Data collection, data processing, and structure determination
Data collection was carried out at beamline 21ID-D of LS-CAT at the
Advanced Photon Source (Argonne National Laboratory). Data were
processed, integrated, and scaled with the programs Mosflm and Scala in
the CCP4 package [52]. Phasing was achieved by using the molecular
replacement method, which was carried out by using the program Phaser
[53] that is implemented in the PHENIX package [54]. The
monomeric structure of the E. coli PPase (PDB code 4um4) was used as a
search model. This structure is chosen as a search model because All
plausible space groups of the P222 family were tried. A single solution
was found in the space group P212121. Interactive model building was
carried out with Coot [55]. Structure refinement was carried out by
PHENIX. Structure determination statistics are shown in Table 1. The
program UCSF Chimera [56] was used to generate all of the molecular
graphic figures. Figures for electrostatic surface rendering were
generated by Chimera using the Coulombic Surface Coloring tool with the
default settings.
Sequence alignments of the eukaryotic soluble PPases with known
structures were carried out by Clustal Omega [57]. Molecular
interactions at the PPA1 homodimerization interface were depicted by
using the program LIGPLOT [58]. Atomic coordinates and diffracÂtion
data have been deposited in the Protein Data Bank with accession code
6C45.
Structural modelling of PPA1 with
various putative
substrates
The determined crystal structure of human PPA1 does not contain
substrate at the putative active site. To gain insights into the
possible catalytic mechanism of human PPA1, modelling studies were
carried out to place various putative substrates at the active site. At
first, a pyrophosphate molecule was docked into the active site by using
AutoDock Vina [59]. Chimera was used to prepare the PPA1 receptor
structure for docking and to visualize the docking results. To explore
whether the PPA1 active site is capable of accommodating peptide
substrates containing phosphorylated tyrosine and/or phosphorylated
threonine residues, a series of phosphotyrosine and/or phosphothreonine
containing peptides were modelled into the PPA1 active site by using
Chimera (including energy minimization of the bound peptide at the
active site). The phosphotyrosine and/or phosphothreonine containing
peptides were initially derived from the phosphorylated MAP kinase Erk2
(PDB code 2erk) [60]. Two Erk2-derived tetrapeptides were used:
184-EYpVA (superscript p indicates phosphotyrosine) and 181-FLTpE
(superscript p indicates phosphothreonine), to model a Yp and a Tp at
the active site respectively. To prepare peptides corresponding to the
JNK1 sequence for modelling studies, the 181-FLTpEYpV sequence in the
Erk2 structure were mutated to JNK1 sequence 181-MMTpPYpV by Chimera. A
pentapeptide 182-MTpPYpV was used to model the Yp at the active site. A
hexapeptide 181-MMTpPYpV was used to model the Tp at the active site.