Abstract
BackgroundPeptidyl-prolyl isomerases (PPIases) are present in all forms of life and play a crucial role in protein folding and regulation. They catalyze the cis-trans isomerization of the peptide bond that precedes proline residues in numerous proteins. The parvulins, which is one family of PPIases, have been extensively investigated in several eukaryotes. However, nothing is known about their expression, function and localization in archaea.ResultsHere, we describe the endogenous expression, molecular structure, function and cellular localization of NmPin, a single-domain parvulin-type PPIase from Nitrosopumilus maritimus. This marine chemolithoautotrophic archaeon belongs to the globally abundant phylum Thaumarchaeota. Using high resolution NMR spectroscopy we demonstrate that the 3D structure of NmPin adopts a parvulin fold and confirmed its peptidyl-prolyl isomerase activity by protease-coupled assays and mutagenesis studies. A detailed topological analysis revealed a positively charged lysine-rich patch on the protein surface, which is conserved in all known parvulin sequences of thaumarchaeotes and targets NmPin to lipids in vitro. Immunofluorescence microscopy confirms that the protein is attached to the outer archaeal cell membrane in vivo. Transmission electron microscopy uncovered that NmPin has a uniform distribution at the membrane surface, which is correlated with a native cell shape of the prokaryote.ConclusionWe present a novel solution structure of a catalytically active thaumarchaeal parvulin. Our results reveal that a lysine-rich patch in NmPin mediates membrane localization. These findings provide a model whereby NmPin is located between the archaeal membrane and the surface layer and hence suggest proteins of the S-layer as the key target substrates of this parvulin.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0274-1) contains supplementary material, which is available to authorized users.
Highlights
Peptidyl-prolyl isomerases (PPIases) are present in all forms of life and play a crucial role in protein folding and regulation
The expected molecular mass and purity were confirmed by mass spectrometry (Fig. 1a), using α-NmPin, an antibody that we generated to analyze the expression of endogenous NmPin by western blot under different osmotic conditions, as the salt concentration may be critical for the marine organism
High salt levels up to 500 mM NaCl disturbed the attachment to the membrane fraction and NmPin was predominantly found in the supernatant of N. maritimus lysates (Fig. 1b)
Summary
Peptidyl-prolyl isomerases (PPIases) are present in all forms of life and play a crucial role in protein folding and regulation. They catalyze the cis-trans isomerization of the peptide bond that precedes proline residues in numerous proteins. Proteins are biomolecules acting as scaffolds, signal transmitters or catalysts of chemical reactions in living cells. Before they can commence their tasks they need to undergo intensive folding steps to adopt their proper threedimensional topologies. By far the most well-studied parvulin is the human Pin, a phosphorylation-dependent molecular switch, which is involved in cell cycle and transcriptional regulation as well as protein quality control [11,12,13,14]. In contrast to eukaryotic Pin-type parvulins found in yeast, metazoans and multicellular archaeplastidae, the prokaryotic representatives lack a recognition site for phosphorylated target residues [20, 25,26,27]
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