Sandbox Reserved 1096: Difference between revisions

The structure of the '''''PAD2 Ca2+ complex''''' in 10mM of CaCl2 differs from apoPAD2. Folding and 3D structure remain unchanged except C3-5 are occupied by Calcium cations when Ca2+ increases<ref name="ART1" />. The ''''apoPAD2'''' enzyme resolved structure shows 2 high-affinity Calcium binding sites that remain consistent. At 10mM Ca3-5 sites are occupied by Calcium cations and, even though there is still only one unoccupied site, the structure is not catalytically competent. This may be explained by the nucleophile active site C647 that is still 12 angstrom away from the catalytic center at 10mM. Yet, the 3 other key catalytic residues: D351, H471 and D473 are properly positioned to promote catalysis and, since they have the same conformation in both the apoenzyme and the holoenzyme, the complex structure PAD2+/Ca2+ represents an intermediate form between the 2 structures. This intermediate structure is stabilized by hydrogen bonds between R347 and Q350 in the active site. These bonds may also inhibit the movement of C647 into the substrate binding pocket<ref name="ART1" /> .  

To obtain the structure of '''''PAD2 holoenzyme''''', a double mutant F221/F222A was engineered to prevent undesired interactions with neighboring hydrophobic pockets that could prevent binding of Calcium at Ca2 site. Structure of PAD2 F221/222A Ca2+ mutant was solved and studied showing an important electron density at all 6 calcium binding sites at 10mM Ca2+. All sites are now binding with calcium. Moreover, The active site cysteine, C647 points toward the catalytic center making the holoenzyme competent for catalysis. Calcium binding to Ca2 site was also found to cause R347 to move out of the active site while W348 moves in to form one wall of the substrate-binding pocket.  

PAD2 is a calcium-dependent enzyme which catalyzes the deimination of [https://en.wikipedia.org/wiki/Arginine Arginine] residues. This reaction occurs only if calcium ions bind to specific sites of PAD2 but they do not directly participate in catalysis : they act as cofactors. Once Calcium bonding is accomplished, a '''''catalytic cysteine residue in position 647''''' in the peptide chain changes its position to carry out a [https://en.wikipedia.org/wiki/Nucleophilic_substitution nucleophilic attack] on guanidium groups of arginine residues. In terms of specific catalysis at the active site of PAD2, four residues are essential for the progress of citrullination: D351, H471, D473 and C647<ref name="ART1" /><ref name="ARTC">DOI:10.1007/s00214-012-1293-9</ref>.  These arginine residues that are substrates in this reaction can bind to PAD2 because calcium binding engenders a move out of the active site for an arginin in position 347 and a move in for a tryptophan in position 348, in order to form a pocket for the substrate<ref name="ART1" />.