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]
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)
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.
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 viperhaemotoxin 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.
^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.
PMID1142424.
^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.
PMID8642605.