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7 Å. The topology is shown schematically using the same colour code. b,
Sequence alignment of hGBP1 (Swissprot accession no. P32455) with Mag-2
(EMBL acccession no. M81128) from mouse and chicken GBP1 (EMBL
accession no. X92112), with the secondary structure assignment as
determined using the programme DSSP^[45]29, with the same colour code
as in a, and aligned with the secondary structure of Ras dot
GppNHp^[46]30 (light blue lines). Contacts between helix alpha 12 and
the rest of the protein are indicated: asterisk, direct; circle,
water-mediated polar interactions; hash sign for both. For brevity,
sequences with lowest homology have been chosen.
[47]High resolution image and legend (233K)
§5§ [48] Table 1: Crystallographic data statistics §5§
[49]Table 1 - Crystallographic data statistics
[50]Full table
The LG domain of GBPs contains the conserved sequence elements of
GTP-binding proteins with modifications. Originally the N/TKxD motif
was believed to be absent in the GBPs^[51]2. An Asp-Asn mutation can
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in complex with GDP(PDB accession no. 1Q21) as a stereo view.
N-terminal residues 1–36 of hGBP1 up to beta 1 have been omitted for
clarity. The colour code is as in [61]Fig. 1; Ras is in cyan. b,
Putative location of nucleotide-binding site in hGBP1. The regions of
hGBP1 potentially involved in binding the guanine nucleotide are shown
as obtained from a structural superimposition of RasGDP (in cyan) with
the corresponding regions in hGBP1 (purple), highlighting functionally
important residues necessary for binding and conformational change as
balls or in ball-and-stick. Whereas Gly 60^ras overlays very well with
Gly 100^hGBP1, residues D119/D184 and T35/T75 do not.
[62]High resolution image and legend (79K)
As GBP is stable in the absence of nucleotide, whereas Ras-like and G
alpha GTP-binding proteins are not, it was of interest to investigate
the effect of the absence of nucleotide on the structure. As all
P-loop-containing proteins^[63]10 bind the beta / gamma -phosphate of
the nucleotide in a similar manner, and as the role of Asp 184 in
binding the guanine base is similar to that of the Asp of the canonical
N/TKxD motif, we can locate the nucleotide-binding site of hGBP1 using
the RasGDP–hGBP1 overlay ([64]Fig. 2b). From this comparison we can
also see that, although part of the binding site is more accessible to
t/data/cp1252/10676968.cp1252 view on Meta::CPAN
[79]Figure 3 : Interaction of the C-terminal helix motif |[alpha]|12/13
with the helical and the LG domains. Unfortunately we are unable to
provide accessible alternative text for this. If you require assistance
to access this image, or to obtain a text description, please contact
npg@nature.com
The electrostatic surface potential shows that the highly charged
regions of the helical and LG domains are masked by an alpha 12/13
motif, as indicated in the lower panel by showing alpha 12/ alpha 13 in
worm representation.
[80]High resolution image and legend (40K)
Mx and dynamins are commonly grouped into a separate class of large
GTP-binding proteins^[81]5. Proteins of this family have several
variations in their domain structure, but they all possess at least a
'GTPase' domain of approx 300 residues, a 'middle' or 'assembly' domain
(150–200 residues) and a 'GED' domain (100 residues). These values are
close to the sizes of the LG, helical bundle and alpha 12/13 domains of
hGBP1, respectively. Biochemically, they share common properties such
as relatively low affinity for nucleotides (K[m] 10–500 micro M), the
ability to form oligomers and a high intrinsic rate of GTP hydrolysis
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multimerization of hGBP1. Unfortunately we are unable to provide
accessible alternative text for this. If you require assistance to
access this image, or to obtain a text description, please contact
npg@nature.com
a, Concentration dependence of the specific GTPase activity of hGBP1.
The data were fitted to a model involving dimer formation yielding 0.6
micro M for the apparent dimerization constant of hGBP1. b,
Size-exclusion chromatography of GDP dot AlF[x]-, GppNHp-, GDP-bound
and nucleotide-free hGBP1.
[88]High resolution image and legend (20K)
The homology to the dynamin family is further corroborated by
demonstrating nucleotide-dependent oligomerization ([89]Fig. 4b). The
elution profiles from standardized analytical size-exclusion
chromatography show that hGBP1 is monomeric in the absence and in the
presence of GDP, whereas it shows higher molecular mass when bound to
the non-hydrolysable GTP analogue GppNHp. The molecular mass is higher
again for hGBP1 in complex with GDP and aluminium fluoride which
together are believed to mimic the transition state of GTP
hydrolysis^[90]8, ^[91]12. It seems that, for efficient GTP hydrolysis
t/data/demos/12169614.utf8 view on Meta::CPAN
operon encode proteins that are involved in the synthesis of cytochrome
c and the cytochrome bc complex, respectively. These proteins are
involved in the electron transport chain and may be up regulated by
sigma-H in response to nutrient-limiting conditions in an attempt to
generate energy. We also find the expression of resABC, which is also
required for cytochrome c synthesis, to be indirectly regulated by
sigma-H. Expression of resABC was previously shown to be induced upon
entry into stationary phase by a putative sigma-A promoter (53).
TABLE 2.
TABLE 2.
Newly identified sigma-H-regulated geneslegend
Expression of the ccdA operon was previously known to coincide with the
time that sigma-H is fully active (33). The ccdA operon transcript
has been mapped by primer extension, and the authors indicated that
sigma-A was likely to drive transcription of the ccdA operon. We have
identified a potential sigma-H promoter that overlaps the putative
sigma-A promoter. Thus, sigma-H could be responsible for additional
regulation of ccdA. ccdA mutant strains are deficient in sporulation at
a very late stage (48), similar to what is observed with spoVS, a
gene controlled by sigma-H.
The best-known role of sigma-H is to activate sporulation. Many of the
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* [126]Download as PowerPoint Slide
Figure 4
Stereoview of coenzyme A binding. a, the F [o] −F [c] simulated
annealing omit map of the CoA binding site of one subunit showing the
bound CoA. The map was calculated from 50.0 to 2.5 Ã… resolution and
contoured at 3.0 Ï‚.b, hydrogen bonds are indicated by solid lines. The
thiol group of coenzyme A is shown in light green, and other atom
colors are as described in the legend to Fig.[127]3 b. His^177 involved
in CoA recognition by stacking interaction with the adenine base and
Asn^282forming a hydrogen-bond with the β-mercaptoethylamine moiety are
not labeled for clarity.
Hydrophobic atoms of the pantothenate moiety of CoA form van der Waals'
contacts with residues Leu^130, Tyr^175, and Ile^281 (Fig. [128]4 b).
The carbonyl oxygen of the pantothenate moiety near the
β-mercaptoethylamine moiety is hydrogen bonded to the hydroxyl group of
Tyr^240 and to the amide group of Asn^282 (Fig. [129]4 b). The amide
nitrogen of the β-mercaptoethylamine moiety is hydrogen-bonded to the
t/data/good/11357122.utf8 view on Meta::CPAN
ends will change with higher resolution. AChBP shares 24% sequence
identity with the ligand-binding domain (LBD) of human alpha [7] (shown
in green), 20–24% with other nicotinic receptors and 15–18% with other
LGICs. The residues conserved in the superfamily are shown in bold with
grey background. Asterisk, beginning and end of the Cys loop. Colouring
of interface residues at the plus (yellow) and minus side (light blue)
shows the lack of sequence conservation in the subunit interface across
the pentameric LGIC family. Nicotinic receptor ligand-binding residues
on the principal (pink) and complementary (dark blue) side are
indicated.
[75]High resolution image and legend (140K)
[76]Top of page
§3§ Structure determination §3§
The crystal structure of AChBP was solved using weak Pb
multiple-wavelength anomalous diffraction (MAD) data in two crystal
forms. The electron density map was improved substantially by
cross-crystal averaging of three crystal forms with 20, 10 and 5 copies
of the protomer in the asymmetric unit ([77]Table 1). The structure was
refined at 2.7 Ã… in space group P4[2]2[1]2, with one AChBP pentamer
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please contact npg@nature.com
a, In this representation each protomer has a different colour.
Subunits are labelled anti-clockwise, with A–B, B–C, C–D, D–E and E–A
forming the plus and minus interface side, with the principal and
complementary ligand-binding sites, respectively (ball-and-stick
representation). b, Viewing the AChBP pentamer perpendicular to the
five-fold axis. The equatorially located ligand-binding site
(ball-and-stick representation) is highlighted only in the A (yellow)–B
(blue) interface.
[88]High resolution image and legend (165K)
[89]Top of page
§3§ The AChBP protomer §3§
Each AChBP protomer is a single domain protein, asymmetric in shape,
with a size of around 62 times 47 times 34 Ã…^3 ([90]Fig. 3a). It
consists of an N-terminal alpha -helix, two short 3[10] helices and a
core of ten beta -strands, which form a beta -sandwich. The order of
beta -strands conforms to a modified immunoglobulin (Ig)
topology^[91]28 ([92]Fig. 3b) with an extra beta -hairpin (f'–f") and
t/data/good/11357122.utf8 view on Meta::CPAN
Topology diagram of the AChBP protomer. For comparison with Ig-folds
the strands have been labelled a–g, showing the additional strand (b')
and hairpin (f'–f"). In this structure, strands have been labelled beta
1– beta 10 with loops (or turns) L1–L10 preceding each strand with the
same number. The beta 5 strand is broken ( beta 5– beta 5') with
internal loop L5'; beta 6 also has a small break, but it is shown
continuously (see Fig. 1). The precise beginnings and ends of strands
may change slightly with increasing resolution, but the topology seen
here will be highly conserved across the entire family of pentameric
LGICs. S: disulphide bridge.
[99]High resolution image and legend (38K)
[100]Top of page
§3§ Positioning of functional regions §3§
In the structure, the N and C termini are located at 'top' and 'bottom'
of the pentamer, respectively. In the ion channels the transmembrane
domains are at the C-terminal end of the LBD, at the 'bottom' of the
AChBP structure ([101]Figs 2b and [102]3), starting directly at the end
of beta -strand beta 10.
t/data/good/11357122.utf8 view on Meta::CPAN
Gln B55/ italic gamma Glu 57, violet), E (Arg B104/ italic gamma
Leu 109, Val B106/ italic gamma Tyr 111, Leu B112/ italic gamma
Tyr 117, Met B114/ italic gamma Leu 119, light blue) and F (Tyr B164,
blue). b, Stereo view of the electron density map displaying a HEPES
buffer molecule in the ligand-binding site. This experimental density
(contoured at 1 sigma ) is derived from cross-crystal averaging. c,
Location of the principal ligand-binding residues (colours as in a,
orientation as in Fig. 2b). d, Location of the complementary
ligand-binding residues (colours as in a, orientation as in Fig. 2b).
Note that loops D, E and F are all on beta -strands.
[132]High resolution image and legend (89K)
All residues in the binding site have been identified by photoaffinity
labelling and mutagenesis studies^[133]6, ^[134]7, ^[135]8, ^[136]9,
^[137]10, ^[138]11, ^[139]12, ^[140]13, ^[141]14, ^[142]15, ^[143]16.
Although the side chain of His A145 is pointing away from the cavity,
its main chain is involved in the binding site. One residue identified
by labelling studies^[144]6, Trp A82 ( alpha [1] Trp 86), is involved
in hydrophobic core formation and located far from the pocket.
Therefore it probably does not participate directly in ligand binding.
Additional residues may be involved in binding large ligands such as
t/data/good/11357122.utf8 view on Meta::CPAN
a, Stereo figure of the dimer interface. Representation of the
interface residues (ball-and-stick) on a schematic secondary structure.
The figure shows the plus face of subunit A (light yellow, orange
interface residues) and the minus face of subunit B (light blue, pink
interface side chains). b, Dimer interface interactions. Note that
owing to the low conservation of these interfaces (Fig. 1) the actual
interactions will not be conserved in any pentameric LGIC interface,
but that in all receptors these topological regions are likely to form
the interface.
[168]High resolution image and legend (58K)
[169]Top of page
§3§ Ligand-gated ion channels §3§
The superfamily of ligand-gated ion channels has highly conserved LBDs,
and the function and location of the conserved residues can now be
analysed in the light of the structure. Most residues that are
conserved in the superfamily ([170]Fig. 1) are hydrophobic and help to
maintain the hydrophobic core of the protomer, grouped into
three clusters ([171]Fig. 6). The first cluster is involved in packing
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Conserved residues are indicated as viewed from the central pore.
Hydrophobic cluster I (red): residues 6, 10, 63, 65, 71, 81, 105, 111;
Cluster II (green): residues 20, 27, 29, 58, 82, 84, 86, 88, 140, 150,
152, 195; Cluster III (pink): residues 33, 35, 38, 41, 48, 52, 123,
125, 136, 138, 165, 171, 173, 199, 201. The hydrophilic conserved
residues (dark blue): Asp 60, Asp 85, Asn 90, Gly 109, Lys 203.
Conserved residues in the ligand-binding site (light blue): His 145,
Tyr 192, or close by: Ala 87. Very few conserved residues are at the
surface. Residues conserved between pentameric LGICs but not AChBP are
indicated by yellow main chain (14, 19, 170 and the Cys-loop124–135).
[178]High resolution image and legend (66K)
Contrary to the above residues, the Cys loop ([179]Fig. 3a) is well
conserved in the pentameric LGIC family but not in AChBP ([180]Fig. 1,
residues 129–141). It is hydrophobic in the receptors, whereas it is
hydrophilic in AChBP. The Cys loop is located at the bottom (membrane)
side of the protein, close to the dimer interface. This position, and
its hydrophobicity in the LGIC family, implies that it could interact
with the membrane or with the transmembrane region of the receptors,
functions that are absent in AChBP.
t/data/good/11493601.utf8 view on Meta::CPAN
domain B are shown, respectively, in blue, gold, and red. The small T1
extension, containing an antiparallel sheet (β16-β17) and an α-helix
(α2), is shown ingreen. The red, yellow, andgreen spheres represent
sodium, calcium, and chloride ions, respectively. Figs. [89]3 and [90]4
were prepared using Molscript ([91]50).
The most striking difference between ι-carrageenase (Fig. [92]3) and
the 11 other β-helix proteins of known structure is the presence, in
the C-terminal region, of two large additional domains (both 67
residues long). Domain A (residues 307–373) replaces the T1 turn
between strands β32 and β35 (see legend of Fig. [93]3 A for
definition). Half of this domain (residues 314–333 and 341–350) could
not be built as no clear electron density was observed. The visible
part of domain A features a sheet of two short antiparallel β-strands,
β33 and β34, edged by one α-helix (residues 358–367) and has an average
B value (37.8 Å^2) twice that of the β-helix core (17.8 Å^2). At the
border of the visible part of domain A is a hydrophobic surface,
suggesting that the nonvisible residues complete a globular-shaped
domain. This domain is weakly bound to the β-helix by only four
hydrogen bonds (Glu^310 O-Lys^252 NZ, Asp^358OD1-Ser^442 OG, Asp^358
OD2-Lys^443 N, and Asp^362 OD1-Tyr^444 OH). Moreover, the visible part
t/data/macroman/10542098.macroman view on Meta::CPAN
is highlighted by yellow stripes. The cell surface position is not to
scale. d, Comparison of DR5 and TNF-R1. DR5 and TNF-R1 (from the TNF
beta −TNF-R1 complex) are depicted schematically in the left and right
panels respectively with equivalent regions in identical colors. The
central panel is based on superposition of DR5 D1 and TNF-R1 D2. The
schematic representation of TNF-R1 is shown in gray while that of DR5
is in green. Disulfide bonds are depicted in yellow as ball-and-stick
representation. e, Portion of the final 2F[o] - F[c] electron density
map contoured at 1 sigma , showing a portion of the TRAIL structure in
the BC loop.
[73]Full Figure [74]Full Figure and legend (68K)
[75]Table 1. Crystallographic Statistics
[76]Table 1 thumbnail
[77]Full Table [78]Full Table
The TRAIL trimer
The TRAIL subunits consist of beta -sandwiches conforming, as expected,
to the jellyroll topology^[79]6. Each TRAIL monomer contains one
cysteine at position 230. Crystallization was performed under
non-reducing conditions and in the crystal a disulfide bond was seen
between two of the three cysteine residues in each trimer. The
formation of this disulfide bridge did not affect receptor binding, as
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defined by pairwise superposition of the TRAIL and TNF beta subunits.
The view of the TRAIL and TNF beta surfaces corresponds to that of
[93]Fig. 1a while the matching DR5 and TNF-R1 surfaces are revealed by
a 180¡ rotation as in the opening of a book. The solvent accessible
surfaces (calculated in isolation) of each component are shown in gray
with areas contributed by residues implicated in the interaction
surface of the complex highlighted in colored patches. Colors for
interaction patches are matched between ligand and receptor for each
complex. Conservation of the color scheme is used to highlight broadly
equivalent regions of interaction in the two complexes.
[94]Full Figure [95]Full Figure and legend (83K)
The DR5 receptor
Members of the TNF-R family are characterized by extracellular repeats
containing three disulfide bridges with a cysteine knot topology^[96]7,
^[97]10. The number of repeats ranges from six in CD30 through four in
TNF-R1 and TNF-R2 to three in Fas and only two in the four TRAIL
receptors. The crystallographically well-ordered portion of the DR5
molecule includes these two repeats (D1 and D2), which form the ligand
binding region in the complex. DR5 starts with an N-terminal cap
containing a partial cysteine knot with a single non-canonical
disulfide ([98]Fig. 1d). This N-terminal cap corresponds to the
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envelopes. b, Comparison of surface charge between TRAIL, TNF alpha ,
TNF beta and their receptors. Blue denotes positive, and red negative;
electrostatic potential is contoured at plusminus 8.0 kT in program
GRASP^[116]31. The views of ligand and receptor are as in [117]Fig. 2b.
c, Interaction of Arg 149 in the AA" loop of TRAIL with Glu 147 in DR5.
d, BIAcore analysis showing binding of DR5 to wild type TRAIL or the
mutant lacking the AA" loop. e, Immunoprecipitation with DR5-Fc in the
presence of wild type (W) or the slightly smaller AA" TRAIL mutant (M).
Lanes 1 and 2 immunoprecipitated material (IP), lanes 3 and 4 material
left in supernatant (SN) following immunoprecipitation.
[118]Full Figure [119]Full Figure and legend (83K)
Features conferring specificity
Inspection of the four TRAIL binding receptor sequences shows 45−81%
identity in D1 and 59−80% identity in D2. Most of the residues involved
in the TRAIL−DR5 interface show conserved characteristics, underscoring
the role played by these residues in receptor−ligand specificity
([120]Fig. 2a). D2 is implicated as a major focus for TRAIL-binding
specificity, with conservation in this receptor subgroup of residues
such as Glu 147, Glu 151, Arg 154 and Asp 175 (DR5 numbering) that
mediate complimentary electrostatic interactions. One of these
distinctive TRAIL-binding residues (Glu 147) makes a salt bridge to Arg
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