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t/data/cp1252/10676968.cp1252  view on Meta::CPAN

   [44]Figure 1 : Primary, secondary and tertiary structure of hGBP1.
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   a, A model of the tertiary structure of hGBP1 presented as a ribbon,
   where the LG domain is in purple, the connecting region in green, the
   helical domain in yellow and alpha 12/ alpha 13 in cyan. Insertions,
   marked I2–I5 in b, are in violet. Dashed lines indicate disordered
   regions in the molecule. The tentative nucleotide-binding area,
   identified by a Ras–GBP overlay, is indicated by a sphere with radius
   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.

t/data/cp1252/10676968.cp1252  view on Meta::CPAN

   a, Superimposition of the LG domain of hGBP1 with the G domain of Ras
   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
   the solvent than in Ras–nucleotide complexes, part of the polypeptide
   chain is in a position that interferes with nucleotide binding. Perhaps
   owing to the absence of nucleotide, the polypeptide chain around the
   binding site is mobile, as no electron density is visible for residues
   69–72 (I1) in the region analogous to switch I, residues 190–193 close
   to the ^181TLRD^184 motif and residues 244–257 in I5, close to the
   SAK/L motif, which is conserved only in the Ras family and is absent in
   GBPs.

   The (phosphate-binding) P loop^[65]10, residues 45–52, adopts a
   structure different from that of the Ras–nucleotide complexes. The
   invariant lysine residue of the P loop does not interact with the
   main-chain carbonyls for stabilization. Instead, in hGBP1 the loop is
   stabilized by interactions with the region analogous to switch II,
   involving hydrogen bonds between Tyr 47 (backbone N) and Asp 103,
   Lys 51 and Thr 98. Furthermore, the structure is not suited for
   nucleotide binding as the phosphates would clash with Tyr 47. The
   region corresponding to switch I in Ras is disordered in hGBP1. Thr 75
   appears to be analogous to Thr 35 in Ras, but is 5 Å away in the
   overlay ([66]Fig. 2b). The ^97DxxG^100 motif of hGBP1 superimposes well
   with that of switch II in Ras. D184 is 6 Å away from the corresponding
   D119 of the canonical N/TKxD motif and would have to move accordingly
   to occupy a similar position in the nucleotide-bound form. In general,
   it appears that the guanine nucleotide-binding site is partly open
   ([67]Fig. 2a, [68]b) such that the incoming nucleotide would enter the
   binding site base first and would then, after a corresponding
   conformational change, bind into the phosphate-binding area, as
   suggested for Ras by the structure of the Ras–Sos complex^[69]11.

   Ras proteins have an intrinsic GTPase reaction rate in the order of

t/data/good/11118459.utf8  view on Meta::CPAN

   Phases were calculated after rigid-body refinement based on the two apo
   SpGlmU molecules present in the asymmetric unit. B, left, ribbon model
   of a SpGlmU subunit, showing the PPase domain (orange), the α-helical
   linker (magenta), the LβH domain (yellow with the unique insertion loop
   inorange), and the C-terminal arm (cyan);right, the SpGlmU trimer with
   bound AcCoA and UDP-GlcNAc (gray bonds with red oxygen, bluenitrogen,
   green sulfur, and purple phosphorus atoms) viewed in the same
   orientation as in the panel on theleft (top) and down the LβH axis
   (bottom); for clarity a single subunit is color-coded as in the panel
   on the left, with the remaining two subunits shown in gray. C, stereo
   view overlay of the Cα trace of apo-SpGlmU (cyan) and SpGlmU·AcCoA
   (orange), with the two respective C termini labeled. The overlap is
   based on a least squares fit of 440 Cα positions.

   The SpGlmU molecule assembles into a trimeric arrangement with overall
   dimensions of 89 × 85 × 90 Å (Fig. [82]2 b). The LβH domains
   (Val-252—Ile-437) are tightly packed against each other in a parallel
   fashion, an α-helical linker (Arg-229—Met-248) sits on top of each
   β-helix and projects the globular pyrophosphorylase domain
   (Ser-2—Asn-227) far away from the trimer interface.

t/data/good/11118459.utf8  view on Meta::CPAN

   snapshots of a highly dynamic system.
   [86]Figure 3
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   Figure 3

   The pyrophosphorylase domain. A, stereo view overlay of the Cα trace of
   E. coli GlmU-Tr (green) and SpGlmU·AcCoA (orange), with the
   pyrophosphorylase signature motif color-coded in cyan. The overlap is
   based on a least squares fit of 104 Cα positions of the central β-sheet
   of the pyrophosphorylase domain. B, stereo view overlay of the PPase
   domain of SpGlmU bound to AcCoA (yellow/cyan) and AcCoA·UDP-GlcNAc
   (orange/green). Backbone regions with associated side chains that
   deviate significantly between the two complex structures are
   highlighted (cyan for the open form, and green for the closed form).
   Secondary structure elements are labeled. C, close-up stereo view of
   the UDP-GlcNAc/Ca^2+ binding site in the closed form; the molecule is
   color-coded as in A with the signature motif incyan; solvent molecules
   are red, and the Ca^2+ ion is green. Hydrogen bonds are shown asdotted
   lines.

t/data/good/11493601.utf8  view on Meta::CPAN

   Se-Met-substituted form of the enzyme ([71]28). After phase extension
   with a native data set at higher resolution, a high quality electron
   density map was obtained (Fig. [72]2) allowing building and refinement
   of the model at 1.6 Ã… resolution. The crystallographic statistics are
   shown in Table [73]I. The asymmetric unit contains two mature
   ι-carrageenase molecules, each containing amino acids 28–491. Residues
   314–334 and 341–350 for molecule A and residues 313–334 and 341–351 for
   molecule B are not visible in the 2F [o] − F [c] electron density map
   and are presumed to exist in disordered or highly flexible
   conformations. Superposition of molecules A and B reveals that the Cα
   atoms overlay with a root mean square deviation of 0.21 Ã….
   Approximately 10 residues in each molecule presented clear alternate
   conformations. The need to refine the occupancy for terminal atoms of
   several residues, such as aspartate, glutamate, or methionine, suggests
   that a fraction of the protein population in the crystal has been
   subjected to radiation damage ([74]29).
   [75]Figure 2
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t/data/macroman/11042188.macroman  view on Meta::CPAN

   complexed with proteinase A, the ε-NH[2] group of Lys^18 of the
   inhibitor hydrogen bonds to one of the carboxyl oxygens of Asp^32 of
   the enzyme (Fig. [153]3). This is one of the two catalytic Asp residues
   that operate the catalytic mechanism of all aspartic proteinases
   ([154]24). The ε-NH[2] group of Lys^18 also hydrogen bonds to one of
   the carboxyl oxygens of the side chain of Asp^22 in the IA[3]
   inhibitory sequence (Fig. [155]3). The other oxygen of the side chain
   COOH of Asp^22 hydrogen bonds to the phenolic OH group of Tyr^75 in the
   enzyme, a residue that is totally conserved in all eukaryotic aspartic
   proteinases and which is positioned almost at the tip of the β-hairpin
   loop or flap that overlays the active site cleft in these enzymes. A
   network of interactions thus cross-links these charged residues of
   IA[3]with the catalytically essential and structurally conserved
   residues of the target enzyme (Fig. [156]3). When the charged side
   chain of Asp^22 in the full-length protein form of IA[3] was replaced
   with the hydrophobic but otherwise almost isosteric side chain of a
   leucine residue, the purified D22L mutant protein had a slightly
   reduced potency both at pH 3.1 and 4.7 (Table [157]I) but nevertheless
   was still an effective inhibitor.
   [158]Figure 3
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