Download SUPPLEMENTARY INFORMATION FOR: The structure of a D

SUPPLEMENTARY INFORMATION FOR:
The structure of a D-lyxose isomerase from the σB regulon of Bacillus subtilis
Jon Marles-Wright, Richard J. Lewis*
Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place,
Newcastle upon Tyne, NE2 4HH.
Supplementary Results and Discussion
Comparison to E. coli z5688 and other cupins
YdaE and its near structural neighbour, E. coli z5688, belong to a class of proteins
known as the cupin phospho-glucose isomerases (cPGIs), which also includes the cPGI from
Thermococcus litoralis (unpublished, PDBid 1J3P). This protein has 17 % sequence identity
to YdaE and aligns over 103 residues giving an RMSD of 1.8 Å. These proteins, and cupins
in general, show relatively low sequence identity, yet the overall fold of the cupin barrel is
very well maintained1 (Fig. S2B/C). Further examples of this conservation of structure over
sequence are seen in the manganese binding protein MncA2 from Synechocystis P6803
(PDBid 2VQA), which shows only 14 % sequence identity to YdaE, yet superimposes over
103 residues with an RMSD of 1.6 Å; similarly the copper binding cupin from the same
bacterium CucA2 (PDBid 2XL7) superimposes with YdaE over 104 residues and an RMSD
of 2.0 Å. These proteins all share the common metal coordination residues, which direct
protein folding around the metal ion. This reinforces the observation that in the cupin family
of proteins have evolved to maintain a high degree of structure conservation, despite
extensive sequence variation and distant evolutionary relationships between different classes
of these proteins1.
The active site of YdaE is essentially identical to that of the E. coli z5688 protein (Fig.
S2A), with the residues coordinating the metal ion and forming the active cleft almost
completely conserved between the two proteins (Fig. S2D). The main residues interacting
with the sugar ligand in z5688 including the glutamic acid residues E110 and E186, lysines
K90 and 108 and arginine R205, are strictly conserved. Two residues differ in this site, F84
which is replaced by an isoleucine and Y32 which is a phenylalanine in z5688. The first
substitution is relatively conservative with the hydrophobic character of the residue
maintained. The hydroxyl of tyrosine 32 in YdaE forms a hydrogen bond to the water
molecule that sits in the position of the O6 of the fructose in the z5688 structure and may act
to stabilise the position of the sugar in the active site. The similarity in the active site would
support the hypothesis that the two proteins have the same ligands and activity in vitro. The
major differences seen between the two proteins are extensions of the cupin fold in z5688
that are absent in YdaE. A stretch of three helices (α2,3,4) and two beta-strands (β7,8) form
an addition to the dimerisation interface of z5688 and contribute the majority of the extra 400
Å2 of buried surface area. These regions are 12 Å from the active site of z5688 at their closest
and are unlikely to significantly influence the substrate binding channel (Fig. S2B/C).
Supplementary References
1.
Dunwell JM, Purvis A, Khuri S. Cupins: The most functionally diverse protein
superfamily? Phytochemistry 2004;65:7-17.
2.
Tottey S, Waldron KJ, Firbank SJ, Reale B, Bessant C, Sato K, Cheek TR, Gray J,
Banfield MJ, Dennison C, Robinson NJ. Protein-folding location can regulate
manganese-binding versus copper- or zinc-binding. Nature 2008;455:1138-1142.
3.
Evans G, Pettifer RF. Chooch: A program for deriving anomalous-scattering factors
from x-ray fluorescence spectra. Journal of Applied Crystallography 2001;34:82-86.
Supplementary Figures
Figure S1. Calculated plots of the anomalous scattering factors, f’ and f’’, for data
collected on a single YdaE crystal and corresponding anomalous difference electron
density maps.
Plots were calculated using Chooch3 with X-ray fluorescence data collected at energies
around the K-edges for arsenic (A1) and zinc (B1). Corresponding anomalous difference
electron density figures are shown for the phased maps at each of the anomalous wavelengths
and contoured at 6σ (A2, B2) and 14σ (A3). These are shown for the metal binding region of
the protein, cyan sticks, with the modelled zinc ion shown in grey and the arsenic in purple,
with two waters shown as red spheres. At the arsenic K-edge, zinc has an f” of 2.7 electrons,
which is about 70 % of arsenic at this wavelength. A peak in the anomalous map calculated
at this wavelength (A2) corresponds to the modelled position of the zinc ion. This peak has
approximately half the height of the peak that corresponds to the modelled arsenic ion (A3).
By contrast, arsenic has no anomalous signal at the zinc K-edge and as a consequence there is
no peak in the zinc anomalous map corresponding to the modelled arsenic ion (B2).
Figure S2. Comparison of the structure of B. subtilis YdaE with other cPGIs.
(A) Comparison of the active site of YdaE and z5688, drawn in wall-eyed stereo; an overlay
of the two structures shows that the active sites are highly similar in these two proteins. The
bound fructose in z5688 is seen at the centre of the image.
(C) and (B) Overlay of B. subtilis YdaE (blue), E. coli z5688 (magenta) and the T. Litoralis
cPGI (green, PDBid 1J3P). The core cupin fold is conserved between all three proteins, with
a number of peripheral extensions unique to each protein.
(D) Structure based alignment of YdaE and z5688. In spite of low overall sequence identity
of 32%, the two proteins align well over the core of the cupin barrel, and residues in the
active site almost completely conserved. Conserved residues are highlighted red, metal coordinating residues are highlighted with stars, conserved active site residues are highlighted
with triangles and dimer interface residues are highlighted with circles.