Mia40 Is Optimized for Function in Mitochondrial Oxidative Protein Folding and Import

Mia40 catalyzes oxidative protein folding in mitochondria. It contains a unique catalytic CPC dithiol flanked by a hydrophobic groove, and unlike other oxidoreductases, it forms long-lived mixed disulfides with substrates. We show that this distinctive property originates neither from particular properties of mitochondrial substrates nor from the CPC motif of Mia40. The catalytic cysteines of Mia40 display unusually low chemical reactivity, as expressed in conventional p<i>K</i> values and reduction potentials. The stability of the mixed disulfide intermediate is coupled energetically with hydrophobic interactions between Mia40 and the substrate. Based on these properties, we suggest a mechanism for Mia40, where the hydrophobic binding site is employed to select a substrate thiol for forming the initial mixed disulfide. Its long lifetime is used to retain partially folded proteins in the mitochondria and to direct folding toward forming the native disulfide bonds

Molecular Determinants of a Regulatory Prolyl Isomerization in the Signal Adapter Protein c‑CrkII

The cellular CT10 regulator of kinase protein (c-CrkII) transmits signals from oncogenic tyrosine kinases to cellular targets. Nuclear magnetic resonance studies had suggested that in chicken c-CrkII a native state prolyl <i>cis</i>–<i>trans</i> isomerization is involved in signal propagation. Corresponding evidence for the closely related human c-CrkII was not obtained. Here we analyzed the kinetics of folding and substrate binding of the two homologues and found that <i>cis</i>–<i>trans</i> isomerization of Pro238 determines target binding in chicken but not in human c-CrkII. A reciprocal mutational analysis uncovered residues that determine the isomeric state at Pro238 and transmit it to the binding site for downstream target proteins. The transfer of these key residues to human c-CrkII established a regulatory proline switch in this protein, as well. We suggest that Pro238 isomerization extends the lifetime of the signaling-active state of c-CrkII and thereby functions as a long-term molecular storage device

Incorporation of an Unnatural Amino Acid as a Domain-Specific Fluorescence Probe in a Two-Domain Protein

The biophysical analysis of multidomain proteins often is difficult because of overlapping signals from the individual domains. Previously, the fluorescent unnatural amino acid <i>p</i>-cyanophenylalanine has been used to study the folding of small single-domain proteins. Here we extend its use to a two-domain protein to selectively analyze the folding of a specific domain within a multidomain protein

PPIase activities of AquaCyp293 and AquaCyp300.

<p>(A) Kinetics of <i>cis</i>/<i>trans</i> isomerization of 3 μM Abz-Ala-Ala-Pro-Phe-pNa followed by fluorescence at 416 nm, without enzyme (black), with 8 nM (blue), 12 nM (green), 16 nM (dark blue) and 20 nM (red) AquaCyp293. (B) Refolding kinetics of RCM-T1 in the presence of increasing concentrations of AquaCyp293, 0 nM (black), 10 nM (blue), 300 nM (green) and 750 nM (red). The kinetics of refolding of 0.1 μM RCM-T1 in 0.1 M Tris/HCl pH 8.0; 2 M NaCl were measured at 15°C in the presence of various concentrations of AquaCyp293. (C, D) Catalytic efficiencies of AquaCyp293 (○) and AquaCyp300 (□) for (C) the <i>cis</i>/<i>trans</i> isomerization of Abz-Ala-Ala-Pro-Phe-<i>p</i>NA and (D) the refolding of RCM-T1. The measured proline-limited refolding rate constants <i>k</i>_app are shown as a function of the PPIase concentration. The <i>k</i>_cat/<i>K</i>_m values derived from the slopes are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157070#pone.0157070.t001" target="_blank">Table 1</a>.</p

Dimerization of AquaCyp300.

<p>(A) Surface representation of the dimeric AquaCyp300 crystal structure, one protomer is colored in light the other in dark grey, the N-terminal-, insertion, and C-terminal structural elements in one protomer are colored blue, green and red, respectively, active site residues of both protomers are colored yellow. (B) Sequence conservation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157070#pone.0157070.ref083" target="_blank">83</a>] within the AquaCyp300 family is mapped onto a cartoon representation of AquaCyp300 protomer2. Residues that are highly conserved (e.g. Phe258, Phe259, Phe260) are shown in blue, sequences with lower identity are shown in white and red.</p

Domain structure and conservation of AquaCyp.

<p>The cyclophilin domains are shown in grey, the additonal N-terminal-, insertion, and C-terminal structural elements are colored in blue, green and red, respectively. The indicated amino acids are conserved among cyclophilins. The two invariant cysteine residues in AquaCyp are shown in bold. Extension and insertion regions are indicated above the sequences.</p

Active site structure of AquaCyp293 (A) and AquaCyp300 (B) in comparison to hCyp18 (C) and EcCypB (D).

<p>Residues that contribute to the active site of the cyclophilin family are labeled and shown in stick representation. (E) Conservation of PPIase active site residues. Residue numbering according to hCyp18 (C).</p

Statistics on diffraction data and structure refinement of the AquaCyp293 and AquaCyp300.

<p>Statistics on diffraction data and structure refinement of the AquaCyp293 and AquaCyp300.</p

Crystal structures of AquaCyp293 and AquaCyp300.

<p>Domain architecture and topology of AquaCyp293 (A) and AquaCyp300 (B). The cyclophilin fold in grey consists of an eight stranded-antiparallel β-barrel and two α-helices covering the top and the bottom of the barrel. The disulfide bridge (yellow) is shown in stick representation. The additional N-terminal-, insertion, and C-terminal structural elements are coloroured in blue, green and red, respectively.</p

Catalytic efficiencies of AquaCyp293 and AquaCyp300 for prolyl isomerization in peptide and protein substrates.

<p>Catalytic efficiencies of AquaCyp293 and AquaCyp300 for prolyl isomerization in peptide and protein substrates.</p