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Figure 1.
Modular architecture of proteins with NTF2-like domains. The
numbers indicate the lengths of the sequences in residues;
the protein names correspond to Figure 1b, accession numbers
are given in parentheses. Abbreviations: LR, leucine-rich repeat;
UBA, ubiquitin-associated domain; RRM, RNA recognition motif;
S_TKc, catalytic domain of serine/threonine protein kinase.
The significant sequence similarity of the C-terminus of TAP
to UBA [Hofmann and Bucher 1996] domains was identified using
PSI-BLAST [Altshul et al. 1997].
Several UBA-like domains were highest ranking in the twilight
zone hits with E values of 0.76 and above. The similarity was
confirmed by MACAW analysis [Schuler et al. 1991] for
about a half length of the alignment (P-values < 1e-50). The
known structure of UBA domains [Dieckmann et al. 1998] suggests a
conserved loop (NWD at position 593 in human TAP) to be involved
in the interactions. A respective D595R mutation indeed
interrupts binding to nucleoporins (see Figure 7).
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Figure 2.
Multiple alignment of selected NTF2-like domains. The sequences are
grouped into four subfamilies according to the domain organization
of entire proteins (Figure 1): (I) TAP; (II) G3BP; (III) plant
protein kinases, and (IV) NTF2. First column, protein names;
second column, species names: At, Arabidopsis thaliana; Ce,
Caenorhabditis elegans; Dm, Drosophila melanogaster;
Hs, Homo sapiens; Nc, Neurospora crassa; Nt,
Nicotiana tabacum; Rn, Rattus norvegicus; Sc,
Saccharomyces cerevisiae; Sp, Schizosaccharomyces
pombe; Xl, Xenopus laevis; third column, positions of the first
aligned residues in each of the sequences; last column, database
accession numbers. The multiple sequence alignment of the sequences
was constructed by CLUSTAL W [Thompson et al. 1994] and
manually refined on the SEAVIEW alignment editor [Galtier
et al. 1996]. The positions conserved in 80% of
the sequences are indicated in the consensus line: c (DEHKR),
charged; h (ACFGHIKLMRTVWY), hydrophobic; l (ILV), aliphatic;
p (CDEHKNQRST), polar; s (ACDGNPSTV), small; t (ACDEGHKNQRST),
turn like; u (AGS), tiny. The positions conserved among the four
subfamilies are indicated in boldfaced characters with colors:
cyan, hydrophobic; green, tiny; blue, hydroxyl; black, polar.
The random mutations of tripeptides to alanines are highlighted
in gray. The designed point mutations that are mentioned in the
text are underlined; the colors of the corresponding asterisks
indicate the phenotypic effect: red, loss of the binding; cyan,
no obvious effect. Mutations and binding assays were performed
as described by Braun et al. [1999]. The known secondary structure
of rat NTF2 is indicated below the consensus line (H, alpha-helix;
E, beta-strand) and agrees well with the predicted secondary
structure [Rost and Sander 1994] of TAP sequences. The surface
accessibility below (0 low, 9 high) was calculated using the DSSP
program [Kabsch and Sander 1983]. The dimer contact (bottom line)
has been derived by subtracting accessible surface area of the dimer
from that of the monomer (i.e. the higher the numbers,
the more involvement in the dimer contact).
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Figure 3.
Proposed model of the interaction between the middle domain
of TAP and p15. The model structure is calculated [Sali and Blundell 1993] based
on the 3D structure of the homodimer of NTF2 [Bullock et al. 1996]. Cyan:
TAP,
green: p15. The side chains are shown for positions where
mutations were introduced: red, loss of the binding; dark
blue/dark green (TAP/p15), same as with the wild-type. Residue
numbers are indicated for the designed point mutations. Numbers
below and above 400 denote residues in p15 and TAP, respectively.
The triple alanine mutations are shown in gray. The figure was
prepared using MOLMOL [Koradi et al. 1996].
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Figure 4. (A) GST pull-down assays were performed with
[35S]methionine labelled TAP or various TAP mutants
and immobilised GST-p15 on glutathione agarose beads.
One tenth of the input (i) and one quarter of the bound
fractions (s) were analysed on SDS-PAGE followed by
fluorography. Lanes (b) show the background obtained
with glutathione agarose beads coated with GST.
(B) GST pull-down assays were performed with
[35S]methionine labelled p15 or various p15 mutants
and immobilised GST-TAP on glutathione agarose beads.
Symbols are as in A. GST pull-down assays were
performed as described by Bachi et al. [2000].
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Figure 5.
Multiple alignment of UBA domains.
See legend of Figure 2 for description of columns.
Species names (other than those in Figure 2): Mm, Mus musculus;
Tt, Thermus thermophilus; Ec, Escherichia coli.
Highly conserved
position are indicated by boldfaced characters and/or colors: blue,
hydrophobic; boldfaced in black, negatively charged; boldfaced in
blue, aromatic.
The mutation, D595R, which interrupts binding to nucleoporins,
are indicated by a red asterisk.
The known secondary structures of 1uba and 1eftu are indicated
below the consensus line (H, alpha-helix; E, beta-strand).
The predicted secondary structure, which follows the known secondary
structures, was calculated by the PHD program [Rost and Sander 1994].
The surface accessibility below (0 low, 9 high) was calculated using
the DSSP program [Kabsch and Sander 1983].
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Figure 6. Structure of 1uba.
The red side chain indicates the position corresponding to
TAP D595.
The figure was prepared using MOLMOL [Koradi et al. 1996].
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Figure 7. Uper panel: GST pull-down assays were performed
with [35S]methionine labelled p15 or the nucleoporins
indicated on the left of the panels and GST, GST-TAP,
GST-TAP D595R or GST-TAP(delta)567-613 as indicated above the lanes.
One tenth of the input and one quarter of the bound fractions
were analysed on SDS-PAGE followed by fluorography.
Lower Panel: Domain organisation of human TAP protein.
TAP domains defined in previous studies are indicated
(Braun et al., 1999; Bachi et al., 2000). The predicted
folding within the LRRs is shown diagramatically. The
transportin binding site defined overlaps with the previously
identified TAP NLS.
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