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   N-terminal domain, and the last 25 C-terminal residues turned out to be
   extremely disordered, if visible at all. A striking improvement of the
   map quality was observed for the AcCoA complex. A continuous model
   could be built comprising residues Ser-2—Gln-459. A crystal lattice
   rearrangement occurred upon soaking of AcCoA complex crystals in the
   solution containing UDP-GlcNAc, and the structure was solved by
   molecular replacement with the program AMoRe ([61]14). Refinement was
   carried out with the programs REFMAC ([62]15) and CNS ([63]16), using
   the maximum likelihood method and incorporating bulk solvent
   corrections, anisotropicF [obs] versus F [calc] scaling, and
   non-crystallography symmetry restraints. 10% of the reflections were
   set aside during refinement for cross-validation purposes. Automated
   correction of the model and solvent building were performed with the
   program ARP/wARP ([64]17). The stereochemistry of the final models was
   verified with the program PROCHECK ([65]18). Refinement statistics are
   summarized in Table[66]II. Coordinates have been deposited in the
   Protein Data Bank under accession reference numbers [67]1HM0 for
   apo-SpGlmU and [68]1HM8 and [69]1HM9 for the AcCoA and the
   AcCoA·UDP-GlcNAc complex, respectively. Fig.[70]1 B was generated with
   Alscript ([71]19), and Figs. [72]2-4 were generated with SPOCK ([73]20)
   and Raster3D ([74]21).
   [75]Previous Section[76]Next Section

 §2§ RESULTS AND DISCUSSION §2§

   The crystal structure of full-length SpGlmU was determined by multiple
   anomalous dispersion techniques. The apo-SpGlmU, SpGlmU-AcCoA, and
   SpGlmU-AcCoA·UDP-GlcNAc structures were refined to 2.3, 2.5, and 1.75
   Ã…, respectively, and have good stereochemistry. The apo-SpGlmU
   structure consists of residues Ser-2 to Val-142 and Val-149 to Glu-447.
   The surface loop Arg-143—Glu-148, located in the pyrophosphorylase
   domain, and the last 12 residues of the acetyltransferase domain,
   Tyr-448—Gln-459, could not be built because of lack of electron
   density. The two complex structures, SpGlmU·AcCoA· and
   SpGlmU·AcCoA·UDP-GlcNAc, consist of residue Ser-2 to Gln-459, and clear
   unbiased electron density could be observed for both AcCoA and
   UDP-GlcNAc prior to the incorporation in the refinement (Fig. [77]2 a).
   [78]Figure 2
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   Figure 2

   Map quality and overall fold of the SpGlmU structure. A, stereo pair of
   the 1.75-Å resolution 2F [o] − F [c]averaged electron density map,
   calculated prior to the incorporation of AcCoA in the refinement and
   contoured at 1.0 (blue) and 3.0 Ï‚ (black) around an AcCoA molecule.
   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.

   The SpGlmU apo-structure, except for the two missing regions
   Arg-143—Glu-148 and Tyr-448—Gln-459, is highly similar to the
   SpGlmU·AcCoA complex structure, with a root mean square deviation of
   0.450 Å for 440 Cα positions (Fig. [83]2 c). The SpGlmU-AcCoA complex
   structure, in turn, is almost identical to the SpGlmU·AcCoA·UDP-GlcNAc
   complex structure in the acetyltransferase domain (root mean square
   deviation of 0.17 Å for 208 Cα positions). However, the two complex
   structures differ greatly in the pyrophosphorylase domain, as discussed
   further below.

   The SpGlmU overall fold for residues Ser-2 to His-330 is similar to
   theE. coli-truncated enzyme ([84]3). However, the relative arrangement
   of the pyrophosphorylase and the acetyltransferase domain differs
   between the crystal structures of SpGlmU and E. coliGlmU-Tr (Fig. [85]3
   a). Indeed, the two GlmU structures present a 20° deviation in the
   direction of the α-helical linker, indicating that this is, in fact, a
   flexible hinge. A direct consequence of this deviation are major
   differences between GlmU-Tr and SpGlmU occurring in the regions of the
   pyrophosphorylase domain neighboring the N-cap of the α-helical linker.
   These conformational changes, together with a high overall mobility of
   the pyrophosphorylase domain, as opposed to the acetyltransferase
   domain, suggest that the presented structures may represent only
   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.

 §5§ The Pyrophosphorylase Domain §5§

   The SpGlmU PPase domain can be divided into two lobes separated by the
   active site pocket. The first hundred residues, containing the
   consensus sequence motif G-X-G-T-(RS)-(X)[4]-P-K, form the nucleotide
   binding lobe, whereas the second lobe, responsible for recognition of
   the sugar moiety, encompasses the remaining residues of the N-terminal
   domain (Fig. [90]3 B).

   Striking differences exist between the PPase domains of apo-SpGlmU and
   the SpGlmU·AcCoA·UDP-GlcNAc complex (root mean square deviation of 2.2
   Å for 226 Cα atoms), indicating that the enzyme undergoes a substantial
   conformational change upon substrate/product binding. In the absence of
   UDP-GlcNAc (apo-SpGlmU and SpGlmU·AcCoA), SpGlmU adopts an open
   conformation, whereas in the UDP-GlcNAc complex two regions within the
   sugar binding lobe move toward each other giving rise to a closed
   conformation (Fig. [91]3 B). Upon product binding the entire region
   encompassing residues Thr-132—Lys-166 moves as a rigid body, making a
   20° tilt resulting in a 7-Å movement of the β5b-β6 surface loop. The
   melting of the last turn of the α-helix α5, facing the β5b-β6 loop,
   transforms the following α5-α6 surface loop (Asn-191—Tyr-197) into an
   extended thumb-shaped hairpin. These movements bring the two above
   surface loops close to each other, such that in the UDP-GlcNAc complex
   the Ala-192 N hydrogen bonds Asp-157 OD1 (Fig. [92]3 b), whereas in the
   unbound form these two residues are 14 Ã… apart. This suggests that the
   two surface loops function like a pair of tongs, closing up upon
   substrate binding and anchoring the sugar deep into the active site
   pocket thereby shielding it from solvent.

   The “breathing” of the PPase domain of SpGlmU could not be observed for
   the E. coli GlmU-Tr enzyme, where the crystal structures reveal a
   closed conformation for both the apo- and UDP-GlcNAc complexed forms
   ([93]3). However, analysis of the crystal packing in the E. coli
   GlmU-Tr structures reveals that the pyrophosphorylase domain is
   constrained into its closed conformation in both the apo-form and the
   GlmU-Tr·UDP-GlcNAc complex by the packing environment, whereas no such
   constraints exist in apo- or complexed SpGlmU crystals.

   The interactions of the enzyme with the nucleotide and the sugar are
   largely conserved within the complex crystal structures from S.
   pneumoniae and E. coli GlmU, yet significant differences reside in the
   surroundings of the pyrophosphate moiety. Whereas in the
   GlmU-Tr·UDP-GlcNAc complex both phosphates are solvent-exposed, in the
   SpGlmU·AcCoA·UDP-GlcNAc complex the α-phosphate is stabilized through
   weak hydrogen bonds to the side chains of sequence-conserved Arg-15 and
   Lys-22, located within the signature motif. Moreover, both phosphate
   groups interact through a calcium ion with Asp-102 and Asn-227,
   situated in the β4-α4 hairpin and in the N-cap of the long α-helical
   linker, respectively (Fig.[94]3 c). This calcium ion exhibits the
   octahedral coordination geometry characteristic of Mg^2+ ions and thus