The goal of the proposed project is to study glycosaminoglycan-mediated maturation process of
procathepsins by computational approaches. Cathepsins, lysosomal proteases, which are the active mature
form of procathepsins, play a crucial role in the extracellular matrix in various processes such as bone
resorption, intracellular proteolysis and regulation of programmed cell death. Their activity can be
mediated by glycosaminoglycans, long unbranched periodic and negatively charged polysaccharides, by
forming complexes with those proteases. The results of this research will bring novel atomistic insights
into this group of complexes.

1. Objectives/hypothesis.
Cathepsins are protein-degrading enzymes that can be found in many living organisms. Most of them
are cysteine proteases (Enzyme Commision number - EC 3.4.22) but are also cathepsins A and G which are
serine proteases (EC 3.4.21) or cathepsins D and E which are aspartyl proteases (EC 3.4.23). To date we
know that there are 15 members of this family with most of their structures experimentally available.
Regardless differences in their aminoacid sequences cathepsins share the same secondary structure pattern
which is reflected in the same fold. Cathepsins play a vital role in diverse of biological processes such as
bone resorption, intracellular proteolysis and regulation of programmed cell death. Changes in their activity
in organism may lead to many serious diseases, either to pycnodysostosis in case of deficiency or to
osteoporosis in case of excessive activity. However, enzymatic activity of the cathepsins can be moderated
by glycosaminoglycans – a group of linear, periodic, negatively charged carbohydrates. This can occur
through various inhibition pathways: i) glycosaminoglycan can bind into active site which makes it
inaccessible for substrate; ii) glycosaminoglycan can bind on already formed complex between protein and
substrate, on a ligand surface, which makes dissociation of substrate unable to occur; iii) glycosaminoglycan
can bind to cathepsin in a way that causes allosteric change in active site which alters its accessiblility for a
substrate.
In order to make cathepsin active, a propeptide has to be cut from procathepsin, the immature form
of the protease. The process of procathepsin maturation can be promoted by the same procathepsin or by
other procathepsin. Moreover, this process can be mediated by glycosaminoglycans. However, the exact
molecular mechanism of procathepsin maturation is still unknown. The main goal of this project is to
characterize the impact of glycosaminoglycans on the activity and maturation process of procathepsins in
terms of the conformational changes in the protein induced by the interactions with glycosaminoglycans.

2. Methodology.
In order to computationally characterize the (pro)cathepsin/glycosaminoglycan complexes, one has
to i) retrieve structures of ligand and receptor; ii) predict the structures of a complexes; iii) examine how the
complexes behave in time; iv) estimate the complexes stabilities. Computational analysis of procathepsin
complexes with glycosaminoglycans requires application of several approaches. First, procathepsin
structures which are accessible from Protein Data Bank will be subjected to molecular dynamics simulations
with the coarse-grained UNRES force field in order to get a deeper view on conformational ensemble of
those proteases. Glycosaminoglycan structures, on the other hand, can be modeled. Using those models,
complex structures can be calculated with use of the molecular docking method. Performing cluster analysis
and picking most representative structures from each of the clusters, the calculated complex structures can be
simulated by the molecular dynamics approach in order to study evolution of a system over time. Post-
processing free energy analysis of the produced molecular dynamics trajectories by various approaches such
as Molecular Mechanics-Poisson Boltzmann with the entropy calculations by normal mode, quasi harmonic
analysis or potential of mean force approach can provide us valuable data on the stability of the complex.
Moreover, additional per-residue analysis of free energy can identify aminoacid residues that contribute
mostly to the interactions between receptor and ligand and in turn – to overall stability of a complex.

3. Impact.
The expected results will contribute to the knowledge on procathepsins as well as on the molecular
interactions of those proteins with GAGs and the effects of GAGs on the conformational aspects in the
process of procathepsins maturation. Therefore, the data planned to be obtained might explain and support
experimental literature data as well as serve as rational basis for guiding further experimental work. The
results expected in this project might add to theoretical basis which can be used in regenerative medicine and
therapies for numerous diseases involving glycosaminoglycan mediated processes.