PA clan of proteases

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PA clan of proteases
TEV protease beta-barrels.png
The double β-barrels that characterise the PA clan are highlighted in red. (TEV protease, PDB: 1lvm ​)
Identifiers
SymbolN/A
Pfam clan CL0124
InterPro IPR009003
SCOP2 50494 / SCOPe / SUPFAM
Membranome 319

The PA clan (Proteases of mixed nucleophile, superfamily A) is the largest group of proteases with common ancestry as identified by structural homology. Members have a chymotrypsin-like fold and similar proteolysis mechanisms but can have identity of <10%. The clan contains both cysteine and serine proteases (different nucleophiles). [1] [2] PA clan proteases can be found in plants, [3] animals, [3] fungi, [3] eubacteria, [4] archaea [5] [6] and viruses. [2]

Contents

The common use of the catalytic triad for hydrolysis by multiple clans of proteases, including the PA clan, represents an example of convergent evolution. [7] The differences in the catalytic triad within the PA clan is also an example of divergent evolution of active sites in enzymes. [2]

History

In the 1960s, the sequence similarity of several proteases indicated that they were evolutionarily related. [8] These were grouped into the chymotrypsin-like serine proteases [9] (now called the S1 family). As the structures of these, and other proteases were solved by X-ray crystallography in the 1970s and 80s, it was noticed that several viral proteases such as Tobacco Etch Virus protease showed structural homology despite no discernible sequence similarity and even a different nucleophile. [2] [10] [11] Based on structural homology, a superfamily was defined and later named the PA clan (by the MEROPS classification system). As more structures are solved, more protease families have been added to the PA clan superfamily. [12] [13]

Etymology

The P refers to Proteases of mixed nucleophile. The A indicates that it was the first such clan to be identified (there also exist the PB, PC, PD and PE clans). [1]

Structure

PA clan structures 10.png
Structural homology in the PA superfamily. The double beta-barrel that characterises the superfamily is highlighted in red. Shown are representative structures from several families within the PA superfamily. Note that some proteins show partially modified structural. Chymotrypsin ( PDB: 1gg6 ​), thrombin ( PDB: 1mkx ​), tobacco etch virus protease ( PDB: 1lvm ​), calicivirin ( PDB: 1wqs ​), west nile virus protease ( PDB: 1fp7 ​), exfoliatin toxin ( PDB: 1exf ​), HtrA protease ( PDB: 1l1j ​), snake venom plasminogen activator ( PDB: 1bqy ​), chloroplast protease ( PDB: 4fln ​) and equine arteritis virus protease ( PDB: 1mbm ​).
PA clan vs C04 family sequence conservation.png
Above, sequence conservation of 250 members of the PA protease clan (superfamily). Below, sequence conservation of 70 members of the C04 protease family. Arrows indicate catalytic triad residues. Aligned on the basis of structure by DALI
Surface structure of TEV protease. The C-terminal extension only present in viral members of the PA clan of chymotrypsin-like proteases as (a) surface with loop in blue (b) secondary structure and (c) b-factor putty (wider regions indicate greater flexibility) for the structure of TEV protease. Substrate in black, active site triad in red. The final 15 amino acids (222-236) of the enzyme C-terminus are not visible in the structure as they are too flexible. (PDB: 1lvm, 1lvb ) Viral protease loop.png
Surface structure of TEV protease. The C-terminal extension only present in viral members of the PA clan of chymotrypsin-like proteases as (a) surface with loop in blue (b) secondary structure and (c) b-factor putty (wider regions indicate greater flexibility) for the structure of TEV protease. Substrate in black, active site triad in red. The final 15 amino acids (222-236) of the enzyme C-terminus are not visible in the structure as they are too flexible. ( PDB: 1lvm, 1lvb ​)

Despite retaining as little as 10% sequence identity, PA clan members isolated from viruses, prokaryotes and eukaryotes show structural homology and can be aligned by structural similarity (e.g. with DALI). [3]

Double β-barrel

PA clan proteases all share a core motif of two β-barrels with covalent catalysis performed by an acid-histidine-nucleophile catalytic triad motif. The barrels are arranged perpendicularly beside each other with hydrophobic residues holding them together as the core scaffold for the enzyme. The triad residues are split between the two barrels so that catalysis takes place at their interface. [14]

Viral protease loop

In addition to the double β-barrel core, some viral proteases (such as TEV protease) have a long, flexible C-terminal loop that forms a lid that completely covers the substrate and create a binding tunnel. This tunnel contains a set of tight binding pockets such that each side chain of the substrate peptide (P6 to P1’) is bound in a complementary site (S6 to S1’) and specificity is endowed by the large contact area between enzyme and substrate. [11] Conversely, cellular proteases that lack this loop, such as trypsin have broader specificity.

Evolution and function

Catalytic activity

Evolutionary divergence of the catalytic triads to use different nucleophiles. Shown are the serine triad of chymotrypsin (clan PA, family S1) and the cysteine triad of TEV protease (clan PA, family C3). Triad Divergence.png
Evolutionary divergence of the catalytic triads to use different nucleophiles. Shown are the serine triad of chymotrypsin (clan PA, family S1) and the cysteine triad of TEV protease (clan PA, family C3).

Structural homology indicates that the PA clan members are descended from a common ancestor of the same fold. Although PA clan proteases use a catalytic triad perform 2-step nucleophilic catalysis, [7] some families use serine as the nucleophile whereas others use cysteine. [2] The superfamily is therefore an extreme example of divergent enzyme evolution since during evolutionary history, the core catalytic residue of the enzyme has switched in different families. [15] In addition to their structural similarity, directed evolution has been shown to be able to convert a cysteine protease into an active serine protease. [16] All cellular PA clan proteases are serine proteases, however there are both serine and cysteine protease families of viral proteases. [7] The majority are endopeptidases, with the exception being the S46 family of exopeptidases. [17] [18]

Biological role and substrate specificity

In addition to divergence in their core catalytic machinery, the PA clan proteases also show wide divergent evolution in function. Members of the PA clan can be found in eukaryotes, prokaryotes and viruses and encompass a wide range of functions. In mammals, some are involved in blood clotting (e.g. thrombin) and so have high substrate specificity as well as digestion (e.g. trypsin) with broad substrate specificity. Several snake venoms are also PA clan proteases, such as pit viper haemotoxin and interfere with the victim's blood clotting cascade. Additionally, bacteria such as Staphylococcus aureus secrete exfoliative toxin which digest and damage the host's tissues. Many viruses express their genome as a single, massive polyprotein and use a PA clan protease to cleave this into functional units (e.g. polio, norovirus, and TEV proteases). [19] [20]

There are also several pseudoenzymes in the superfamily, where the catalytic triad residues have been mutated and so function as binding proteins. [21] For example, the heparin-binding protein Azurocidin has a glycine in place of the nucleophile and a serine in place of the histidine. [22]

Families

Within the PA clan (P=proteases of mixed nucleophiles), families are designated by their catalytic nucleophile (C=cysteine proteases, S=serine proteases). Despite the lack of sequence homology for the PA clan as a whole, individual families within it can be identified by sequence similarity.

FamilyExamplesKnown structure?
C03 polio-type picornain 3C ( poliovirus )Yes
C04 tobacco etch virus protease ( tobacco etch virus )Yes
C24rabbit hemorrhagic disease virus 3C-like peptidase ( rabbit hemorrhagic disease virus)No
C30 porcine transmissible gastroenteritis virus-type main peptidase ( transmissible gastroenteritis virus )Yes
C37 calicivirin ( Southampton virus )Yes
C62gill-associated virus 3C-like peptidase ( gill-associated virus )No
C74pestivirus NS2 peptidase ( bovine viral diarrhea virus 1)No
C99iflavirus processing peptidase ( Ectropis obliqua picorna-like virus )No
C107alphamesonivirus 3C-like peptidase ( Cavally virus )No
S01 chymotrypsin A ( Bos taurus )Yes
S03 togavirin ( Sindbis virus )Yes
S06 IgA specific serine endopeptidase ( Neisseria gonorrhoeae )Yes
S07 flavivirin ( yellow fever virus )No
S29 hepacivirin ( hepatitis C virus )Yes
S30 potyvirus P1 peptidase ( plum pox virus )No
S31 pestivirus NS3 polyprotein peptidase ( bovine viral diarrhea virus 1)No
S32 equine arterivirus serine peptidase ( equine arteritis virus )Yes
S39 sobemovirus peptidase ( cocksfoot mottle virus )Yes
S46 dipeptidyl-peptidase 7 ( Porphyromonas gingivalis )No
S55 SpoIVB peptidase ( Bacillus subtilis )No
S64Ssy5 peptidase ( Saccharomyces cerevisiae )No
S65picornain-like cysteine peptidase (Breda-1 torovirus )No
S75White bream virus serine peptidase ( White bream virus )No

See also

Related Research Articles

<span class="mw-page-title-main">Chymotrypsin</span> Digestive enzyme

Chymotrypsin (EC 3.4.21.1, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the S1 position) of the enzyme. It is activated in the presence of trypsin. The hydrophobic and shape complementarity between the peptide substrate P1 side chain and the enzyme S1 binding cavity accounts for the substrate specificity of this enzyme. Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing leucine at the P1 position.

<span class="mw-page-title-main">Protease</span> Enzyme that cleaves other proteins into smaller peptides

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism, and cell signaling.

In biology and biochemistry, protease inhibitors, or antiproteases, are molecules that inhibit the function of proteases. Many naturally occurring protease inhibitors are proteins.

<span class="mw-page-title-main">Serine protease</span> Class of enzymes

Serine proteases are enzymes that cleave peptide bonds in proteins. Serine serves as the nucleophilic amino acid at the (enzyme's) active site. They are found ubiquitously in both eukaryotes and prokaryotes. Serine proteases fall into two broad categories based on their structure: chymotrypsin-like (trypsin-like) or subtilisin-like.

<span class="mw-page-title-main">Metalloproteinase</span> Type of enzyme

A metalloproteinase, or metalloprotease, is any protease enzyme whose catalytic mechanism involves a metal. An example is ADAM12 which plays a significant role in the fusion of muscle cells during embryo development, in a process known as myogenesis.

<span class="mw-page-title-main">Catalytic triad</span> Set of three coordinated amino acids

A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.

<span class="mw-page-title-main">Cysteine protease</span> Class of enzymes

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

<span class="mw-page-title-main">Aspartic protease</span>

Aspartic proteases are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.

Serine hydrolases are one of the largest known enzyme classes comprising approximately ~200 enzymes or 1% of the genes in the human proteome. A defining characteristic of these enzymes is the presence of a particular serine at the active site, which is used for the hydrolysis of substrates. The hydrolysis of the ester or peptide bond proceeds in two steps. First, the acyl part of the substrate is transferred to the serine, making a new ester or amide bond and releasing the other part of the substrate is released. Later, in a slower step, the bond between the serine and the acyl group is hydrolyzed by water or hydroxide ion, regenerating free enzyme. Unlike other, non-catalytic, serines, the reactive serine of these hydrolases is typically activated by a proton relay involving a catalytic triad consisting of the serine, an acidic residue and a basic residue, although variations on this mechanism exist.

<span class="mw-page-title-main">TEV protease</span> Highly specific protease

TEV protease is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). It is a member of the PA clan of chymotrypsin-like proteases. Due to its high sequence specificity, TEV protease is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo.

MEROPS is an online database for peptidases and their inhibitors. The classification scheme for peptidases was published by Rawlings & Barrett in 1993, and that for protein inhibitors by Rawlings et al. in 2004. The most recent version, MEROPS 12.4, was released in late October 2021.

<span class="mw-page-title-main">Subtilase</span>

Subtilases are a family of subtilisin-like serine proteases. They appear to have independently and convergently evolved an Asp/Ser/His catalytic triad, like in the trypsin serine proteases. The structure of proteins in this family shows that they have an alpha/beta fold containing a 7-stranded parallel beta sheet.

<span class="mw-page-title-main">Threonine protease</span> Class of enzymes

Threonine proteases are a family of proteolytic enzymes harbouring a threonine (Thr) residue within the active site. The prototype members of this class of enzymes are the catalytic subunits of the proteasome, however, the acyltransferases convergently evolved the same active site geometry and mechanism.

<span class="mw-page-title-main">Kazal domain</span>

The Kazal domain is an evolutionary conserved protein domain usually indicative of serine protease inhibitors. However, kazal-like domains are also seen in the extracellular part of agrins, which are not known to be protease inhibitors.

<span class="mw-page-title-main">3C-like protease</span> Class of enzymes

The 3C-like protease (3CLpro) or main protease (Mpro), formally known as C30 endopeptidase or 3-chymotrypsin-like protease, is the main protease found in coronaviruses. It cleaves the coronavirus polyprotein at eleven conserved sites. It is a cysteine protease and a member of the PA clan of proteases. It has a cysteine-histidine catalytic dyad at its active site and cleaves a Gln–(Ser/Ala/Gly) peptide bond.

A protein superfamily is the largest grouping (clade) of proteins for which common ancestry can be inferred. Usually this common ancestry is inferred from structural alignment and mechanistic similarity, even if no sequence similarity is evident. Sequence homology can then be deduced even if not apparent. Superfamilies typically contain several protein families which show sequence similarity within each family. The term protein clan is commonly used for protease and glycosyl hydrolases superfamilies based on the MEROPS and CAZy classification systems.

<span class="mw-page-title-main">Pacifastin</span>

Pacifastin is a family of serine proteinase inhibitors found in arthropods. Pacifastin inhibits the serine peptidases trypsin and chymotrypsin.

<span class="mw-page-title-main">Glutamic protease</span>

Glutamic proteases are a group of proteolytic enzymes containing a glutamic acid residue within the active site. This type of protease was first described in 2004 and became the sixth catalytic type of protease. Members of this group of protease had been previously assumed to be an aspartate protease, but structural determination showed it to belong to a novel protease family. The first structure of this group of protease was scytalidoglutamic peptidase, the active site of which contains a catalytic dyad, glutamic acid (E) and glutamine (Q), which give rise to the name eqolisin. This group of proteases are found primarily in pathogenic fungi affecting plant and human.

Asparagine peptide lyase are one of the seven groups in which proteases, also termed proteolytic enzymes, peptidases, or proteinases, are classified according to their catalytic residue. The catalytic mechanism of the asparagine peptide lyases involves an asparagine residue acting as nucleophile to perform a nucleophilic elimination reaction, rather than hydrolysis, to catalyse the breaking of a peptide bond.

<span class="mw-page-title-main">Papain-like protease</span> Protein family of cysteine protease enzymes

Papain-like proteases are a large protein family of cysteine protease enzymes that share structural and enzymatic properties with the group's namesake member, papain. They are found in all domains of life. In animals, the group is often known as cysteine cathepsins or, in older literature, lysosomal peptidases. In the MEROPS protease enzyme classification system, papain-like proteases form Clan CA. Papain-like proteases share a common catalytic dyad active site featuring a cysteine amino acid residue that acts as a nucleophile.

References

  1. 1 2 Rawlings ND, Barrett AJ, Bateman A (January 2012). "MEROPS: the database of proteolytic enzymes, their substrates and inhibitors". Nucleic Acids Research. 40 (Database issue): D343-50. doi:10.1093/nar/gkr987. PMC   3245014 . PMID   22086950.
  2. 1 2 3 4 5 Bazan JF, Fletterick RJ (November 1988). "Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications". Proceedings of the National Academy of Sciences of the United States of America. 85 (21): 7872–6. Bibcode:1988PNAS...85.7872B. doi: 10.1073/pnas.85.21.7872 . PMC   282299 . PMID   3186696.
  3. 1 2 3 4 Laskar A, Rodger EJ, Chatterjee A, Mandal C (May 2012). "Modeling and structural analysis of PA clan serine proteases". BMC Research Notes. 5: 256. doi: 10.1186/1756-0500-5-256 . PMC   3434108 . PMID   22624962.
  4. ↑ Barbosa JA, Saldanha JW, Garratt RC (July 1996). "Novel features of serine protease active sites and specificity pockets: sequence analysis and modelling studies of glutamate-specific endopeptidases and epidermolytic toxins". Protein Engineering. 9 (7): 591–601. doi: 10.1093/protein/9.7.591 . PMID   8844831.
  5. ↑ "MEROPS - Archaeal S01 proteases".
  6. ↑ Ruiz-Perez F, Nataro JP (March 2014). "Bacterial serine proteases secreted by the autotransporter pathway: classification, specificity, and role in virulence". Cellular and Molecular Life Sciences. 71 (5): 745–70. doi:10.1007/s00018-013-1355-8. PMC   3871983 . PMID   23689588.
  7. 1 2 3 Buller AR, Townsend CA (February 2013). "Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad". Proceedings of the National Academy of Sciences of the United States of America. 110 (8): E653-61. Bibcode:2013PNAS..110E.653B. doi: 10.1073/pnas.1221050110 . PMC   3581919 . PMID   23382230.
  8. ↑ de Haën C, Neurath H, Teller DC (February 1975). "The phylogeny of trypsin-related serine proteases and their zymogens. New methods for the investigation of distant evolutionary relationships". Journal of Molecular Biology. 92 (2): 225–59. doi:10.1016/0022-2836(75)90225-9. PMID   1142424.
  9. ↑ Lesk AM, Fordham WD (May 1996). "Conservation and variability in the structures of serine proteinases of the chymotrypsin family". Journal of Molecular Biology. 258 (3): 501–37. doi:10.1006/jmbi.1996.0264. PMID   8642605.
  10. ↑ Gorbalenya AE, Blinov VM, Donchenko AP (January 1986). "Poliovirus-encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families". FEBS Letters. 194 (2): 253–7. doi: 10.1016/0014-5793(86)80095-3 . PMID   3000829. S2CID   23268152.
  11. 1 2 Phan J, Zdanov A, Evdokimov AG, Tropea JE, Peters HK, Kapust RB, Li M, Wlodawer A, Waugh DS (December 2002). "Structural basis for the substrate specificity of tobacco etch virus protease". The Journal of Biological Chemistry. 277 (52): 50564–72. doi: 10.1074/jbc.M207224200 . PMID   12377789.
  12. ↑ Allaire M, Chernaia MM, Malcolm BA, James MN (May 1994). "Picornaviral 3C cysteine proteinases have a fold similar to chymotrypsin-like serine proteinases". Nature. 369 (6475): 72–6. Bibcode:1994Natur.369...72A. doi:10.1038/369072a0. PMID   8164744. S2CID   4312593.
  13. ↑ Snijder EJ, Wassenaar AL, van Dinten LC, Spaan WJ, Gorbalenya AE (March 1996). "The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases". The Journal of Biological Chemistry. 271 (9): 4864–71. doi: 10.1074/jbc.271.9.4864 . PMID   8617757.
  14. ↑ Dougherty WG, Parks TD, Cary SM, Bazan JF, Fletterick RJ (September 1989). "Characterization of the catalytic residues of the tobacco etch virus 49-kDa proteinase". Virology. 172 (1): 302–10. doi:10.1016/0042-6822(89)90132-3. PMID   2475971.
  15. ↑ Laskar A, Rodger EJ, Chatterjee A, Mandal C (May 2012). "Modeling and structural analysis of PA clan serine proteases". BMC Research Notes. 5 (1): 256. doi: 10.1186/1756-0500-5-256 . PMC   3434108 . PMID   22624962.
  16. ↑ Shafee T, Gatti-Lafranconi P, Minter R, Hollfelder F (September 2015). "Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease". ChemBioChem. 16 (13): 1866–1869. doi:10.1002/cbic.201500295. PMC   4576821 . PMID   26097079.
  17. ↑ Suzuki Y, Sakamoto Y, Tanaka N, Okada H, Morikawa Y, Ogasawara W (March 2014). "Identification of the catalytic triad of family S46 exopeptidases, closely related to clan PA endopeptidases". Scientific Reports. 4: 4292. Bibcode:2014NatSR...4E4292S. doi:10.1038/srep04292. PMC   3944710 . PMID   24598890.
  18. ↑ Sakamoto Y, Suzuki Y, Iizuka I, Tateoka C, Roppongi S, Fujimoto M, Inaka K, Tanaka H, Masaki M, Ohta K, Okada H, Nonaka T, Morikawa Y, Nakamura KT, Ogasawara W, Tanaka N (May 2014). "S46 peptidases are the first exopeptidases to be members of clan PA". Scientific Reports. 4: 4977. Bibcode:2014NatSR...4E4977S. doi:10.1038/srep04977. PMC   4021333 . PMID   24827749.
  19. ↑ Salvesen G (2013). Rawlings N (ed.). Handbook of proteolytic enzymes. Boston: Academic Press. ISBN   9780123822192.
  20. ↑ Polgár L (October 2005). "The catalytic triad of serine peptidases". Cellular and Molecular Life Sciences. 62 (19–20): 2161–72. doi:10.1007/s00018-005-5160-x. PMID   16003488. S2CID   3343824.
  21. ↑ Todd AE, Orengo CA, Thornton JM (October 2002). "Sequence and structural differences between enzyme and nonenzyme homologs". Structure. 10 (10): 1435–51. doi: 10.1016/s0969-2126(02)00861-4 . PMID   12377129.
  22. ↑ Iversen LF, Kastrup JS, Bjørn SE, Rasmussen PB, Wiberg FC, Flodgaard HJ, Larsen IK (April 1997). "Structure of HBP, a multifunctional protein with a serine proteinase fold". Nature Structural Biology. 4 (4): 265–8. doi:10.1038/nsb0497-265. PMID   9095193. S2CID   19949043.
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