![]() ![]() Firstly, the CL1-cystatin/stefin complexes were built through molecular docking and then atomistic molecular dynamics studies of the binding mode of all nine complexes were performed. In this paper the detailed molecular mechanisms behind the diverse inhibitory interaction of human family 1 & 2 cystatins, namely stefin A, B & cystatin C, D, F, M/E, S, SA, SN, with cathepsin L1 (CL1) were investigated. Various reports suggests the levels of cystatin C, cystatin M/E and stefin A in tissue and extracellular fluids can serve as relatively reliable markers for diagnosis and prognosis of a variety of diseases while cathepsin B & L levels may well be used as a potential indicator of tumor aggressiveness and metastasis. Substantial reduction in stefin B inhibitory activity and concomitant increase in cathepsins S and L activity is observed in EPM1 pathogenesis. In vivo studies further elucidate the considerable correlation of cathepsin B & L activities with the inhibitory activities of stefin A & B in breast carcinoma and head & neck carcinoma patients, respectively. Apart from three conserved regions, C-terminal residues also takes part significantly in papain, cathepsin B & H interaction of stefin B. For SD-type cystatins, the QXVXG region exhibits main effect on papain inhibition, whereas all three conserved regions participate for cathepsin CPs. The L1 and L2 loop of cystatin C is recognized to hold the major share in total free energy of binding of papain, actinidin, cathepsins B and H. In contrast to the previous observations, N-terminal segment of human stefin A and B is reported to contribute about 40% of the total free energy of binding for papain, cathepsin B & L although the first β hairpin loop fails to account for stefin A in CP binding but credited for stefin B-papain interaction and the second loop of stefin A shows variable affinity depending on the CP, responsible for 20–30% of total binding energy in bovine cystatin B-papain, -cathepsin H and–cathepsin B interaction. ![]() N-terminal, L1 & L2 β-hairpin loops, also vary appreciably. Furthermore, the relative contributions of three conserved regions, viz. Even in case for human S-type cystatins, where more than 90% sequence similarity is observed, cystatin S comes out as a significantly poorer inhibitor in comparison to cystatin SA & SN. Human stefin B, cystatin F appears to be 100 fold poorer inhibitor of cathepsin L in comparison to cystatin C while stefin A, cystatin M/E are found to be 1000 fold, cystatin SN shows 10 5 fold and cystatin S & SA exhibits 10 6 fold lower inhibitory activity. In case of human stefins and cystatins, cystatin C emerges as the best overall inhibitor of C1 family of CPs whereas SD type of cystatins are the poorest of the lot. The inhibitory affinities of the human family 1 & 2 cystatins, even towards a particular CP, differ notably. īut the conservation of tertiary structure or similar mechanism of inhibition does not tally well with the inhibition profile of cystatins. Structural studies have recognized three conserved regions in cystatin fold, the exposed L1 loop containing highly conserved (Q-X-V-X-G) region flanked between projecting N-terminal segment and C-terminal L2 loop comprising equally conserved PW segment (with an exception of human stefins), altogether form a tripartite, largely hydrophobic, wedge-shaped edge complementary to the active site of papain-like CPs. Stefins lack AS loop and the second α-helix (α2). The connectivity within the cystatin fold is: (N)-β1-α1-β2-L1-β3-(AS)-α2-β4-L2-β5-(C) where AS is a broad “appending structure” positioned at the opposite side relative to the N-terminus and β hairpin loops L1 and L2. In spite of substantial difference in protein sequence, all members of cystatin superfamily shares the characteristic cystatin fold formed by five-stranded anti-parallel β-sheets (β1-β5) wrapped around a core of a five-turn α-helix (α1) lying almost perpendicular to the sheets (S1 Fig). Affiliation: Department of Biochemistry & Biophysics, University of Kalyani, Kalyani, West Bengal, IndiaĬystatins are typical emergency inhibitors of C1 and C13 family of cysteine proteases (CPs), classified into four groups, namely family 1 or stefins, family 2 or cystatins, family 3 or kininogens and family 4 or cystatins devoid of CP activity. ![]()
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