Protein structure lectures

1. Structure and Function of Proteases

2. Cysteine proteases
In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the
nucleophile that attacks the peptide bond
Cysteine proteases are commonly encountered in fruits including the papaya, pineapple, fig
and kiwifruit. The proportion of protease tends to be higher when the fruit is unripe.
Cysteine proteases are used as an ingredient in meat tenderizers.

3. Papain in complex with its
covalent inhibitor E-64 (PDB id:
1PE6)
Examples of cysteine proteases: Papain, Cathepsins, Caspases, Actinidain, Bromelain,
Calpains, TEV protease
Cleavage site: [AVLIFWY]-[KR]-|-{V}
Papain also cleave the Fc portion of
immunoglobulins from the Fab portion.
Optimal temp: 60-70 C.

4. Mammalian proteases cathepsins have a role in the immune and other systems.
Activated at the low pH found in
lysosomes.
Cathepsins have a vital role in
mammalian cellular turnover, e.g.
bone resorption.

5. Caspases: play a major role in apoptosis. Active site is similar to that of papain, but their
overall structures are unrelated.
Substrate binding requires breaking a salt
bridge

6. Catalytic mechanism of cysteine proteases

7. Biological importance of cysteine proteases
In plants, cysteine proteases play multi-faceted roles such as physiology and development,
signalling pathways, senescence, apoptosis, storage of proteins in seeds, biotic and abiotic
stresses.
In humans, they are responsible for apoptosis, MHC class II immune responses, prohormone
processing, extracellular matrix remodeling, inflammation.
In traditional medicines, these are used to treat intestinal worm infections.

9. Aspartyl proteases
One aspartic acid residue (deprotonated) activates the attacking water molecule by poising it
for deprotonation, whereas the other aspartic acid residue (protonated) polarizes the peptide
carbonyl, increasing its susceptibility to attack.
Aspartyl proteases cleave preferably dipeptide bonds that have hydrophobic residues as well
as a β-methylene group.

10. Members of this class include renin, pepsin and HIV protease.
Why do two aspartic acids show different protonation states?

11. HIV protease: an unfused dimeric aspartyl protease that is similar to the fused protein.

12. Proposed catalytic mechanism for aspartic proteases
Unlike the closely related serine proteases these proteases do not form a covalent intermediate
during cleavage.

14. Metalloproteases
The active site of such a protein contains a bound metal ion, almost always zinc, that activates
a water molecule to act as a nucleophile to attack the peptide carbonyl group.
The ligands co-ordinating the metal ion can vary with histidine, glutamate, aspartate, lysine,
and arginine. The fourth coordination position is taken up by a labile water molecule.

15. Examples: thermolysin and meltrin. Thermolysin specifically catalyzes the hydrolysis of
peptide bonds containing hydrophobic amino acids.
Thermolysin is also widely used for peptide bond formation through the reverse reaction of
hydrolysis.

16. The digestive enzyme carboxypeptidase A is another classic examples of the zinc proteases.
Hydrolyzes carboxyl terminal peptide bond (Prefer bulky and aliphatic residues)

17. Structure and active site of carboxypeptidase A

18. Catalytic mechanism of carboxypeptidase A

19. Matrix metalloproteinases catalyze the reactions in tissue remodeling and degradation.
sp: signal sequence; pro: pro-domain; cat: catalytic domain; FNII: fibronectin type II motif; L1&L2:
linkers; Hpx: hemopexin domain; Mb: plasma membrane; TM: transmembrane domain; Cy: cytoplasmic
tail; CysR: cysteine rich; Ig: immunoglobulin domain; GPI: glycosylphosphatidilyinositol anchor.

21. Reaction pathway investigated for the peptide bond cleavage by MMPs

23. An isopeptide bond is an amide bond which forms between the carboxyl terminus of one
protein and the amino group of a lysine residue on another (target) protein.
Isopeptide bonds can occur between the side
chain amine of lysine and the side chain
carboxyl groups of either glutamate or
aspartate.
Bond formation can be (1) enzyme catalyzed (transglutaminases), (2) Spontaneous (HK97
bacteriophage capsid formation and Gram-positive bacterial pili).
Examples of isopeptide bond: glutathione, ubiquitin.
Deubiquitinating enzyme

24. Biological Roles of Isopeptide Bonds:
Signalling: influencing protein function, chromatin condensation, or protein half-life).
Structural: blood clotting, ECM modeling, apoptosis pathway, formation of pathogenic pilin,
actin skeleton remodeling.
Applications of spontaneous isopeptide bond formation
Peptide tag called SpyTag can spontaneously and irreversibly react with its binding partner
(SpyCatcher) through a covalent isopeptide bond.
The molecular tool can be used for in vivo protein targeting, fluorescent microscopy and
irreversible attachment for a protein microarray.

25. Deubiquitinating enzymes (DUBs) are a large group of proteases that cleave ubiquitin from
proteins and other molecules.
In humans there are nearly 100 DUB genes, which can be classified into two main classes:
cysteine proteases and metalloproteases.
These ubiquitin modifications are added to proteins by the ubiquitination machinary;
ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin
ligases (E3s).

27. Mechanisms of protease regulation
Protease
regulation
Gene expression,
activation of
zymogens
Targeting to
lysosomes
Regulation by post-
translational
modification
Endogenous inhibitors, Self-
cleavage

29. A total of 183 genes encoding protease inhibitors have been annotated in the rat genome,
which markedly contrasts with the more than 600 protease genes present in this species.
Protease inhibitors are important drugs
Protease inhibitors classification according to their mechanism of inhibition
1. Canonical inhibitors: In a virtually substrate-like manner (e.g. serpins).
2. Exosite-binding inhibitors: Binding to the adjacent region of the active site (e.g.
cystatins) .
3. TIMPs (tissue inhibitors of metalloproteinases): combination of the canonical and exosite
binding mechanisms.
4. Allosteric inhibitors: bind a region that is distantly located from the active site (e.g. X-
linked inhibitor of apoptosis protein, a caspase inhibitor).

30. Serpin superfamily: α1-antitrypsin, C1-inhibitor, antithrombin, α1-antichymotrypsin,
plasminogen activator inhibitor-1 and neuroserpin.

31. Most natural protease inhibitors are similar in structure to the peptide substrates of the enzyme
that each inhibits.
Renin-angiotensin-aldosterone system

32. Angiotensinogen
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-...
Angiotensin I
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu | Val-Ile-...
Angiotensin II
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe | His-Leu
Angiotensin III
Asp | Arg-Val-Tyr-Ile-His-Pro-Phe
Angiotensin IV
Arg | Val-Tyr-Ile-His-Pro-Phe
Captopril, an inhibitor of the metalloprotease angiotensin-converting enzyme (ACE)

33. Crixivan, an inhibitor of the HIV protease, is used in the treatment of AIDS

34. Structure of pepstatin in the binding pocket of pepsin
Pepstatin: aspartyl proteases inhibitor
Isovaleryl-Val-Val-Sta-Ala-Sta

35. Exercise:
List out the names of protease inhibitors and their target proteins routinely used in the
laboratory.