Papain-like cysteine peptidases are found in all domains of life. They degrade proteins by hydrolysis of peptide bonds. In animals, they are called cysteine cathepsins and are located primarily in lysosomes. Active cysteine cathepsins are monomeric, single-domain proteins with the active site at the top of the molecule. The latter contains a Cys-His catalytic diad (colored yellow and blue, respectively).
Three-dimensional structure of a papain-like peptidase. Cys and His residues of the catalytic diad are shown as yellow and blue spheres, respectively.
The principal and best-known endogenous regulators of cysteine cathepsins are proteinaceous inhibitors that bind into the active site. However, allosteric regulation has been emerging in recent years as an important mode of cysteine cathepsin regulation in vivo and targeting sites outside of the active site is a promising strategy in drug development. The most studied cysteine cathepsin in this respect is cathepsin K, the principal peptidase involved in bone resorption by osteoclasts.
We are investigating evolutionarily conserved mechanisms of allosteric regulation in papain-like peptidases using a combination of experimental and computational methods. We identified the presence of one protein sector in these enzymes (shown as blue spheres in the image below). The protein sector, as defined by Halabi et al. & Ranganathan (Cell. 2009. 138(4):774-86), is a network of residues that transmits allosteric communication between the active site (yellow spheres) and distant regulatory (allosteric) sites. Initial work was done on human cathepsin K as the model enzyme, but we have since expanded our work to other cathepsins.
The protein sector (blue) and highly conserved residues (active site, yellow) of papain-like peptidases, illustrated on the structure of human cathepsin K.
By our current interpretation, allosteric regulation in papain-like peptidases can be described by a simple two-state model. The enzyme exists in equilibrium between two states – low-affinity T state and high-affinity R state. Allosteric inhibitors shift the equilibrium towards the T state and activators shift it towards the R state.
Allosteric two-state model.
Structurally, the two states differ in the width of the active site cleft in its narrowest region (S1 and S2 subsites) that binds the P1 and P2 residues of the substrate. Allosteric inhibitors stabilize the T state (narrow S1-S2 cleft), whereas allosteric activators stabilize the R state (wide S1-S2 cleft). We proposed this model initially for cathepsin K, where multiple allosteric sites are known. MD simulations indicate that the same mechanism applies for all allosteric sites. Evidence suggests that other papain-like peptidases operate by the same mechanism.
(left) The T and R states differ in the width of the active site cleft around subsites S1 and S2. (right) Ensemble of conformations of loops lining the S1-S2 cleft in human cathepsin K.