Ssp1, a Site-specific Parvulin Homolog from Neurospora crassa Active in Protein Folding

Abstract

Peptidyl-prolyl cis-trans-isomerasesHere we describe the purification and characterization of Ssp1, an abundant parvulin homolog from Neurospora crassa, which is unique among the known eucaryotic parvulins in containing a polyglutamine stretch between the N-terminal WW domain and the C-terminal PPIase domain. Ssp1 is a site-specific PPIase with respect to the amino acid N-terminal to the proline residue. Peptides with glutamate, phosphoserine, or phosphothreonine in the ؊1-position proved to be the best substrates. Ssp1 is not only able to isomerize small peptides but is also active in protein folding, as shown with mouse dihydrofolate reductase. Using the substrate specificity of Ssp1, we could identify Glu 81 -Pro 82 as a PPIase-sensitive site in folding of dihydrofolate reductase.These results demonstrate that Ssp1 is a potent mediator of protein folding and that parvulins can serve as tools to elucidate rate-limiting steps in protein folding reactions.Peptidyl-prolyl cis-trans-isomerases (PPIases; 1 EC 5.2.1.8.) are enzymes able to catalyze the cis-trans-isomerization of Xaa-Pro bonds in oligopeptides and proteins (for reviews, see Refs.

The Arabidopsis thaliana PIN1At Gene Encodes a Single-domain Phosphorylation-dependent Peptidyl Prolylcis/trans Isomerase

“…The described PIN1At protein lacks the N-terminal domain but contains a sequence that is highly homologous to the PIN1 ␣1 helix. Furthermore, preliminary structural data by circular dichroism and conformational 13 C␣ chemical-shift deviations from random coil values are consistent with this stretch being in a helical conformation. Functionally, the role of a protein-protein interaction module was initially recognized for the WW domains (14), but only very recently has it been shown that the PIN1 WW domain interacts specifically with a number of proteins phosphorylated on one or more serine or threonine residues (17).…”

“…Spectra were recorded from 180 to 250 nm using a 0.1-and 1-mm path length cell at 20°C. Backbone carbon and hydrogen resonances were assigned from a set of triple resonance NMR experiments (19) recorded on a 1-mM 13 C, 15 N-labeled PIN1At sample in 50 mM deuterated Tris-Hcl, pH 6.3, 100 mM NaCl, 1 mM dithiothreitol, 1 mM PMSF, and 0.5 mM EDTA using a DMX 600-MHz spectrometer (Bruker, Karlsruhe, Germany).…”

“…For clarity, the phosphorylation-dependent class of eukaryotic parvulin PPIases will be designated PIN-type PPIases. Further members of the PIN-type PPIase class are the DODO from Drosophila melanogaster (12), the PINA from Aspergillus nidulans (7), and the SSP1 from Neurospora crassa (13).…”

Functional Conservation of Phosphorylation-specific Prolyl Isomerases in Plants

“…Interestingly, the dephosphorylation of a protein or peptide by PP2A can be dramatically influenced by the PPIase activity of Pin1, which apparently converts cis pSer/pThr-Pro bonds in the substrate into PP2A-accessible trans peptide bonds (31). Furthermore, a function in protein folding has been suggested for the N. crassa protein Ssp1, because it is able to accelerate the refolding of urea-denatured dihydrofolate reductase in vitro (15).…”

“…Among this family are the highly conserved Pin1-type PPIases (10,11), which are the only PPIases that seem to be essential for cell survival, at least in budding yeast and HeLa cells. The Pin1-type PPIases, such as the human Pin1 (11), Saccharomyces cerevisiae ESS1/Ptf1 (10,12,13), Drosophila Dodo (14), Neurospora crassa Ssp1 (15), Xenopus Pin1 (16), Aspergillus nidulans Pin1 (17), and others, consist of an amino-terminal WW domain and a carboxyl-terminal PPIase domain. WW domains, which are characterized by two invariant tryptophans, are present in a variety of signaling and regulatory proteins and were originally identified as protein interaction modules that bind to proline-rich regions in protein targets (18,19).…”