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   ([62]24), including bulk solvent and anisotropic B-factor corrections.
   NCS restraints were used only for the apo GNA1 model. High temperature
   factors and weak electron density maps are associated with residues
   Gln-52 to Lys-57 in the two models. The 3 intertwined dimers of the apo
   GNA1 model have an average root mean square deviation of 0.7 Ã… for all
   Cα atoms. In the GNA1-AcCoA complex model, the root mean square
   deviation value between the 2 dimers is 0.4 Å for all Cα atoms. The
   GNA1-CoA-GlcNAc6P complex structure was obtained from a rigid body
   refinement using the GNA1-AcCoA complex as a starting model. Fourier
   difference maps clearly revealed the location of the bound CoA and
   GlcNAc6P in two of the four molecules. The structure of the
   GNA1-CoA-GlcN6P complex was also solved at 2.5 Ã…; superimposition of
   the two ternary complexes (GNA1-CoA-GlcNAc6P and GNA1-CoA-GlcN6P)
   revealed that GlcN6P (the substrate) and GlcNAc6P (the reaction
   product) were positioned similarly. Because the GNA1-CoA-GlcNAc6P
   structure was obtained at a higher resolution than that of
   GNA1-CoA-GlcN6P, we only considered in the analysis the
   GNA1-CoA-GlcNAc6P complex structure. The stereochemistry of the refined
   models was analyzed by PROCHECK ([63]25); no residue was found in the
   disallowed regions of the Ramachandran plot. The coordinates of apo,
   AcCoA-, and CoA-GlcNAc6P-complexed GNA1 have been deposited in the
   Protein Data Bank (accession codes [64]1I21, [65]1I12, and [66]1I1D).

   Figs. [67]1, 2, 4, and 5 were generated with SPOCK ([68]26) and
   Raster3D ([69]27) except for Fig. [70]1 A, which was computed with
   Alscript ([71]28).
   [72]Previous Section[73]Next Section

 §2§ RESULTS AND DISCUSSION §2§

 §5§ Overall Structure §5§

   The three-dimensional structures of GNA1 in its apo state and complexed
   forms with AcCoA or with CoA and GlcNAc6P have been solved and refined
   at 2.4, 1.3, and 1.8 Ã… resolution, respectively. Overall, the electron
   density is well defined for these structures (Fig. [74]1 B) except for
   a surface loop comprising residues Gln-52 to Lys-57 (cf.“Experimental
   Procedures”). As predicted from sequence analysis, GNA1 shares
   structural similarities with other GNAT superfamily members ([75]1,
   [76]11). The GNA1 fold consists of a central core, composed of a mixed
   5-stranded β-sheet flanked by 4 α-helices, and a COOH-terminal strand
   β6, which is projected away from the central core (Fig.[77]2 A).
   [78]Figure 2
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   Figure 2

   Structure of GNA1 and AcCoA, GlcNAc6P binding sites. A, ribbon
   representations of the GNA1 fold (left) and the intertwined GNA1 dimer
   (right). In subunit 1, the GNA1 secondary structure elements forming
   the structurally conserved GNAT core are shown ingreen, the exchanged
   β-strand in yellow, and the remaining structural elements in cyan.
   Subunit 2 is shown in magenta with its exchanged strand β6 inred. The
   molecular surface of AcCoA-(B) and CoA-GlcNAc6P-(C) complexed GNA1,
   oriented as in Fig.[82]1 B (left view) and color-coded (B) as in Fig.
   [83]1 B, with the regions undergoing small structural rearrangements
   upon AcCoA binding displayed under a transparent surface (the cyan and
   yellow bonds refer to the apo and AcCoA-complexed GNA1 models,
   respectively). AcCoA is shown with carbon (white), nitrogen (blue),
   sulfur (green), oxygen (red), and phosphorous (purple) atoms. C, the
   color code is according to the electrostatic potential with positive
   and negative charges shown inblue and red, respectively. The essential
   catalytic Tyr-143 is displayed through a transparent surface. CoA and
   GlcNAc6P are shown with yellow carbon atoms. D, stereoview of the
   GlcNAc6P binding site with residues from subunit 1 and 2 shown in cyan
   and magenta, respectively. The dotted lines indicate hydrogen bonds.
   Residues within the GNAT conserved β-bulge are displayed in green.

   The GNA1 structure is dimeric in the crystal as well as in solution, as
   attested from gel filtration data (not shown). The crystalline dimer is
   made of two intertwined GNA1 monomers in which strand β6 of one subunit
   exchanges with the identical strand from the other subunit (Fig. [84]2
   A). A β-strand exchange between subunits in a dimer is an unusual
   feature among GNATs and has been observed only in the HAT Hpa2
   structure ([85]9). In all other structurally characterized GNAT, except
   Hat1 that lacks a β6 strand ([86]5), the hinge loop preceding strand β6
   folds back onto its own subunit. This difference is reminiscent of
   three-dimensional domain-swapped proteins in which the loop that
   precedes the exchanged domain can switch from a closed to an opened
   conformation thereby leading to either a monomeric or a dimeric form
   ([87]29). In the case of GNA1 or Hpa2, the α4–β6 loop is too small to
   undergo such a conformational switch, and the dimeric assembly is
   further stabilized by a hydrophobic interface, two features that make
   three-dimensional domain swapping unlikely. Nonetheless, the monomeric
   GNATs and the intertwined dimers of GNA1 and Hpa2 are most probably
   related by divergent evolution from a common ancestor, and the
   evolutionary mechanisms that have led to dimer formation may have
   included three-dimensional domain swapping.

 §5§ The Cofactor Binding Site §5§

   In each subunit of the GNA1 dimer, AcCoA is positioned in a large
   hydrophobic cleft located at the site where the two parallel strands,
   β4 and β5, diverge because of a β-bulge in strand β4 that positions the
   side chains of Glu-98 and Asp-99 on the same face of the β-sheet. The
   presence of this β-bulge is remarkably well conserved among GNATs,
   which suggests a critical role for this structural element in the
   formation of the AcCoA binding site.

   AcCoA adopts a conformation similar to that described in other
   AcCoA-complexed GNAT structures ([88]1). The acetyl group of AcCoA,
   which marks the active site, is located between strand β5 and the
   β-bulge and is largely stabilized by contacts with the protein; the two
   carbon atoms contract hydrophobic interactions with residues Ile-100,
   Leu-133, and Tyr-143, and the carbonyl oxygen inserts into an oxyanion
   hole formed by the backbone amides of residues Asp-99 and Ile-100. Such
   an oxyanion hole has been observed in the structurally
   relatedN-myristoyltransferase ([89]30) but is a unique feature within
   the structurally characterized GNATs.

   Superimposition of the apo and AcCoA-complexed GNA1 structures shows
   that AcCoA binding induces subtle structural rearrangements that are
   confined to the edges of the cleft and result in a slightly narrower
   cleft. Residues 102–109 in the α3–β5 loop and 134–143 in the β5–α4 loop
   plus the N-cap of α4 move by ∼1.3 and 1.1 Å, respectively, toward the
   center of the cleft (Fig. [90]2 A). Whether these conformational
   changes, induced upon cofactor binding, are a prerequisite for acceptor
   substrate binding as shown for other GNATs ([91]31-33) needs to be
   ascertained by kinetic studies. A detailed comparison with other GNATs
   reveals that these rearrangements differ from those reported for (i)
   tGCN5, in which the cofactor-binding cleft opens slightly upon AcCoA
   binding to accommodate the histone tail ([92]7); and (ii) AANAT, in
   which a major rearrangement of the α1-loop-α2 region occurs upon AcCoA