Nuclear targeting by Nuclear Localization Signals (NLS) · The IBB cargo binds the C-terminal half...
Transcript of Nuclear targeting by Nuclear Localization Signals (NLS) · The IBB cargo binds the C-terminal half...
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Nuclear targeting by Nuclear Localization Signals (NLS)
Richardson and Laskey (1988)
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The nuclear import pathway of proteins containinga classical Nuclear Localization Signal (NLS)
Uptake of NLS-containing protein in the cytosol
Importin β (Kapβ) - transport factor- binds the NPC
Importin α (Kapα) - adaptor - binds the cargo (NLSs)
βimp
RanGTP
βimp
αimp
αimp
NPC
RanGDP
Release of NLS-containing protein in the nucleus
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βimp
αimpImportin α (Kap α) recognizes diverse NLSs
Nuclear Localization Signals:short sequences (5 to 18 aa)no strict consensuspositively-charged
KKKRKKKRSK
PAAKRVKLD
KRPAATKKAGQAKKKK
SV40 TagHistone H2Bc-myc
Nucleoplasmin
KTKRK
What determines the recognition at the atomic level?
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radiation sample
MICROSCOPE
lightelectrons
cell,organelleviruses, ribosomes
10-6 m (micron)10-8 m (nanometers)
X-RAY CRYSTALLOGRAPHY
electron density
10-10 m (Angstrom)X-rays protein crystals
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0.4 mm 0.15 mm 0.05 mm
Protein crystals contain 1012 - 1014 molecules
The molecules have to pack in an orderly fashionfor the crystals to diffract to high resolution
2Å resolution diffraction pattern(ESRF synchrotron, Grenoble)
We obtain the structure of the average molecule
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The experimental data: electron density
3Å resolution 4Å resolutionaccuracy
We build and refine a model in the data:
2Å resolutionaccuracy + precision
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βimp
RanGTP
βimp
αimp
αimp
NPC
RanGDP
cargo uptake
translocation
cargo release
How are NLSs recognized with both versatility and specificity?
How does the transport factor dock to the NPC?
How does RanGTP (but not RanGDP) releases the cargo?
Questions we can address by crystallography
Questions we cannot address by crystallographyDynamics of the processFlexible conformational states
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Cargo uptake by importin α (karyopherin α)
IBB ARMADILLO REPEAT DOMAINN C
NLS bindingImportin β binding
Impα (Kapα)
ARM repeat: ~40 residue3 α-helices
The NLS-binding domain is built by 10 ARM repeats
2.2 Å resolutionRfree 27.5%
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The NLS-binding domain of importin α has anelongated shape with a long surface groove
0% 100%surface coloured by sequence identity
The surface groove is linedby conserved residues
The surface groove isthe site of NLS binding
SV40 Tag NLS binding
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NLS larger binding site
SV40 NLS: KKKRKV
Trp residues
Asn residues
charged/polar residues
5 Lys/Arg are bound
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NLS smaller binding site
Two Lys are bound
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The bipartite nucleoplasmin NLS binds along the whole surface groove
smaller site larger site
K R P A A T K K A G Q A K K K K
upstreamcluster
downstreamcluster
linker
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NLS binding
Polar interactions
Hydrophobic
Electrostatic
precise chemical recognition
versatile recognition
Conti et al., 98; Conti and Kuriyan 00; Fontes et al. 00
KRPAATKKAGQAKKKK
KKKRKVPAAKRVKLD
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Kobe, 99
Full-length importin α is autoinhibited
The entire IBB domain binds importin β
Part of the IBB domain binds to the larger NLS binding site of α
βimp
RanGTP
βimp
αimp
αimp
NLSs bind imp α better when imp β is present
IBB domain
ARM domain
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Importin β is built by 19 tandem HEAT repeats
N
C
123
4
5
6
7
8
9
1011 12 13
14
15
161718
19
N
IBB domain ofimportin α
Cingolani et al., ‘99
βimp
RanGTP
βimp
αimp
αimp
Overall snail-like conformation of importin β, with its C-terminal half wrapped around the IBB cargo
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ARM and HEAT repeats are structurally similar
ARM repeat HEAT repeat
G P
C-termW N
N-term
P
WC-term
N-term
W WW
importin β(19 HEAT repeats)
NLS-binding domain of α(10 ARM repeats)
WW
W WW
W
The C-terminal helix of ARM and HEAT repeatsline the inner cargo-binding surface of α and β
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The import complex docks to the NPCvia the binding of importin β to nucleoporins
HEAT 5, 6
HEAT 7
NN
HEAT 7 HEAT 19
The IBB cargo binds the C-terminal half of β at its inner concave surface
The FxFG repeats of a nucleoporinbind β at its outer convex surface
The FxFG nucleoporin and the IBB cargo bind different regions of β
β - IBB domain of α(Cingolani et al., ‘99)
β fragment - FxFG of Nsp1(Bayliss et al., ‘00)
βimp
RanGTP
βimp
αimp
αimp
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In the nucleus RanGTP binds to importin βand displaces the IBB cargo
RanGTP and the IBB cargo have overlapping binding sites at the acidic loop of β
β - IBB domain of α(Cingolani et al., ‘99)
β fragment - RanGTP(Vetter et al., ‘00)
N
HEAT 7 HEAT 19
HEAT 1
N
HEAT 8
The IBB cargo binds the inner surfaceof β between HEAT 7 and 19
RanGTP binds the inner surface of β between HEAT 1 and 8
acidic loop
βimp
RanGTP
βimp
αimp
αimp
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In the cytosol RanGDP is unable to bind to importin β
RanGTPswitch I and II move close to the GTP γPC-teminal helix is displaced from the core domain
RanGDPswitch I and II move away from nucleotideC-teminal helix binds to the core domain
(Scheffzek et al., ‘95)β fragment -RanGTP(Vetter et al., ‘99)
switch II
switch I
C-terminal helix
switch I
switch II
acidic loop
In RanGDP, the conformation of the C-terminal helix is sterically incompatible with β
C-terminal helixis disordered
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Similar binding of RanGTPto importin β (Kap β1) and to transportin (Kap β2)
transportin - RanGTP(Chook and Blobel, ‘99)
β fragment - RanGTP(Vetter et al., ‘99)
switch I
switch II
acidic loop(62 residues)
switch II
acidic loop(11 residues)
switch I
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Ran binding to importin β-like transport factors:shape recognition mechanism
L
EW
NL
EW
N switch Iswitch II
C-terminal helix
RanGDP
Importin β fragment - RanGTP
Transportin - RanGTP
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CYTOSOL
NUCLEUS
imp β (∆C) - RanGTPβ2 - RanGTP
RanGTP - RanBD
RanGDP
imp α - NLS imp β - IBB
imp α
Structural studies of NLS-nuclear import: a “dynamic” view
NPC NPC
imp β (∆C) - FxFG
Scheffzek et al. ‘95
Vetter et al. ‘99, Seewald et al., ‘02
Cingolani et al. ‘99Conti et al. ‘98
Kobe, ‘99
Bayliss et al., ‘00
Vetter et al. ‘99Chook et al. ‘99
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Structural studies of NLS-nuclear import: a”dynamic” view
imp α
imp β (∆C) - RanGTPtransportin - RanGTP
RanGDP
CYTOSOL
NUCLEUS
RanGTP - RanBD
imp β (∆C) - FG
NPC
imp α - NLS imp β - IBB
NLS-protein
RanGTP�
RanGDP-NTF2
RanGAP-RanGTP - RanBD
NPC
Ran-RCC1
chrom
atin
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At least 22 karyopherins in the human genome
IMPORT
EXPORTXpo-t - tRNA
- importin α
- snurportin- NES domain (Leu-rich) of proteins with RanBP3
- EIF4A
CAS
Importin 13
Crm1
Importin β
Transportin
Importin 13
- IBB domain (Arg-rich) of importin α (adaptor for NLS import)and snurportin (adaptor for U snRNP import)
- BIB domain (Arg/Lys rich) of ribosomal proteins
- M9 domain (Gly/Met rich) of hnRNP A1- TAP N-term domain (Gly/Arg rich)- BIB domain (Arg/Lys rich) of ribosomal proteins
- mago/Y14, etc.
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nuclear exportnuclear import
cargo releasecargo uptake βexpβimp
cargo uptakecargo release
RanGTP
βimp
RanGTP
βexp
RanGTP
RanGDP
Karyopherins can bind multiple substrates
Substrates can bind multiple karyopherins
Karyopherins can behave as importins and exportins
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Different overall conformations of two importins:Importin β + IBB cargo Transportin + Ran
frontside frontside
Conformational changes as they carry out an import cycle?
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Small angle X-ray scattering (SAXS)Dmitri Svergun, EMBL-Hamburg
front view
top view
4 independent reconstructions oftransportin - RanGTP
average
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Conformational changes of nuclear import factors
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Import-mediators vs Export-mediators
Importin β ( Kap β1) 96 kDa, 19 repeatsTransportin ( Kap β2) 98 kDa, 19 repeats
Xpo-t 106 kDaCAS 106 kDa
Crm1 118 kDa
IMP
EXP
Importin β Exportin-tImportin β − RanGTP Exportin-t RanGTP tRNA
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Conformational variability of karypherins
Side view
Front view
Xpo-t Los1 Xpo1 Cse1
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Elena Conti
SAXS studies: Noemi FukuharaElena FernandezJudith Ebert
Dmitri Svergun (EMBL-Hamburg)
NLS studies: John Kuriyan (Rockefeller Univ.)