Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Abstract

Cyclophilin catalyzes the ubiquitous process ''peptidyl-prolyl cis-trans isomerization,'' which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1 H-15 N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

Structural and Biophysical Characterization of Rab5a from Leishmania Donovani

“…It suggests the existence of three different stages of unfolding of Rab5aMDCD60-79C107S, with two intermediates. The size exclusion chromatography (SEC) data for Rab5aMDCD60-79C107S is comparable to previously reported SEC data for carbonic anhydrase, b-lactamase, and CPR3, which have two intermediates in the unfolding pathway (60)(61)(62).…”

“…Although both proteins belong to Ras superfamily and possess a characteristic GTPase fold, the difference in their unfolding profile could also arise because of differences in their primary sequences ($73% similar). Such differences in the protein unfolding pathway are not restricted to the GTPase proteins but is also reported for other proteins, including members of the GST family (PfGST and PvGST) (68), cyclophilins (CPR3, LdCyp, and PPiA) (49,62,69), etc. Earlier studies suggest that the difference in the number of hydrophobic residues at the folding initiation site may give rise to such difference in the unfolding pathway of proteins that belong to same class (70).…”

A staphylococcal anti-sigma factor possesses a single-domain, carries different denaturant-sensitive regions and unfolds via two intermediates

“…Different regions/domains of various proteins have shown different sensitivity to the denaturing agents [ 49 – 51 ]. An examination of our data reveals that upon increasing [urea] from 4 M to 7 M, rRsbW dimer content falls from ~95% to ~35% ( Fig 4D ), while kinase activity drops from ~80% to ~5% ( Fig 4F ).…”

A staphylococcal cyclophilin carries a single domain and unfolds via the formation of an intermediate that preserves cyclosporin A binding activity

“…The unfolding mechanism of drug-bound/unbound rCyp shows some similarity and dissimilarity with those of other drug-bound/unbound cyclophilins [40, 41, 43]. A yeast-encoded cyclophilin (CPR3) in the presence of urea was unfolded by means of the creation of two structurally different intermediates [43]. Of the CPR3 intermediates, the intermediate formed at relatively less urea concentration has the characteristics of a molten globule [55].…”

“…The three-dimensional structures of the cyclophilins are conserved but their amino acid sequences are not identical [1–3], indicating that the unfolding mechanism of these proteins may be different. Thus far, unfolding mechanisms of only a few cyclophilins [40–43] were studied though these proteins were considered as the promising drug targets [2, 3, 10, 11].…”