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==General Description==
==General Description==
<Structure load='4N20' size='450' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
<Structure load='4N20' size='450' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
'''''Protein Arginine Deiminase type 2 ''''' also known as '''''PAD2''''', is a calcium-dependent [https://en.wikipedia.org/wiki/Enzyme enzyme] that catalyzes in humans the conversion of [https://en.wikipedia.org/wiki/Arginine Arginine] residues into [https://en.wikipedia.org/wiki/Citrulline Citrulline] in a [https://en.wikipedia.org/wiki/Post-translational_modification post-translational modification] referred to as [https://en.wikipedia.org/wiki/Citrullination Citrullination]. The structure of '''''PAD2 Apoenzyme''''' described here was elucidated at a calcium concentration of 0mM (Ca2+) with a resolution of 1.657 Å by [https://en.wikipedia.org/wiki/X-ray_crystallography x-ray diffraction cristallography]<ref name="PDB">[https://www.rcsb.org/structure/4N20</ref> . The biological assembly of PAD2 consists of a head-to-tail dimer with immunoglobin-like domains and a nucleophilic [https://en.wikipedia.org/wiki/Cysteine cysteine] residue responsible of catalytic activity in the active site <ref name="ART1">DOI:10.1021/cb500933j</ref>. In humans, five genes clustered in a single locus code for Arginine deiminases: ''PADI1,PADI2,PADI3,PADI4,PADI6''<ref name="ARTFr">DOI:10.1051/medsci/201127149</ref>. Expression of different [https://en.wikipedia.org/wiki/Protein_isoform isoforms] of PAD seem to depend strongly on cell types and tissues even though PAD2 may be an ubiquist protein <ref name="ARTFr" />. Peptidyl Arginine Deiminase type 2 appears to have an essential role in the development of [https://en.wikipedia.org/wiki/Breast_cancer Breast Cancer] <ref name="ART1" />, [https://en.wikipedia.org/wiki/Multiple_sclerosis Multiple Sclerosis(MS)]<ref name="ARTMS">DOI:10.1051/medsci/201127149</ref> and other degenerative disorders such as [https://en.wikipedia.org/wiki/Rheumatoid_arthritis Rheumatoid Arthritis] <ref name="ARTFr" /> thus making it a potential target for inhibitor design treatments. Related structures for PAD2 include: [[4n2b]] and [[4n2c]].  
'''''Protein Arginine Deiminase type 2 ''''' also known as '''''PAD2''''', is a calcium-dependent [https://en.wikipedia.org/wiki/Enzyme enzyme] that catalyzes in humans the conversion of [https://en.wikipedia.org/wiki/Arginine Arginine] residues into [https://en.wikipedia.org/wiki/Citrulline Citrulline] in a [https://en.wikipedia.org/wiki/Post-translational_modification post-translational modification] referred to as [https://en.wikipedia.org/wiki/Citrullination Citrullination]. The structure of '''''PAD2 Apoenzyme''''' described here was elucidated at a calcium concentration of 0mM (Ca2+) with a resolution of 1.657 Å by [https://en.wikipedia.org/wiki/X-ray_crystallography x-ray diffraction cristallography]<ref name="PDB">[https://www.rcsb.org/structure/4N20</ref> . The biological assembly of PAD2 consists of a head-to-tail dimer with immunoglobin-like domains and a nucleophilic [https://en.wikipedia.org/wiki/Cysteine cysteine] residue responsible of catalytic activity in the active site <ref name="ART1">DOI:10.1021/cb500933j</ref>. In humans, five genes clustered in a single locus code for Arginine deiminases: ''PADI1,PADI2,PADI3,PADI4,PADI6''<ref name="ARTFr">DOI:10.1051/medsci/201127149</ref>. Expression of different [https://en.wikipedia.org/wiki/Protein_isoform isoforms] of PAD seem to depend strongly on cell types and tissues even though PAD2 may be an ubiquist protein <ref name="ARTFr" />. Peptidyl Arginine Deiminase type 2 appears to have an essential role in the development of [https://en.wikipedia.org/wiki/Breast_cancer Breast Cancer] <ref name="ART1" />, [https://en.wikipedia.org/wiki/Multiple_sclerosis Multiple Sclerosis(MS)]<ref name="ARTMS">DOI:10.1051/medsci/201127149</ref> and other degenerative disorders such as [https://en.wikipedia.org/wiki/Rheumatoid_arthritis Rheumatoid Arthritis] <ref name="ARTFr" /> thus making it a potential target for inhibitor design treatments. Related structures for PAD2 include: [[4n2b]] and [[4n2c]].
 
==Structural Features==
==Structural Features==
==='''Primary, secondary and tertiary structure'''===
==='''Primary, secondary and tertiary structure'''===
==='''Calcium binding sites and active site'''===
The structure of the '''''apoenzyme''''' '''''apoPAD2''''' shows a stable head to tail dimer.The monomer is formed by 2 immunoglobulin-like domains and a C-term catalytic domain calcium binding site. There are six different calcium binding sites '''''C1 to C6''''' C2-5 are unoccupied in apoPAD2 but there is an electron density on C1 and C6 so those are occupied by calcium.
The structure of the '''''PAD2.Ca2+ complex''''' in 10mM of CaCl2 differs from the apoPAD2 one's. There is the same folding except C3-5 are occupied. Even though just one site is unoccupied, the structure is not catalytically competent, it is explained by the active site nucleophile C647, indeed, it is just 12 angstrom away from the catalytic center. Yet, the 3 other key catalytic residues, D351, H471, 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+ represent an intermediaire between the 2 structures. This intermediaire structure is stabilized thanks to hydrogen bonds between R347 and Q350 who are in the active site. It also inhibits the movement of C647 who is in the substrate binding pocket.
To obtain the structure of the holoenzyme '''''PAD2''''', the scientists had engineer a double mutant, because the F221 and F222 binds in a hydrophobic pocket, this prevents the calcium-binding at Ca2.
So they studied the structure of the PAD2 F221/222A.Ca2+ mutant. It shows an important electron density at all 6 calcium binding sites. All sites are now binding with calcium. Moreover, The active site cysteine, C647 points toward the catalytic center. this conformation is competent for catalysis. It is not the only effect of calcium binding to Ca2 site, it also causes R347 to move out of the active site while W348 moves in to form one wall of the substrate-binding pocket.
==='''Catalysis of deimination'''===


== Citrullination of Arginine residues ==
In humans, PAD2 is involved in a type of post-translational modification called citrullination. Indeed, this calcium-dependent enzyme catalyzes a deimination reaction : PAD2 uses one molecule of water to replace the terminal nitrogen of Arginine by an oxygen and a ketone group is formed in place of a ketimine one.
The transformation of arginyl residues ([https://fr.wikipedia.org/wiki/Arginine Arginine]), which are positively charged, into citrullyl residues, which are neutral, leads to the '''''modification of the global charge''''' of the targeted protein. These residue modifications are the result of hydrolysation of guanidinium groups in side chains and may produce important conformational changes in proteins by increasing the hydrophobicity. The catalytic residue which allows hydrolyzation of the guanidium group is a '''''nucleophilic cysteine in position 647'''''. The proper positioning of C647 is due to the binding of calcium ions to PAD 2, essential for deamination reaction.
== Role in Human Health ==
==='''Citrullination of Myelin Basic Protein (MBP) and Multiple Sclerosis'''===
Two main types of Arginine Deiminases are extensively expressed in the [https://en.wikipedia.org/wiki/Central_nervous_system Central Nervous System (CNS)]:PAD2 and PAD4<ref name="ARTMS"/>. PAD2, mainly produced by oligodendrocytes was found to be implicated in pathological citrullination of [https://en.wikipedia.org/wiki/Myelin_basic_protein Myelin Basic Protein (MBP)]. MBP along with oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP) play an essential role in [https://en.wikipedia.org/wiki/Myelin myelin sheath] stabilization by maintaining adequate compaction and adhesion between axonal cytoplasmic surfaces and negatively charged lipids of [https://en.wikipedia.org/wiki/Myelin myelin sheath]. [https://en.wikipedia.org/wiki/Citrullination Citrullination] of MBP is thought to be a naturally occuring post-translational modification of the immature CNS<ref name="ARTMS" /><ref>PMID: 7518827</ref>, for instance around 20% of isolated MBP is citrullinated in normal human adults whereas an average of 45% citrullinated MBP was detected in chronic MS patients and up to 80% for fulminating MS (Marburg's Syndrome)<ref>PMID: 7518827</ref><ref>PMID: 8687186</ref>. Given that hyper-citrullination of MBP marks important stages in the development of the CNS, MBP's citrullination by PAD2 in MS patients suggests a switch to immaturity as a repair mechanism for neurological damage<ref name="ARTMS" />. This switch to immaturity could however promote or initiate pathological effects in MS.
Numerous post-translational modifications in myelin sheath-related proteins can alter folding and 3D configuration of polypeptides thus modifying functional and structural properties of the tissue <ref>PMID: 22420465</ref>. These modifications may naturally occur as regulatory processes in cells but, in the case of MBP's citrullination, they may promote/initiate pathological states<ref name="ARTMS" />. Two main consequences regarding Myelin Basic Protein citrullinaton have been proposed: 1) Change of arginine residues to citrulline by PADs could trigger the generation of [https://en.wikipedia.org/wiki/Neoepitope neo-epitopes] for which no tolerance exists; 2) Citrullination may induce MBP's misfolding exposing [https://en.wikipedia.org/wiki/Immunodominance immunodominant] [https://en.wikipedia.org/wiki/Epitope epitopes]<ref name="ARTMS"/>. Both effects result in auto-immune responses towards CNS tissues in the first case due to the newly generated neo-epitopes that trigger 'new' immunological responses. In the second case, pertubation of internal electrostatic interations within MBP as a consequence of citrullination may generate misfolded versions of the protein in the myelin sheath that were proven to be more extended<ref>PMID: 12898292</ref>. These newly generated versions of MBP enhance the exposure of a central-membrane-binding fragment that represents a primary immunodominant epitope in the [https://en.wikipedia.org/wiki/Cytoplasm cytoplasm] and is thus its [https://en.wikipedia.org/wiki/Proteolysis proteolysis] is capable of generating more immunodominant species that trigger immunological responses<ref>PMID:16537438</ref><ref>PMID: 17531841</ref>. Modification of MBP's functional properties may have a direct impact in adhesion and compaction of the myelin sheath promoting demyelination in Multiple Sclerosis. Related structures of immunological associations with Myelin Basic Protein have been studied in: [[1k2d]] and [[1bx2]].
 
==='''PAD2 and ER Target-gene Expression in Breast Cancer'''===
PAD2 functions as an [https://en.wikipedia.org/wiki/Estrogen_receptor Estrogen Receptor] (ER) coactivator in [https://en.wikipedia.org/wiki/Breast_cancer Breast cancer] cells, using the citrullination of histone tail arginine residues at ER binding sites. This makes it an attractive therapeutic target, yet, the mechanisms that regulates it are for a major part unknown. Indeed, it is used as a component of ER-related gene expression that is positively correlated with [https://proteopedia.org/wiki/index.php/Herceptin_-_Mechanism_of_Action HER2] protein levels in breast cancer cell lines, and in primary HER2+ breast tumors<ref>PMID:25621824<ref/>. This shows that PAD2 activity plays a role in breast cancer progression. The inhibition of PAD2 decreases ER target-gene expression. Moreover, when in presence of CL-amidine, which is PAD inhibitor, the tumor burden in a breast cancer.
='''Human Peptidylarginine Deiminase Type 2''' =
==General Description==
<Structure load='4N20' size='450' frame='true' align='right' caption='Insert caption here' scene='Insert optional scene name here' />
'''''Protein Arginine Deiminase type 2 ''''' also known as '''''PAD2''''', is a calcium-dependent [https://en.wikipedia.org/wiki/Enzyme enzyme] that catalyzes in humans the conversion of [https://en.wikipedia.org/wiki/Arginine Arginine] residues into [https://en.wikipedia.org/wiki/Citrulline Citrulline] in a [https://en.wikipedia.org/wiki/Post-translational_modification post-translational modification] referred to as [https://en.wikipedia.org/wiki/Citrullination Citrullination]. The structure of '''''PAD2 Apoenzyme''''' described here was elucidated at a calcium concentration of 0mM (Ca2+) with a resolution of 1.657 Å by [https://en.wikipedia.org/wiki/X-ray_crystallography x-ray diffraction cristallography]<ref name="PDB">[https://www.rcsb.org/structure/4N20</ref> . The biological assembly of PAD2 consists of a head-to-tail dimer with immunoglobin-like domains and a nucleophilic [https://en.wikipedia.org/wiki/Cysteine cysteine] residue responsible of catalytic activity in the active site <ref name="ART1">DOI:10.1021/cb500933j</ref>. In humans, five genes clustered in a single locus code for Arginine deiminases: ''PADI1,PADI2,PADI3,PADI4,PADI6''<ref name="ARTFr">DOI:10.1051/medsci/201127149</ref>. Expression of different [https://en.wikipedia.org/wiki/Protein_isoform isoforms] of PAD seem to depend strongly on cell types and tissues even though PAD2 may be an ubiquist protein <ref name="ARTFr" />. Peptidyl Arginine Deiminase type 2 appears to have an essential role in the development of [https://en.wikipedia.org/wiki/Breast_cancer Breast Cancer] <ref name="ART1" />, [https://en.wikipedia.org/wiki/Multiple_sclerosis Multiple Sclerosis(MS)]<ref name="ARTMS">DOI:10.1051/medsci/201127149</ref> and other degenerative disorders such as [https://en.wikipedia.org/wiki/Rheumatoid_arthritis Rheumatoid Arthritis] <ref name="ARTFr" /> thus making it a potential target for inhibitor design treatments. Related structures for PAD2 include: [[4n2b]] and [[4n2c]].   
==Structural Features==
==='''Primary, secondary and tertiary structure'''===
==='''Calcium binding sites and active site'''===
==='''Calcium binding sites and active site'''===
The structure of the '''''apoenzyme''''' '''''apoPAD2''''' shows a stable head to tail dimer.The monomer is formed by 2 immunoglobulin-like domains and a C-term catalytic domain calcium binding site. There are six different calcium binding sites '''''C1 to C6''''' C2-5 are unoccupied in apoPAD2 but there is an electron density on C1 and C6 so those are occupied by calcium.
The structure of the '''''apoenzyme''''' '''''apoPAD2''''' shows a stable head to tail dimer.The monomer is formed by 2 immunoglobulin-like domains and a C-term catalytic domain calcium binding site. There are six different calcium binding sites '''''C1 to C6''''' C2-5 are unoccupied in apoPAD2 but there is an electron density on C1 and C6 so those are occupied by calcium.
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==='''PAD2 and ER Target-gene Expression in Breast Cancer'''===
==='''PAD2 and ER Target-gene Expression in Breast Cancer'''===
PAD2 functions as an [https://en.wikipedia.org/wiki/Estrogen_receptor Estrogen Receptor] (ER) coactivator in [https://en.wikipedia.org/wiki/Breast_cancer Breast cancer] cells, using the citrullination of histone tail arginine residues at ER binding sites. This makes it an attractive therapeutic target, yet, the mechanisms that regulates it are for a major part unknown. Indeed, it is used as a component of ER-related gene expression that is positively correlated with [https://proteopedia.org/wiki/index.php/Herceptin_-_Mechanism_of_Action HER2] protein levels in breast cancer cell lines, and in primary HER2+ breast tumors <ref>PMID:25621824<ref/>. This shows that PAD2 activity plays a role in breast cancer progression. The inhibition of PAD2 decreases ER target-gene expression. Moreover, when in presence of CL-amidine, which is PAD inhibitor, the tumor burden in a breast cancer.
PAD2 functions as an [https://en.wikipedia.org/wiki/Estrogen_receptor Estrogen Receptor] (ER) coactivator in [https://en.wikipedia.org/wiki/Breast_cancer Breast cancer] cells, using the citrullination of histone tail arginine residues at ER binding sites. This makes it an attractive therapeutic target, yet, the mechanisms that regulates it are for a major part unknown. Indeed, it is used as a component of ER-related gene expression that is positively correlated with [https://proteopedia.org/wiki/index.php/Herceptin_-_Mechanism_of_Action HER2] protein levels in breast cancer cell lines, and in primary HER2+ breast tumors. This shows that PAD2 activity plays a role in breast cancer progression. The inhibition of PAD2 decreases ER target-gene expression. Moreover, when in presence of CL-amidine, which is PAD inhibitor, the tumor burden in a breast cancer.


==Publication Abstract from ACS Publications <ref name="ART1" />==
==Publication Abstract from ACS Publications <ref name="ART1" />==
Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.
Protein arginine deiminases (PADs) are calcium-dependent histone-modifying enzymes whose activity is dysregulated in inflammatory diseases and cancer. PAD2 functions as an Estrogen Receptor (ER) coactivator in breast cancer cells via the citrullination of histone tail arginine residues at ER binding sites. Although an attractive therapeutic target, the mechanisms that regulate PAD2 activity are largely unknown, especially the detailed role of how calcium facilitates enzyme activation. To gain insights into these regulatory processes, we determined the first structures of PAD2 (27 in total), and through calcium-titrations by X-ray crystallography, determined the order of binding and affinity for the six calcium ions that bind and activate this enzyme. These structures also identified several PAD2 regulatory elements, including a calcium switch that controls proper positioning of the catalytic cysteine residue, and a novel active site shielding mechanism. Additional biochemical and mass-spectrometry-based hydrogen/deuterium exchange studies support these structural findings. The identification of multiple intermediate calcium-bound structures along the PAD2 activation pathway provides critical insights that will aid the development of allosteric inhibitors targeting the PADs.


== References ==
==References==
<references/>
<references/>
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