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. 2001 Nov 15;20(22):6191-202.
doi: 10.1093/emboj/20.22.6191.

Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture

C Peneff  1 , P Ferrari, V Charrier, Y Taburet, C Monnier, V Zamboni, J Winter, M Harnois, F Fassy, Y Bourne
Affiliations

Crystal structures of two human pyrophosphorylase isoforms in complexes with UDPGlc(Gal)NAc: role of the alternatively spliced insert in the enzyme oligomeric assembly and active site architecture

C Peneff et al. EMBO J. .

Abstract

The recently published human genome with its relatively modest number of genes has highlighted the importance of post-transcriptional and post-translational modifications, such as alternative splicing or glycosylation, in generating the complexities of human biology. The human UDP-N-acetylglucosamine (UDPGlcNAc) pyrophosphorylases AGX1 and AGX2, which differ in sequence by an alternatively spliced 17 residue peptide, are key enzymes synthesizing UDPGlcNAc, an essential precursor for protein glycosylation. To better understand the catalytic mechanism of these enzymes and the role of the alternatively spliced segment, we have solved the crystal structures of AGX1 and AGX2 in complexes with UDPGlcNAc (at 1.9 and 2.4 A resolution, respectively) and UDPGalNAc (at 2.2 and 2.3 A resolution, respectively). Comparison with known structures classifies AGX1 and AGX2 as two new members of the SpsA-GnT I Core superfamily and, together with mutagenesis analysis, helps identify residues critical for catalysis. Most importantly, our combined structural and biochemical data provide evidence for a change in the oligomeric assembly accompanied by a significant modification of the active site architecture, a result suggesting that the two isoforms generated by alternative splicing may have distinct catalytic properties.

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Figures

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Fig. 1. Sequence alignment of pyrophosphorylases. The sequence alignment of AGX1 and AGX2 with other eukaryotic UAPs and two human UDPGlc Ppases (UDP1 and UDP2) is presented as well as that with GlmU, based on a structural comparison. Conserved and homologous residues between the eukaryotic enzymes are highlighted with a red and yellow background, respectively. The AGX secondary structure elements are shown above the sequences, with those forming the central core in yellow, and those part of the N- and C-terminal domains in green and blue, respectively. AGX residues involved in nucleotide-sugar binding are indicated by blue circles, blue triangles and a letter P for those contacting the nucleotide, the sugar and the phosphate moiety, respectively. The Ppase consensus sequence motif is boxed. Pink triangles identify residues buried at the dimer interface.
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Fig. 2. The nucleotide sugar-complexed structures of AGX1. (A) Ribbon representation of the structure of UDPGlcNAc-complexed AGX1, colour-coded as in Figure 1. UDPGlcNAc is shown in its final 2Fo – Fc electron density map (in cyan, contoured at 2σ) with white carbon, blue nitrogen, red oxygen and purple phosphorus atoms. The loop (NB loop) found in all Ppases is highlighted in red. The α12–β14 loop (I loop), which contains the site of insertion (indicated by a red arrow) of the AGX2 17 amino acid peptide, is shown in purple. (B) Stereo view of the active site of the AGX1–UDPGlcNAc complex structure colour-coded as in (A) with residues involved in nucleotide-sugar binding displayed with orange carbon atoms. The superimposed UDPGalNAc molecule from the AGX1–UDPGalNAc complex structure is shown in thin dark green sticks. Green dotted lines indicate hydrogen bonds. (C) Stereo view of the final 2Fo – Fc electron density map (in cyan, contoured at 2σ) of AGX1 in complex with UDPGalNAc (shown as in B). The positive and negative peaks in the Fo – Fc electron density map (–3.5σ in green and 3.5σ in red) clearly indicate the partial substitution of UDPGlcNAc (shown as in A) by UDPGalNAc in the crystal. Hydrogen bonds found between protein residues and the sugar-O4 in the equatorial (UDPGlcNAc) or axial (UDPGalNAc) conformations are indicated by green and orange dotted lines, respectively.
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Fig. 3. The AGX1 dimer. (A) Ribbon representation of the AGX1 dimer. Each subunit is coloured as in Figure 2A. One subunit is shown with a transparent molecular surface. The red arrows indicate the site of insertion of the 17 residue peptide in AGX2 in each subunit. (B) Stereo view of the AGX1 UDPGlcNAc-binding pocket of one subunit occluded by the I loop of the other subunit. I loop residues Lys455 and Arg453 (purple carbon atoms) are hydrogen-bonded to UDPGlcNAc phosphate groups. Arg115, which lies at the dimer interface, is shown under a transparent surface with orange carbon atoms. An asterisk indicates the site of insertion of the 17 residue peptide in AGX2. The protein surface corresponding to the central core and the N-terminal domains is coloured in beige and green, respectively.
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Fig. 4. Structural alignment of the central domain of AGX1 and the Ppase domain of GlmU. Ribbon representations of the superimposed structures of the UDPGlcNAc-complexed forms of AGX1 and EcGlmU, which aligned with an r.m.s.d. of 1.8 Å over 180 Cα. AGX1 is coloured as in Figure 2A whilst GlmU is shown in cyan with bound UDPGlcNAc in green thin sticks and the NB loop highlighted in green.
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