Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Abstract

BackgroundCystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis.ResultsStarting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability.ConclusionA combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing

“…This value is consistent with data described by [ 67 ], who report that the recombinant human cystatin C inhibits human cathepsin B activity, with a K i value of 0.38 nM. Furthermore, [ 52 , 68 ] respectively obtained K i values of 0.5 nM and 0.83 nM for CaneCPI-4 against recombinant human cathepsin B. Cystatins are competitive and reversible inhibitors of cysteine peptidases [ 69 ]. The structure of cathepsin B-like cysteine peptidases has an occluding loop that blocks the cleft of the enzyme active site [ 62 ] and hinders the access of cystatin to this site, resulting in high K i values for most cystatins against cathepsin B when compared to other cysteine peptidases that lack this loop [ 70 , 71 , 72 ].…”

X‐ray crystallography and NMR studies of domain‐swapped canecystatin‐1

“…The reason that most cystatins have low inhibitory activity towards cathepsin B is that there is a large occluding loop in the protease, which leads to steric hindrance with the inhibitor when present as a correctly folded monomer. Perturbation of the closed interface may favor a partially unfolded monomer, in which the three components of the inhibitory site are uncoupled, leading to sufficient structural malleability to reduce steric hindrance and allow access to the protease active site . Moreover, our data show that canecystatin‐1 is present in solution predominantly as domain‐swapped dimers, which are unable to inhibit cysteine proteases, owing to the absence of the appropriate conformation for inhibitory loop 1.…”

Review: The future of cystatin engineering

“…Such increased flexibility will reduce steric hindrance with better direct binding of the two inhibitory hairpin loops to the catalytic site [36]. Future research is necessary though, to extend these findings to demonstrate the overall potential of the DNA shuffling technology with other plant cystatins and the cysteine protease strategy.…”

“…A new and interesting strategy, with regards to cystatin engineering, is DNA shuffling [36]. The approach can be applied to recombine two cystatins to produce a new hybrid cystatin with improved inhibitory activity.…”

“…DNA shuffling has been recently successfully applied by Valadares et al. [36] with a sugarcane cystatin (CaneCPI-1) and rice OC-I with both sequences having a 56% DNA similarity. A clone with higher inhibitory activity towards cathepsin B was obtained with two unanticipated point mutations as well as an N-terminal deletion.…”