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This Sandbox is Reserved from 25/11/2019, through 30/9/2020 for use in the course "Structural Biology" taught by Bruno Kieffer at the University of Strasbourg, ESBS. This reservation includes Sandbox Reserved 1091 through Sandbox Reserved 1115.
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Contents

Human Peptidylarginine Deiminase Type 2

General Description

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Protein Arginine Deiminase type 2 also known as PAD2, is a calcium-dependent enzyme that catalyses in humans the conversion of Arginine residues into Citrulline in a post-translational modification referred to as 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 x-ray diffraction crystallography[1] . The biological assembly of PAD2 consists of a head-to-tail dimer with and a nucleophilic cysteine residue responsible of catalytic activity in the [2]. In humans, five genes clustered in a single locus code for Arginine deiminases: PADI1,PADI2,PADI3,PADI4,PADI6[3]. Expression of different isoforms of PAD seem to depend strongly on cell types and tissues even though PAD2 may be an ubiquist protein [3]. Peptidyl Arginine Deiminase type 2 appears to have an essential role in the development of Breast Cancer [2], Multiple Sclerosis(MS)[4] and other degenerative disorders such as Rheumatoid Arthritis [3] 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

In terms of primary structure, the apoenzyme described under PDB's 4N20 contains 4 key catalytic residues that are essential for citrullination in the (, D351, H471 and D473). The cysteine residue is involved in nucleophilic attacks that promote deimination of Arginine residues whereas the other residues are mainly responsible for stabilization of substrates entering the [2]. Other key residues include D125, D127, E131 and E354 which may play an important role in Calcium binding for sites and given that side-chains are around 5Å away from Calcium cations. Considering both Aspartate and Glutamate are negatively charged at pH 7, electrostatic interactions may exist between these residues and Ca2+ in binding sites. In the other hand, the structure of apoPAD2 shows some common secondary motifs such as superimposed anti-parallel beta sheets that generate beta sandwiches. Closer to the C-terminal, both alpha and beta secondary structures may be observed with 17 alpha helixes and multiple parallel and anti-parallel beta sheets that make up an alpha/beta propeller[2]. Tertiary structure of apoPAD2 monomer is well described by Slade et al. as being composed of three distinct domains: ( and ) at positions (1-115)(116-295) respectively and a at (296-665) that contains the . Both comprise typical beta-sandwiches and the generates an alpha/beta propeller. Finally, with respect to the quaternary structure, PAD2 Apoenzyme crystallises as a stable dimer of identical subunits (monomers). The biological assembly of the enzyme was thus observed to be an association of two monomers whose structural highlights were described above.

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 ( to ), Ca2-5 are unoccupied in apoPAD2 but there is an electron density on and so those are occupied by calcium in the apoenzyme.

The structure of the PAD2 Ca2+ complex in 10mM of CaCl2 (4n2b) differs from apoPAD2. Folding and 3D structure remain unchanged except Ca3-5 are occupied by Calcium cations when Ca2+ increases[2]. The apoPAD2 enzyme resolved structure shows 2 high-affinity Calcium binding sites ( and ) 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 that is still 12 angstroms away from the catalytic centre 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 into the substrate binding pocket[2] .

To obtain the structure of PAD2 holoenzyme (4n2c), a double mutant F221/F222A was engineered to prevent undesired interactions with neighbouring 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, points toward the catalytic centre 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.

Main transformations of apo and holo-enzymes that explain PAD2's activity. Obtained from https://doi.org/10.1021/cb500933j for educational use
Main transformations of apo and holo-enzymes that explain PAD2's activity. Obtained from https://doi.org/10.1021/cb500933j for educational use


Catalysis of deimination

PAD2 is a calcium-dependent enzyme which catalyses the deimination of 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 binding is accomplished, a catalytic cysteine residue in position 647 in the peptide chain changes its position to carry out a nucleophilic attack on guanidinium groups of arginine residues. In terms of specific catalysis at the active site of PAD2, four residues are essential for the progress of citrullination: and [2][5]. Histidine 471 and Cysteine 647 are directly involved in deimination catalysis while Aspartates 351 and 473 facilitate the reaction by interacting with Arginine residues (substrates) holding them in place inside the active site[5]. A mechanism of reaction has been proposed in which the nucleophilic attack of thiolate in C647 generates the cleavage of a carbon-nitrogen bond to form Ammonia. A water molecule will then replace ammonia and, after hydrolysis of thiouronium intermediate, citrulline product is formed and the enzyme's active site is regenerated[5]. Arginine residue substrates can bind to PAD2 because calcium binding engenders a move out of the active site for an arginine in position 347 and a move in for a tryptophan in position 348 in order to form a pocket for the substrate[2].

Citrullination of Arginine residues

In humans, PAD2 is involved in a type of post-translational modification called citrullination. Indeed, this calcium-dependent enzyme catalyses 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 [2].

Deimination of Arginine into Citrulline
Deimination of Arginine into Citrulline

The transformation of arginyl residues (Arginine), which are positively charged at a neutral pH, 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 whose catalysis has been explained previously. As a result, the deimination reaction catalysed by PAD2 may produce important conformational changes in proteins by increasing the hydrophobicity.

Role in Human Health

Citrullination of Myelin Basic Protein (MBP) and Multiple Sclerosis

Two main types of Arginine Deiminases are extensively expressed in the Central Nervous System (CNS):PAD2 and PAD4[4]. PAD2, mainly produced by oligodendrocytes was found to be implicated in pathological citrullination of Myelin Basic Protein (MBP). MBP along with oligodendrocyte glycoprotein (MOG) and proteolipid protein (PLP) play an essential role in myelin sheath stabilization by maintaining adequate compaction and adhesion between axonal cytoplasmic surfaces and negatively charged lipids of myelin sheath. Citrullination of MBP is thought to be a naturally occurring post-translational modification of the immature CNS[4][6], 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)[6][7]. 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[4]. 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 [8]. These modifications may naturally occur as regulatory processes in cells but, in the case of MBP's citrullination, they may promote/initiate pathological states[4]. 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 neo-epitopes for which no tolerance exists; 2) Citrullination may induce MBP's misfolding exposing immunodominant epitopes[4]. 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, perturbation of internal electrostatic interactions 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[9]. These newly generated versions of MBP enhance the exposure of a central-membrane-binding fragment that represents a primary immunodominant epitope in the cytoplasm and is thus its proteolysis is capable of generating more immunodominant species that trigger immunological responses[10][11]. 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 Estrogen Receptor (ER) coactivator in Breast cancer cells, using the citrullination of histone tail arginine residues at ER binding sites. This makes it an attractive therapeutic target, yet, the regulatory mechanisms are mainly unknown. Indeed, it is used as a component of ER-related gene expression that is positively correlated with HER2 protein levels in breast cancer cell lines, and in primary HER2+ breast tumours[2]. This shows that PAD2 activity plays a role in breast cancer progression. The inhibition of PAD2 decreases ER target-gene expression. Moreover, in presence of CL-amidine, which is PAD inhibitor, the tumour burden decreases in breast cancer.

Publication Abstract from ACS Publications [2]

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

  1. [https://www.rcsb.org/structure/4N20
  2. 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 Slade DJ, Fang P, Dreyton CJ, Zhang Y, Fuhrmann J, Rempel D, Bax BD, Coonrod SA, Lewis HD, Guo M, Gross ML, Thompson PR. Protein Arginine Deiminase 2 Binds Calcium in an Ordered Fashion: Implications for Inhibitor Design. ACS Chem Biol. 2015 Jan 26. PMID:25621824 doi:http://dx.doi.org/10.1021/cb500933j
  3. 3.0 3.1 3.2 Mechin MC, Nachat R, Coudane F, Adoue V, Arnaud J, Serre G, Simon M. [Deimination or citrullination, a post-translational modification with many physiological and pathophysiological facets]. Med Sci (Paris). 2011 Jan;27(1):49-54. doi: 10.1051/medsci/201127149. PMID:21299962 doi:http://dx.doi.org/10.1051/medsci/201127149
  4. 4.0 4.1 4.2 4.3 4.4 4.5 Mechin MC, Nachat R, Coudane F, Adoue V, Arnaud J, Serre G, Simon M. [Deimination or citrullination, a post-translational modification with many physiological and pathophysiological facets]. Med Sci (Paris). 2011 Jan;27(1):49-54. doi: 10.1051/medsci/201127149. PMID:21299962 doi:http://dx.doi.org/10.1051/medsci/201127149
  5. 5.0 5.1 5.2 McCoy, R.S., Braun-Sand, S.B. Semimicroscopic investigation of active site pK a values in peptidylarginine deiminase 4. Theor Chem Acc 131, 1293 (2012) DOI:10.1007/s00214-012-1293-9
  6. 6.0 6.1 Moscarello MA, Wood DD, Ackerley C, Boulias C. Myelin in multiple sclerosis is developmentally immature. J Clin Invest. 1994 Jul;94(1):146-54. doi: 10.1172/JCI117300. PMID:7518827 doi:http://dx.doi.org/10.1172/JCI117300
  7. Wood DD, Bilbao JM, O'Connors P, Moscarello MA. Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein. Ann Neurol. 1996 Jul;40(1):18-24. doi: 10.1002/ana.410400106. PMID:8687186 doi:http://dx.doi.org/10.1002/ana.410400106
  8. Zhang C, Walker AK, Zand R, Moscarello MA, Yan JM, Andrews PC. Myelin basic protein undergoes a broader range of modifications in mammals than in lower vertebrates. J Proteome Res. 2012 Oct 5;11(10):4791-802. doi: 10.1021/pr201196e. Epub 2012 Sep, 21. PMID:22420465 doi:http://dx.doi.org/10.1021/pr201196e
  9. Bates IR, Harauz G. Molecular dynamics exposes alpha-helices in myelin basic protein. J Mol Model. 2003 Oct;9(5):290-7. doi: 10.1007/s00894-003-0145-x. Epub 2003 Jul, 24. PMID:12898292 doi:http://dx.doi.org/10.1007/s00894-003-0145-x
  10. Musse AA, Boggs JM, Harauz G. Deimination of membrane-bound myelin basic protein in multiple sclerosis exposes an immunodominant epitope. Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4422-7. doi:, 10.1073/pnas.0509158103. Epub 2006 Mar 9. PMID:16537438 doi:http://dx.doi.org/10.1073/pnas.0509158103
  11. Musse AA, Harauz G. Molecular "negativity" may underlie multiple sclerosis: role of the myelin basic protein family in the pathogenesis of MS. Int Rev Neurobiol. 2007;79:149-72. doi: 10.1016/S0074-7742(07)79007-4. PMID:17531841 doi:http://dx.doi.org/10.1016/S0074-7742(07)79007-4
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