Structure and dynamics of UDP-glucose pyrophosphorylase from Arabidopsis thaliana with bound UDP-glucose and UTP

Abstract

The structure of the UDP-glucose pyrophosphorylase encoded by Arabidopsis thaliana gene At3g03250 has been solved to a nominal resolution of 1.86 Angstroms. In addition, the structure has been solved in the presence of the substrates/products UTP and UDP-glucose to nominal resolutions of 1.64 Angstroms and 1.85 Angstroms. The three structures revealed a catalytic domain similar to that of other nucleotidyl-glucose pyrophosphorylases with a carboxy-terminal beta-helix domain in a unique orientation. Conformational changes are observed between the native and substrate-bound complexes. The nucleotide-binding loop and the carboxy-terminal domain, including the suspected catalytically important Lys360, move in and out of the active site in a concerted fashion. TLS refinement was employed initially to model conformational heterogeneity in the UDP-glucose complex followed by the use of multiconformer refinement for the entire molecule. Normal mode analysis generated atomic displacement predictions in good agreement in magnitude and direction with the observed conformational changes and anisotropic displacement parameters generated by TLS refinement. The structures and the observed dynamic changes provide insight into the ordered mechanism of this enzyme and previously described oligomerization effects on catalytic activity.

Figure 1

Fold of UDPGP. The substrate complexes (2icy/2icx) are used to illustrate the location of the active site and UDP-glucose/UTP is shown as orange stick model.

1. Ribbon diagram of UDPGP monomer. Domains are color coded as follows: central domain (blue), carboxy-terminal domain (red), amino-terminal domain (magenta), sugar binding domain (green).

2. Electrostatic surface diagram illustrating the positively charged substrate cavity.

3. Apparent UDPGP dimer. The amino-terminal domain of each monomer resides near the active site of the other respective monomer.

Figure 1

Fold of UDPGP. The substrate complexes (2icy/2icx) are used to illustrate the location of the active site and UDP-glucose/UTP is shown as orange stick model.

1. Ribbon diagram of UDPGP monomer. Domains are color coded as follows: central domain (blue), carboxy-terminal domain (red), amino-terminal domain (magenta), sugar binding domain (green).

2. Electrostatic surface diagram illustrating the positively charged substrate cavity.

3. Apparent UDPGP dimer. The amino-terminal domain of each monomer resides near the active site of the other respective monomer.

Figure 1

Fold of UDPGP. The substrate complexes (2icy/2icx) are used to illustrate the location of the active site and UDP-glucose/UTP is shown as orange stick model.

1. Ribbon diagram of UDPGP monomer. Domains are color coded as follows: central domain (blue), carboxy-terminal domain (red), amino-terminal domain (magenta), sugar binding domain (green).

2. Electrostatic surface diagram illustrating the positively charged substrate cavity.

3. Apparent UDPGP dimer. The amino-terminal domain of each monomer resides near the active site of the other respective monomer.

Figure 2

Residue numbering throughout UDPGP. The ribbon color corresponds to the residue number starting with blue for the amino-terminal and ending in red for the carboxy-terminal. Every tenth alpha carbon is shown as a sphere.

Figure 3

The active site of the UDPGP enzyme. Residues are colored according to the structural domain they belong in (central domain - blue, carboxy-terminal domain - red, amino-terminal domain - magenta, sugar binding domain - green). UDP-glucose and UTP are shown as an orange stick model.

1. UDP-glucose complex (2icy).

2. UTP complex (2icx).

Figure 4

Overlay of the native (magenta), UTP complex (cyan), and the UDP-glucose complex (green). The open and closed forms of the sugar binding and nucleotide binding loops are illustrated and the residues shown in Figure 3 are displayed as stick models. UDP-glucose and UTP are shown as orange and blue stick models respectively.

Figure 5

Displacement models

1. Motion in the Arabidopsis UDPGP as predicted by normal mode analysis. Displacement vectors are represented by red arrows. Trajectories were obtained from model 1 chain A of the UDP-glucose complex (2icy).

2. Plot of normalized positional variance for UDPGP from the B-factors in the final refinement (blue), TLS refinement (red), Normal Mode Analysis model (black) and observed atomic displacements (dashed magenta). The correlation coefficient is 0.76 between TLS and B-factors, 0.81 between TLS and NMA, 0.56 between NMA and B-factors, 0.58 between TLS and observed atomic displacements, and 0.34 between B-factors and observed atomic displacements.

3. Matrix of correlations in positional deviation for UDPGP computed from 100 modes of NMA. Red indicates a positive correlation between residues, blue indicated anti-correlation, and green indicates lack of correlation. Arrows point to strong correlation between the nucleotide binding loop (residues 87–100) and residues around Lys360 of the carboxy-terminal domain.

Figure 6

Differences between human AGX1 and Arabidopsis UDPGP.

1. The AGX1 dimer.

2. Overlay of the AGX1 (magenta) and UDPGP (cyan) monomers. The β-helix of UDPGP prevents dimerization similar to that of AGX1.