingwerf and R. Kort,
Blue Light Activates the ?B Dependent Stress
Response of Bacillus subtilis via YtvA, J.
Bacteriol., 2006, 188, 6411-6414.
1B
The loss and overproduction of YtvA abolish and
enhance, respectively, the increase in ?B
activity at moderate light intensities. These
effects were absent in the dark and in red light
but present under blue-light illumination.
B. subtilis strain overproducing YtvA (?B
activity via a reporter gene)
Conclusions YtvA is a blue-light receptor that
enhances ?B activity and has probably a link to
the sporulation pathway. How does YtvA work from
a molecular point of view?
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Gel-chromatography suggests a common interface
for LOV-LOV and intraprotein interactions
Ytva-LOV25-126
LOV
YtvA
Mw 2.6 ? MWLOV elongated dimer
LOV
Negligible light effect
Mw 1.6 ? MWYtvA elongated Monomer
YtvA-LOV is dimeric in solution. LOV dimerization
is not prevented from the N-terminus YtvA is
monomeric indicating that the LOV-LOV and the
LOV-(linker)-STAS interfaces are partially or
totally overlapped
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Model of the LOV-LOV dimer large involvement of
the ?-sheet, confirmed by circular dichroism
experiments.Mainly apolar interactions. In black
residues at the LOV-LOV interface (within 4Å)
LOV
Model of YtvA25-254 LOV-coreJ? linkerSTAS
domain. The LOV core makes contact with both
linker and STAS, via polar and apolar
interactions. In black and blue residues at the
interface on the LOV core and on the
J?-linkerSTAS domain respectively
STAS
J?-linker
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Is YtvA-STAS an NTP-binding domain?
On the STAS domain interactions with the LOV core
(in gold) and with the J?-linker (darker gold)
comprise highly conserved motifs within the
YtvA-like protein family The A?S strand makes
contact with the F?-G? loop and G? on the LOV
core and with the J?-linker The J?-linker
interacts with DLSG a classical GTP-binding
motif (?-phosphate). On other STAS domains the
corresponding DSSG sequence is transiently
phosphorylated and has a key regulative role.
A?S
DLSG
A general NTP-binding role has been anticipated
for STAS domains and demonstrated for SPOIIAA (a
STAS domain that regulates sporulation in
Bacillus)
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Topology of small GTP binding proteins (SRP54,
signal recognition particles) and YtvA STAS
In green region of high sequence and structural
homology
??
DTxG
TKHD
DLSG
N
P
6
4
5
1
4
6
5
7
2
3
3
C
N
C
Orientation of GTP may not be the same as in
SRP54 Additional stabilizing factors (e.g. the
N-cap, the J-linker) The (intact) LOV core does
not participate in GTPTR binding.
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A fluorescent derivative of GTP (GTPTR) binds to
YtvA
Competition between GTPTR and GTP/ATP
Red-shift and gt GTPTR ?F
(red shift lt polarity and/or gt polarizability)
Guanosine
e-
Fluorescence quenched in solution gt ?F upon
binding
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1-1 simple binding (when no apoprotein is
present). The variation in the Absorption maximum
may be of choice, given that the variation in
fluorescence is small and internal quenching is
operating . Very evident with ATPTR (loss of F
upon binding), because Adenosine is worse as an
e- donor quencher
GTPTR KD 10 38 ?M ATPTR KD 8 36 ?M
Comparable affinity for both NTP.
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Are the Light-driven conformational changes
transmitted from the LOV core to the NTP-binding
cavity?
FMN
GTPTR
FMN
ATPTR
?
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For both NTPs the affinity appears higher in the
dark state or/and the binding site undergoes a
conformational change that alters the
polarizability of the microenvironment The light
induced change is fully reversible in the dark
GTPTR
ATPTR
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YtvA binds ATP and GTP confirming a general
NTP-binding activityof STAS domains Possible
involvement in signal transduction 1. a drop in
ATP is suggested to be the trigger to activate ?B
in B. Subtilis (Zhang et. Al. 2005, J.
Bacteriology, 187, 7554) 2. A drop in GTP is
concomitant to the initiation of
sporulaton (Piggot et. al. Curr. Op. Microbiol. ,
2004, 7, 579)
Blue news NTP binding properties of the
blue-light sensitive YtvA protein from Bacillus
subtilis Valentina Buttani, Aba Losi, Eugenia
Polverini, Wolfgang Gärtner FEBS Lett. , 2006,
580, 3818-3822 Conformational analysis of the
blue-light sensing protein YtvA reveals a
competitive interface for LOV-LOV dimerization
and interdomain interactionsValentina Buttani,
Aba Losi, Thorsten Eggert, Ulrich Krauss,
Karl-Erich Jaeger, Zhen Cao , Wolfgang Gärtner
2006 (submitted)
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Acknowledgements
Parma team Valentina Buttani
Spectroscopy and analysis Eugenia Polverini
Structural modeling Roberta Bedotti
Technical help Mülheim Team Sven Jansen
Mutagenesis Helen Steffen
Technical help Zhen Cao
Bacterial LOV proteins and constructs Ulrich
Krauss Photophysiology, P. putida
LOVs Special thanks to Wolfgang Lubitz Silvia
Braslavsky Supported in part by the DFG
(Forschergruppe FOR526).