Quantum Mechanics/Molecular Mechanics Study on Caspase-2 Recognition by Peptide Inhibitors

Petar M Mitrasinovic

doi:10.17344/acsi.2020.5847

Abstract

For a variety of biological and medical reasons, the ongoing development of humane caspase-2 inhibitors is of vital importance. Herein, a hybrid (Quantum Mechanics/Molecular Mechanics - QM/MM), two-layered molecular model is derived in order to understand better the affinity and specificity of peptide inhibitor interaction with caspase-2. By taking care of both the unique structural features and the catalytic activity of human caspase-2, the critical enzyme residues (E217, R378, N379, T380, and Y420) with the peptide inhibitor are treated at QM level (the Self-Consistent-Charge Density-Functional Tight-Binding method with the Dispersion correction (SCC-DFTB-D)), while the remaining part of the complex is treated at MM level (AMBER force field). The QM/MM binding free energies (BFEs) are well-correlated with the experimental observations and indicate that caspase-2 uniquely prefers a penta-peptide such as VDVAD. The sequence of VDVAD is varied in a systematic fashion by considering the physicochemical properties of every constitutive amino acid and its substituent, and the corresponding BFE with the inhibition constant (Ki) is evaluated. The values of Ki for several caspase-2:peptide complexes are found to be within the experimental range (between 0.01 nM and 1 ?M). The affinity order is: VELAD (Ki=0.081 nM) > VDVAD (Ki=0.23 nM) > VEIAD (Ki=0.61 nM) > VEVAD (Ki=3.7 nM) > VDIAD (Ki=4.5 nM) etc. An approximate condition needed to be satisfied by the kinetic parameters (the Michaelis constant - KM and the specificity constant - kcat/KM) for competitive inhibition is reported. The estimated values of kcat/KM, being within the experimentally established range (between 10-4 and 10-1 ?M-1 s-1), indicate that VELAD and VDVAD are most specific to caspase-2. These two particular peptides are nearly 1.5, 3 and 4 times more specific to the receptor than VEIAD, VEVAD and VDIAD respectively. Additional kinetic threshold, aimed to discriminate tightly bound inhibitors, is given.

Highlights

Homologues that make up the caspase family of cysteine proteases are essential mediators of cellular processes, such as apoptosis, proliferation, and differentiation.[1,2] They are synthesized and stored as inactive zymogens, as well as divided into inflammatory and apoptotic caspases according to their function and pro-domain structure
The sequence of the penta-peptide inhibitor was varied using single point mutations generated by applying the Mutagenesis engine of PyMol-v0.99 to the experimental structure 3R6G.PDB in a backbone-dependent fashion.[11]
The salt bridge between Glu[217] and Arg[378], which is present in the apo caspase-2 (3.37 Å, Figure 2a), is broken in the caspase-2:VDVAD complex (8.05 Å, Figure 2b), because Thr[380] and Tyr[420] in P5 recognition move 2.1 and 3.6 Å, respectively (Figure 2d)

Summary

Introduction

Homologues that make up the caspase (casp) family of cysteine proteases are essential mediators of cellular processes, such as apoptosis, proliferation, and differentiation.[1,2] They are synthesized and stored as inactive zymogens, as well as divided into inflammatory (caspase-1, -4, -5, -12 in humans and caspase-1, -11, and -12 in mice) and apoptotic (caspase-3, -6, -7, -8, and -9 in mammals) caspases according to their function and pro-domain structure. The functions of caspase-2, -10, and -14 can not be categorized. Apoptotic caspases are further subclassified by their mechanism of action as initiators (caspase-8 and -9) and executioners (caspase-3, -6, and -7). The first identified mammalian member is caspase-2 and its physiological role is not quite clear. Caspase-2, one of the most evolutionarily conserved caspases, is inclined to behave as either executioner or initiator. In terms of substrate specificity, caspase-2 is similar to caspase-3 and

Methods

Results

Conclusion