NmPin from the marine thaumarchaeote Nitrosopumilus maritimus is an active membrane associated prolyl isomerase
- Lukas Hoppstock†1,
- Franziska Trusch†2,
- Christoph Lederer1,
- Pieter van West2,
- Martin Koenneke3 and
- Peter Bayer1Email authorView ORCID ID profile
© Hoppstock et al. 2016
Received: 3 March 2016
Accepted: 14 June 2016
Published: 27 June 2016
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. 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.
Here, 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.
We 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.
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 three-dimensional topologies. The cis-trans isomerization of peptide bonds in Xaa-Pro moieties (Xaa being any amino acid) is essential but also a rate-limiting step in such protein folding processes . Due to a high energy barrier (~20 kcal/mol) between the two almost isoenergetic conformers, the rate of interconversion is extremely slow [1, 2]. However, an important group of proteins, called the peptidyl-prolyl cis-trans isomerases (PPIases), catalyze and accelerate this reaction and thereby essentially control the folding of proteins [3, 4]. PPIases are grouped in three classes – the cyclophilins (CYP) , FK506-binding proteins (FKBP) , and the parvulins  – according to their topology .
Parvulins, a group of small globular proteins with a distinctive βα3βαβ2-fold, are found in all kingdoms of life [9, 10]. By far the most well-studied parvulin is the human Pin1, a phosphorylation-dependent molecular switch, which is involved in cell cycle and transcriptional regulation as well as protein quality control [11–14]. Pin1 is reported to influence ageing, cancer development and neurodegenerative processes in Alzheimer’s and Parkinson’s diseases (reviewed in ). Prokaryotic parvulins, such as the structurally characterized SurA [16–19] and PpiD [20, 21] from Escherichia coli, PrsA from Bacillus subtilis [22–24] or PrsA from Staphylococcus aureus , are involved in folding and maturation of extracellular, periplasmic and outer membrane proteins. 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, 25–27].
Except for the smallest member and archetype of the parvulin family, Par10 from E. coli [7, 9, 27], all proteins mentioned above contain N- or C-terminal extensions/domains in addition to the parvulin domain. Functional studies have shown that the parvulin domain of PpiD and the N-terminal domain of SurA both lack cis-trans isomerase activity, but possess chaperone activity [20, 28]. Due to their function, some parvulins are tightly linked to membranes: PrsA, a foldase for secreted proteins and essential for cell wall assembly in B. subtilis is connected with a lipid-anchor at an N-terminal cysteine residue to the outer leaflet of the cell membrane [22, 29, 30] and PpiD, the periplasmic foldase of outer membrane proteins, is embedded in the lipid double-layer via an N-terminal transmembrane helix . The PrsA of L. monocytogenes is also attached to the membrane by a lipid-anchor and supports the correct folding of secreted proteins during infection and hence plays an important role for the virulence of the pathogen .
During the last decade, the number of sequenced and annotated archaeal genomes has increased, with some of them including parvulin homologue genes . In contrast to the multi-domain parvulins described above, all identified archaeal parvulins consist of a single domain (sdPar)  and exhibit strong homologies to Par10 . The first and only structurally characterized archaeal parvulin CsPinA  originates from the psychrophilic thaumarchaeote Cenarchaeum symbiosum, which lives as a symbiont of the marine sponge Axinella mexicana and therefore eludes pure cultivation. Hence, its expression has not been demonstrated in vivo and no further studies regarding the localization or the cellular role of CsPinA have been performed. Only the 3D structure of the archaeal representative CsPinA has been characterized after expressing the protein recombinantly in E. coli. Furthermore, neither data of its catalytic activity nor its substrate specificity are available so far.
More recently, Könneke et al.  reported the isolation of the first thaumarchaeote into pure culture. Nitrosopumilus maritimus is 0.17–0.22 μm in diameter and 0.5–0.9 μm in length and grows chemoautotrophically by oxidizing ammonia to nitrite and by fixing carbon dioxide as a sole carbon source. Due to their ubiquity and high abundance, ammonia-oxidizing thaumarchaeotes have become recognized as major nitrifiers in a wide range of habitats . Here, we provide novel insight into the cellular localization of the endogenous parvulin NmPin in N. maritimus and present a detailed high resolution structure. NmPin turned out to be a catalytically active prolyl-isomerase with a parvulin-type fold that is associated to the archaeal cell membrane.
NmPin is endogenously expressed and is a catalytically active sdPar
The solution structure of NmPin shows a parvulin-fold and exhibits a novel lysine-rich patch
NMR and refinement statistics for NmPin (residue 4–93)
NOE-based distance restraints
Medium range (2 ≤ Ii-jI ≤ 4)
Long range (Ii-jI ≥ 5)
2 Distances, 3 vdW
Φ + ψ dihedral restraints
Hydrogen bond restraints
Coordinate precision (Å)
0.30 ± 0.04
0.80 ± 0.03
First-generation packing quality
2.918 ± 1.251
Second-generation packing quality
4.617 ± 1.785
Ramachandran plot appearance
0.886 ± 0.227
χ1/χ2 rotamer normality
−3.471 ± 0.486
1.647 ± 0.186
Ramachandran plot (%)
Most favored regions
Generously allowed regions
Pairwise root mean square deviation (RMSD) of NmPin to parvulin homologs. RMSD values were calculated with YASARA using the alignment tool Mustang 
RMSD (Cα) [Å]
% Seq. identity
However, regarding the folding and the special dimensions of the active site, the eukaryotic cis-/trans-isomerase Par14 from human (PDB ID: 3UI4, RMSD of 1.72 Å) shows the highest similarity to NmPin . Hence, we have used the structure of Par14 to deduce and assign the catalytic tetrad of NmPin (C/D-H-H-T/S) . It is built up by residues His10 and His87 (common dual histidine motif) , which are flanked by Asp42 and Ser82. To confirm the functionality of this derived tetrad, we mutated the two flanking residues to alanines, yielding the mutants NmPinD42A and NmPinS82A (Fig. 2c). The two mutant enzymes showed a significant decrease in catalysis rate when compared to the wildtype protein with residual activities of 0.7 % and 3.5 %, respectively (Fig. 2d). As the circular dichroism (CD) spectra of the mutant proteins are comparable with the wild type spectrum (Fig. 2e), structural changes as the reason for the loss of PPIase activity can be excluded. To localize the substrate binding pocket on NmPin a 1H-15N-HSQC-based NMR chemical shift perturbation experiment was performed using the -R-P- substrate peptide as a ligand. HN resonances comprising shift differences ≥ 0.04 ppm (D42, K47, G53, M60, V61, A68, E83, Y86, I88) exclusively belong to residues of the active site or to surrounding amino acids (Fig. 2f). This strongly suggests that substrates are bound and converted within the proposed catalytic groove. In addition, we have used a D42C mutant of NmPin to gain further information about the recognition of the residues N-terminal to the proline in the substrate (Fig. 2g). The NmPinD42C has the same catalytic efficiency as wildtype NmPin (data not shown), but prefers short amino acids (Ala, Ser) compared to the wildtype (Arg, Leu). Interestingly, opposite to the catalytic cleft is a highly positively charged surface motif composed of eight lysines (K5, K7, K31, K34, K37, K47, K48, K90) (Fig. 2h). This prominent feature (K9, K11, K35, K38, K41, K51, R52, K94) is also found in CsPinA (Fig. 2i).
NmPin binds to lipids and is located at the outer membrane side of N. maritimus
NmPin is a novel functional parvulin from a marine Archaeon
In this study, we describe the endogenous expression of a novel archaeal parvulin and confirm its cellular occurrence as a single-domain protein. Our high resolution NMR structure of recombinant NmPin reveals a typical parvulin fold with all residues present to form a catalytically active site. NmPin, as well as its homologue CsPinA, show isomerase activity towards a series of tested model substrates (Fig. 1d). In contrast to eukaryotic Pin representatives but similar to other prokaryotic parvulins studied so far [50, 51], archaeal PPIases do not catalyze the isomerization of peptides containing a phosphorylated serine residue preceding the proline. However, protein phosphorylation as a regulatory mechanism is not generally excluded as numerous genomes of archaeal organisms contain open reading frames for potential protein kinases and protein phosphatases in homology to known eukaryotic proteins . The inability of NmPin to isomerize phosphorylated peptides is structurally reflected by the absence of a phosphate-binding domain or phosphate-binding protein extension, which is present in all known phospho-specific prolyl isomerases such as the WW-domain in human Pin1 and ESS1 from C. albicans [37, 53–55] or a distinct four-amino acid insertion in several plant representatives . The phosphate recognition by NmPin becomes also very unlikely considering the predominantly negative surface potential around the active site due to the exposed Glu83, which is also involved in substrate binding as shown by NMR chemical shift perturbation experiments (Fig. 2a). The substrate selectivity of NmPin for amino acids with long side chains (Arg, Leu) preceding the proline moiety is mainly determined by the catalytic site Asp42. Despite the structural differences between the sdPar NmPin and the N-terminally extended human Par14, and the difference in the catalytic efficiency (Par14: k cat /K M = 1.01 × 103 M–1s–1 , NmPin: k cat /K M = 5.66 × 105 M–1s–1 for Suc-A-L-P-F-pNA), their substrate selectivity is nearly identical. This observation supports recent studies, where the TACK superphylum, including the Thaumarchaeota, were put in a phylogenetic sister relationship with Eukaryotes [58–61]. Although NmPin and Par14 seem to have the same substrate selectivity, their cellular localization seem to determine their different function in the cell since NmPin is important for the folding of extracellular proteins while the human Par14 is involved in signal transduction  and the maturation of ribosomal RNA .
The surface layer provides N. maritimus cells with a rod-shaped structure
The vast majority of archaea possesses a single membrane and most of these membranes are covered by a protein layer, which consists of a single protein species often modified by glycosylation . Our TEM data clearly indicate that the rod-shaped cells of N. maritimus are also enveloped by a surface layer (S-layer). The precise composition of this S-layer is unknown. However, upon stress, either mechanical (centrifugation) or osmotic (salt concentration), the cell shape is altered to a spheroidal form, which is accompanied by a ruptured S-layer (Fig 4c, f ). Additionally, some cells eject parts of their cytoplasm due to damage (Fig. 4h). Only a small number of cells are able to avoid the stress and remain intact. The S-layer maintains the cellular shape of the prokaryote. However, the exact physiological role of the S-layer in N. maritimus under natural conditions needs to be investigated. It is important for the shape of the organism but may also provide protection against natural enemies and viruses.
NmPin is located on the outer membrane surface
The cytoplasmic membrane of N. maritimus consist of intact polar lipids with negatively charged phosphatidic, glycosidic or phosphoglycosidic head groups [43–45], which present an interface suitable for binding to the lysine-rich patch provided by NmPin. The use of eukaryotic lipids as a model system for our lipid sedimentation assays strengthens the pure electrostatic character of the NmPin-lipid interaction since no further anchoring seems to be required. This type of attaching PPIases to a membrane simply by electrostatic interactions might be a general feature of archaeal parvulins since the patch is also conserved in CsPinA from C. symbiosum. In addition, the flat shape of the lysine cluster provides ideal conditions for an electrostatic membrane interaction, in contrast to DNA binding proteins, which mostly possess basic patches with significant curvature [64, 65]. A mutation of the highly conserved residue Lys7  to Glu7 hampers binding of NmPin to lipids. For endogenously expressed NmPin, no integral lipid anchors or transmembrane helices for proper membrane binding were found. This is in contrast to the two known membrane-bound bacterial parvulins, where PrsA of B. subtilis is connected to the outer leaflet of the cell membrane with a lipid-anchor attached at an N-terminal cysteine residue [22, 29, 30] and PpiD of E. coli, which is embedded in the double-layer via an N-terminal transmembrane helix . However, in human cells, several examples of peripheral membrane proteins are found where a nonspecific electrostatic interaction between a cluster of basic residues of the protein and acidic phospholipids in the membrane is required for activity and regulation . In N. maritimus cells with an intact membrane, NmPin is observed and highly abundant on the surface (Fig. 4b, g). In contrast, the amount of NmPin in cells treated with PBS is significantly reduced. The hypoosmotic stress leads to a complete removal of the S-layer as well as a swelling of N. maritimus and, concomitantly, a different surface curvature which can interfere with lipid-NmPin complex formation. The multivalent negative phosphate ion, which is a strong competitor for the interaction with charged lipids , may have an additional effect. Both effects in combination could lead to the release of NmPin from the membrane under low salt conditions. Therefore, we assume that NmPin is located in the ‘quasi-periplasmic space’ between the membrane and the S-layer [67, 68] (Fig. 4i). Several ways for the secretion of folded and unfolded proteins in archaea have been reported. For example, the general secretion (Sec) pathway or the Twin arginine translocase (Tat) pathway that might also be responsible for the translocation of NmPin [69, 70].
Functional role of NmPin
We monitored the localization of NmPin under different osmotic conditions and observed a strong accumulation of the isomerase next to the cell membrane under marine-like salt conditions (Fig. 4b, e). Under low salt stress, NmPin levels are significantly reduced (Fig. 4g), which contradicts an involvement in stress-related pathways and suggests a similar cellular role of NmPin as observed for the PPIases PrsA , PpiD  and SurA  in bacteria. The latter are located at the membrane and are involved in the maturation of extracellular, periplasmic and outer membrane proteins. We therefore assume NmPin to be involved in the folding of S-layer proteins and in maintaining the correct structure of the cell envelope. However, we do not yet have any information about the S-layer protein of N. maritimus. Nevertheless, our hypothesis of an pseudo-periplasmic space located PPIase is supported by the fact that the S-layer glycoprotein complex (tetrabrachion) of the related Crenachaeote Staphylothermus marinus contains a unique proline residue inside a V-I-P-K-F motif which separates the right-handed and left-handed supercoil parts [71, 72]. Obviously, the cis-/trans-conformation of the proline affects the structure of the glycoprotein complex and thereby influences the structure of the whole S-layer. Indeed, we could find a peptidylprolyl isomerase in Staphylothermus marinus with a high proportion of lysines (12.0 %) similar to NmPin (16.1 %) pointing towards a potential lysine patch for membrane binding whereby no transmembrane helices could be predicted. This indicates the importance of folding-assisting PPIases such as NmPin at the outside of the cell membrane for archaeal organisms.
In this study, we determined the solution structure of the parvulin NmPin from the ubiquitous and ecological relevant thaumarchaeote N. maritimus. NmPin represents the first prolyl isomerase of the domain Archaea whose parvulin-like activity as well as membrane-associated location in vivo were characterized. Its structure revealed a lysine-rich patch, which was identified as a membrane-binding interface in vitro. In vivo NmPin is located at the outer surface of the membrane. The endogenous cellular expression level of the protein and its uniform distribution is highest in the presence of an intact cell envelope, precisely between the membrane and S-layer. Membrane association has been previously reported for multi-domain parvulins in bacteria [18, 21, 22] as well as in eukaryotes . For NmPin we present a novel type of membrane association of a single-domain parvulin without any need for anchoring modifications or transmembrane domains, which might be a more general feature of archaeal parvulins since the same kind of membrane interaction was observed for CsPinA. Considering recent studies, which suggest that the archaeal ancestors of eukaryotes are affiliated with the TACK superphylum, including the Thaumarchaeotes [58–61], NmPin likely represents a highly original member of the parvulin family. This assumption is supported by our results, showing that the NmPin folding topology and substrate selectivity are still conserved in the human Par14 protein [8, 57].
Cloning and mutagenesis
The nmpin gene was PCR-amplified with oligonucleotides forward (5′-CATTCGGGCCCTCAAACAAAATCAAATGTTCACAC-3′) and reverse (5′-TGCAGGGATCCTTATCCGAATCTCTTGATAATATG-3′) (Metabion) using genomic DNA of N. maritimus as a template. The resulting fragment containing the restriction sites for ApaI and BamHI (NEB, Fermentas) was cloned into a modified pET-41b(+) (Addgene) vector as described elsewhere . Mutants of nmpin were designed by site-directed mutagenesis using the QuikChange™ Lightning or the Q5 site-directed mutagenesis kit (Stratagene, NEB) and confirmed by Sanger sequencing (GATC GmbH).
Protein expression and purification
For protein production, plasmids were transformed into E. coli BL21(TL3)T1r (Sigma). Unlabeled protein was produced in 1 L of 2 × YT medium, grown to an optical density at 600 nm (OD600) of 0.8 at 37 °C. For isotopically-labelled protein, cells of a culture of 1 L in LB medium (OD600 of 0.8) were transferred to 4 L M9 minimal medium supplemented with 1 g/L [15N]ammonium chloride and, if required, 4 g/L [13C]glucose and further grown to an OD600 of 1.0. Subsequently, protein expression was induced by addition of 0.2 mM IPTG and incubated overnight at 25 °C prior to centrifugation (3700 × g, 20 min, 4 °C). Cell lysis of the resuspended pellet in PBS at pH 8.0 was performed by sonification (Bandelin Sonopuls). Cell debris were removed by ultracentrifugation (95,800 × g, 4 °C, 60 min) and the supernatant was applied to a GSH-sepharose column (GE Healthcare) and eluted with 10 mM glutathione. The GST-tag was cleaved off by PreScission protease and the resulting NmPin protein purified by size exclusion chromatography (SEC) on a Superdex 75PG 26/600 (GE Healthcare) in 50 mM Tris and 150 mM NaCl at pH 8.0. Finally, the protein was dialyzed against buffers as indicated.
Recombinant NmPin (10 μg) was desalted with C18-tips (Supel-Tips C18, Supelco), eluted with 2 μL of 50 % (v/v) acetonitrile/0.1 % (v/v) trifluoroacetic acid and acidified with an equal volume of 2.0 % (v/v) trifluoroacetic acid. The sample was embedded in a saturated dihydroxyacetophenone matrix in ethanol with 25 % (v/v) aqueous ammonium citrate dibasic solution (18 mg/mL). Analysis was performed using an autoflex speed (Bruker) operated in a positive ionization and reflector mode. Spectra were recorded with flexcontrol and the dataset processed with flexanalysis (Bruker).
N. maritimus cultivation and harvesting
N. maritimus cultivation was performed as described previously [35, 47, 75] in synthetic crenarchaeota media (SCM) with a starting concentration of 1 mM ammonium chloride as an energy source. Batches of 5 L were inoculated with 5 % (v/v) culture of N. maritimus, incubated at 29 °C in the dark without stirring and growth was monitored via nitrite concentration. Cells were harvested by centrifugation (4800 × g, 25 °C, 60 min) and pellets resuspended in PBS at pH 8.0.
α-NmPin antibody for detection of the endogenous protein was produced in rabbits with recombinant NmPin from E. coli as the antigen (Eurogentec). The final bleed was affinity purified against recombinant NmPin. For western blot analysis cells were washed in cold PBS at pH 8.0 with variable salt concentrations as indicated, supplemented with a protease inhibitor cocktail (Roche mini) and lysed by sonification (5 × 15 s at 60 % intensity, Sonopuls, Bandelin) prior to ultracentrifugation at 100,000 × g, 4 °C for 50 min. The supernatant was removed and the pellet resuspended in the same volume of PBS. Samples were subjected to SDS-PAGE and transferred by a semi-dry blot to a nitrocellulose membrane (30 min, 80 mA). Blocking of the membrane was done in PBST150 (PBS, pH 8.0, 150 mM NaCl, 0.1 % (v/v) Tween20) with 3 % milk powder at 4 °C overnight; 1:1000 diluted α-NmPin was incubated in the same buffer for 3 h at room temperature (RT). Washing of the membrane was done with one step PBST500 (PBS, pH 8.0, 500 mM NaCl, 0.1 % (v/v) Tween20) followed by two steps with PBST150, each 15 min at RT under shaking. Incubation of HRP-coupled α-rabbit IgG secondary antibody (Sigma) was done in a 1:2000 dilution in PBST150 with 3 % milk powder for 30 min at RT. The membrane was washed as before and subjected to chemoluminescent detection with SuperSignal West Femto Kit (Thermo) on a CL-XPosure film (Thermo).
Catalytic activity of NmPin was measured using a conformer-specific protease coupled assay as described previously [5, 76]. The chromogenic peptide substrates, following the scaffold Suc-Ala-Xaa-Pro-Phe-paranitroaniline (pNA), were pre-incubated overnight in 0.5 M LiCl in 2,2,2-trifluoroethanol at a concentration of 15 mM. α-chymotrypsin (35 μM; Sigma) was equilibrated with variable concentrations of NmPin for 5 min in PBS buffer at pH 6.8 at 10 °C. Subsequently, the assay was started by addition of 75 μM peptide and the reaction was spectrophotometrically monitored via pNA cleavage at 390 nm. For each substrate, the cis-content of the peptide and the reaction rate constant of chymotrypsin were determined on the basis of the uncatalyzed digestion. Using these constants the observed curves were fitted globally to a bi-exponential reaction equation with GraphPad Prism 5.04. The k cat /K M was obtained from conditions with linear reaction rate to PPIase concentration correlation by using the following equation: k cat /K M = (kobs-kuncat)/[PPIase].
NMR spectroscopy and resonance assignment
Spectra were recorded on a Bruker Ultrashield 700 MHz spectrometer equipped with a cryoprobe unit at 300 K. For sample preparation, 1 mM NmPin was dissolved in 600 μL of 50 mM KPi buffer, pH 6.5, 10 %/90 % (v/v) D2O/H2O or 100 % D2O containing 0.02 % (w/v) NaN3 and 50 μM DSS as calibration standard. Spectra were usually recorded using pulse sequences from the Bruker standard library (except 1H-15N-HSQC-NOESY). A set of a 1H-15N-HSQC, an HNCACB and a CBCACONH spectrum was sufficient to trace the chain of the protein sequence and to assign HN, NH, Cα and Cβ atoms of NmPin to their respective frequencies in the spectrum. The assignment of carbon atoms was completed using HCCH-TOCSY and -COSY spectra and the 1H assignment by 1H-13C- and 1H-15N-HSQC-TOCSY spectra. Aromatic hydrogen atoms were assigned by 2D spectra (TOCSY, COSY, NOESY in H2O and D2O). NOESY distance constraints were retrieved from 2D-NOESY and 3D 1H-15N-HSQC-NOESY spectra. Processing and evaluation of spectra was performed with Topspin 3.0 (Bruker), assignment was done with the CcpNmr-Analysis 2.3.1 software package .
NOE restraints were identified and transformed into distance constraints using the automated standard protocol of Cyana. For each hydrogen bond, retrieved from a series of 1H-15N-HSQC spectra after lyophilizing NmPin and dissolving it in 100 % D2O, two lower limit constraints were set for the distances from N to O and from HN to O. The structure of NmPin was calculated using Cyana 3.0 . Owing to the high sequence identity (78.4 %) of NmPin to CsPinA (PDB ID: 2RQS) a homology model was calculated using the software YASARA (Nova forcefield) and set as guide structure for a first cycle of calculation. Water refinement was performed with the software package YASARA using the structure module and the YASARA Nova forcefield . Structural coordinates and NMR shift data were deposited in the RCSB databank (entry ID: 2MO8) and in the BMRB databank (entry ID: 18801), respectively.
Chemical shift perturbation analysis
To a 200 μM sample of 15N-labelled NmPin in 50 mM KPi, pH 6.5, the peptide Suc-A-R-P-F-pNA in the same buffer was added stepwise to a final concentration of 15 mM. For each step, a 1H-15N- SOFAST-HMQC spectrum at 25 °C was recorded. All residues exhibiting chemical shifts ≥ 0.04 ppm were used to map the substrate binding interface on the surface of the NmPin structure using YASARA .
Lipid sedimentation assays
Lipid-binding of NmPinwt, NmPinK7E/K34E and NmPinK7E was carried out as described previously with modifications [80, 81]. Brain lipid extracts from bovine (Folch fraction I, Sigma) were resuspended in HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) to a concentration of 5 mg/mL under continuous stirring. The protein samples (15 μM) were incubated with varying liposome concentrations for 15 min at 37 °C and 350 rpm in a total volume of 40 μL and subsequently centrifuged (50 min, 100,000 × g, 4 °C). The supernatant was removed and the pellet resuspended in the equivalent volume HEPES buffer prior to analysis by SDS-PAGE.
CD spectra were measured in 0.1 cm cuvettes with 0.15 mg/mL protein in 50 mM NaPi, pH 7.4 at 20 °C as a sum of 50 single spectra with a Jasco J-710 spectropolarimeter (Jasco, Gross-Umstadt). A buffer baseline was subtracted from all datasets, units were converted to mean residue ellipticity and the mutant spectra normalized to the wildtype spectrum.
For localization of endogenous NmPin, 10 mL N. maritimus suspension in SCM medium, with or without addition of 4 % PFA, were centrifuged for 60 min at 4800 × g at 25 °C. The supernatant was discarded and the pellet resuspended in 200 μL SCM medium or PBS as indicated. Cells were incubated for 1 h at 29 °C for attachment to poly-lysine coated cover slips. Fixation was done with ice-cold 4 % PFA in PBS for 15 min at RT. For permeabilization, the PFA was supplemented with 0.1 % Triton. Five washing steps (each 3 min) with PBS were performed to stop the cross-link reaction. Blocking was done with 5 % goat serum in PBST (PBS, 0.1 % (v/v) Tween20) for 1 h at RT. α-NmPin antibody (1:50, rabbit) was incubated in 3 % goat serum/PBST overnight at 4 °C followed by four washing steps with PBST (each 3 min). Alexa488-coupled secondary α-rabbit IgG antibody from goat (Invitrogen) was diluted in 3 % goat serum/PBST (2 μg/mL) and incubated for 1 h at RT in the dark followed by four washing steps with PBST (each 3 min). DNA was stained with DAPI contained in the mounting medium (Vectashield, Vector Laboratories). Pictures were taken with a Zeiss Imager M2 with metal halide light source and the corresponding filter set (Alexa488: 495 nm/517 nm, exposure time: 500 ms, DAPI: 395 nm/461 nm, exposure time: 200 ns). Data processing was done with ZEN 2012 SP blue edition.
Sample preparation for TEM microscopy was modified as described previously ; 10 mL N. maritimus suspension in SCM medium, with or without addition of 4 % PFA, were centrifuged for 60 min at 4800 × g at 25 °C. The supernatant was discarded and the pellet resuspended in 20 μL SCM medium or PBS as indicated for attachment to lacy carbon grids with ultrathin Formvar (200 nm mesh, Ladd Research Industries, Burlington, VT) for 30 min at RT. Cells were washed twice with SCM or PBS for 5 min at RT. Fixation was done with 2 % glutaraldehyde in PBS for 5 min at RT followed by three washing steps with PBS, 5 min each. Permeabilization was done with 2.5 % Triton in PBS for 5 min at RT followed by three washing steps with PBS, 5 min each. Blocking was performed with 5 % goat serum in PBST for 30 min at RT (three washing steps with PBST, 5 min each). α-NmPin antibody (1:20, rabbit) was incubated in 2 % goat serum/PBST for 3 h at RT followed by three washing steps with PBST, 5 min each. Secondary α-rabbit IgG antibody conjugated with 5 nm colloidal gold particles (1:20, goat, Sigma) in 2 % goat serum/PBST was incubated for 1 h at RT (three washing steps with PBST, 5 min each). The antigen-antibody complex was fixed again with 2 % glutaraldehyde in PBS for 5 min at RT followed by three washing steps in PBST, 5 min each. A final washing step was done for 15 min in ddH2O and subsequent air drying overnight. Pictures were taken with a JEOL 1400 plus (AMT UltraVUE camera) at 80 kV. Data processing was done with ImageJ.
We cordially thank Alma Rute for excellent technical assistance and the DFG (GRK 1431-1 and 1431-2) for financial support (PB). We thank the Microscope and Histology Facility of the University of Aberdeen for providing their equipment. We also thank Jacob Hargreaves for proofreading the manuscript.
The experiments were conceived by all authors. LH, FT and CL performed the practical work. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Brandts JF, Halvorson HR, Brennan M. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. Biochemistry. 1975;14:4953–63.View ArticlePubMedGoogle Scholar
- Zimmerman SS, Scheraga HA. Stability of cis, trans, and nonplanar peptide groups. Macromolecules. 1976;9:408–16.View ArticlePubMedGoogle Scholar
- Stein RL. Mechanism of enzymatic and nonenzymatic prolyl cis-trans isomerization. Adv Protein Chem. 1993;44:1–24.View ArticlePubMedGoogle Scholar
- Schmid FX, Mayr LM, Mücke M, Schönbrunner ER. Prolyl isomerases: role in protein folding. Adv Protein Chem. 1993;44:25–66.View ArticlePubMedGoogle Scholar
- Fischer G, Bang H, Mech C. Nachweis einer Enzymkatalyse für die cis-trans-Isomerisierung der Peptidbindung in prolinhaltigen Peptiden. Biomed Biochim Acta. 1984;43:1101–11.PubMedGoogle Scholar
- Siekierka JJ, Hung SH, Poe M, Lin CS, Sigal NH. A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature. 1989;341:755–7. doi:10.1038/341755a0.View ArticlePubMedGoogle Scholar
- Rahfeld JU, Schierhorn A, Mann K, Fischer G. A novel peptidyl-prolyl cis/trans isomerase from Escherichia coli. FEBS Lett. 1994;343:65–9.View ArticlePubMedGoogle Scholar
- Sekerina E, Rahfeld JU, Müller J, Fanghänel J, Rascher C, Fischer G, et al. NMR solution structure of hPar14 reveals similarity to the peptidyl prolyl cis/trans isomerase domain of the mitotic regulator hPin1 but indicates a different functionality of the protein. J Mol Biol. 2000;301:1003–17. doi:10.1006/jmbi.2000.4013.View ArticlePubMedGoogle Scholar
- Rahfeld JU, Rücknagel KP, Schelbert B, Ludwig B, Hacker J, Mann K, et al. Confirmation of the existence of a third family among peptidyl-prolyl cis/trans isomerases. Amino acid sequence and recombinant production of parvulin. FEBS Lett. 1994;352:180–4.View ArticlePubMedGoogle Scholar
- Rulten S, Thorpe J, Kay J. Identification of eukaryotic parvulin homologues: a new subfamily of peptidylprolyl cis-trans isomerases. Biochem Biophys Res Commun. 1999;259:557–62. doi:10.1006/bbrc.1999.0828.View ArticlePubMedGoogle Scholar
- Lu KP, Hanes SD, Hunter T. A human peptidyl-prolyl isomerase essential for regulation of mitosis. Nature. 1996;380:544–7. doi:10.1038/380544a0.View ArticlePubMedGoogle Scholar
- Ranganathan R, Lu KP, Hunter T, Noel JP. Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell. 1997;89:875–86.View ArticlePubMedGoogle Scholar
- Zheng H, You H, Zhou XZ, Murray SA, Uchida T, Wulf G, et al. The prolyl isomerase Pin1 is a regulator of p53 in genotoxic response. Nature. 2002;419:849–53. doi:10.1038/nature01116.View ArticlePubMedGoogle Scholar
- Pastorino L, Sun A, Lu P, Zhou XZ, Balastik M, Finn G, et al. The prolyl isomerase Pin1 regulates amyloid precursor protein processing and amyloid-beta production. Nature. 2006;440:528–34. doi:10.1038/nature04543.View ArticlePubMedGoogle Scholar
- Lee TH, Pastorino L, Lu KP. Peptidyl-prolyl cis-trans isomerase Pin1 in ageing, cancer and Alzheimer disease. Expert Rev Mol Med. 2011;13:e21. doi:10.1017/S1462399411001906.View ArticlePubMedGoogle Scholar
- Bitto E, McKay DB. Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins. Structure. 2002;10:1489–98.View ArticlePubMedGoogle Scholar
- Bitto E, McKay DB. The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins. J Biol Chem. 2003;278:49316–22. doi:10.1074/jbc.M308853200.View ArticlePubMedGoogle Scholar
- Hennecke G, Nolte J, Volkmer-Engert R, Schneider-Mergener J, Behrens S. The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition. J Biol Chem. 2005;280:23540–8. doi:10.1074/jbc.M413742200.View ArticlePubMedGoogle Scholar
- Ricci DP, Schwalm J, Gonzales-Cope M, Silhavy TJ. The activity and specificity of the outer membrane protein chaperone SurA are modulated by a proline isomerase domain. MBio. 2013;4(4):e00540–13. doi:10.1128/mBio.00540-13.View ArticlePubMedPubMed CentralGoogle Scholar
- Weininger U, Jakob RP, Kovermann M, Balbach J, Schmid FX. The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity. Protein Sci. 2010;19:6–18. doi:10.1002/pro.277.PubMedGoogle Scholar
- Matern Y, Barion B, Behrens-Kneip S. PpiD is a player in the network of periplasmic chaperones in Escherichia coli. BMC Microbiol. 2010;10:251. doi:10.1186/1471-2180-10-251.View ArticlePubMedPubMed CentralGoogle Scholar
- Jacobs M, Andersen JB, Kontinen V, Sarvas M. Bacillus subtilis PrsA is required in vivo as an extracytoplasmic chaperone for secretion of active enzymes synthesized either with or without pro-sequences. Mol Microbiol. 1993;8:957–66.View ArticlePubMedGoogle Scholar
- Tossavainen H, Permi P, Purhonen SL, Sarvas M, Kilpeläinen I, Seppala R. NMR solution structure and characterization of substrate binding site of the PPIase domain of PrsA protein from Bacillus subtilis. FEBS Lett. 2006;580:1822–6. doi:10.1016/j.febslet.2006.02.042.View ArticlePubMedGoogle Scholar
- Wahlström E, Vitikainen M, Kontinen VP, Sarvas M. The extracytoplasmic folding factor PrsA is required for protein secretion only in the presence of the cell wall in Bacillus subtilis. Microbiology. 2003;149:569–77.View ArticlePubMedGoogle Scholar
- Heikkinen O, Seppala R, Tossavainen H, Heikkinen S, Koskela H, Permi P, et al. Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA--implications for the catalytic mechanism of parvulins. BMC Struct Biol. 2009;9:17. doi:10.1186/1472-6807-9-17.View ArticlePubMedPubMed CentralGoogle Scholar
- Jaremko Ł, Jaremko M, Elfaki I, Mueller JW, Ejchart A, Bayer P, et al. Structure and dynamics of the first archaeal parvulin reveal a new functionally important loop in parvulin-type prolyl isomerases. J Biol Chem. 2011;286:6554–65. doi:10.1074/jbc.M110.160713.View ArticlePubMedGoogle Scholar
- Kühlewein A, Voll G, Hernandez Alvarez B, Kessler H, Fischer G, Rahfeld J, et al. Solution structure of Escherichia coli Par10: The prototypic member of the Parvulin family of peptidyl-prolyl cis/trans isomerases. Protein Sci. 2004;13:2378–87. doi:10.1110/ps.04756704.View ArticlePubMedPubMed CentralGoogle Scholar
- Behrens S, Maier R, de Cock H, Schmid FX, Gross CA. The SurA periplasmic PPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO J. 2001;20:285–94. doi:10.1093/emboj/20.1.285.View ArticlePubMedPubMed CentralGoogle Scholar
- Vitikainen M, Lappalainen I, Seppala R, Antelmann H, Boer H, Taira S, et al. Structure-function analysis of PrsA reveals roles for the parvulin-like and flanking N- and C-terminal domains in protein folding and secretion in Bacillus subtilis. J Biol Chem. 2004;279:19302–14. doi:10.1074/jbc.M400861200.View ArticlePubMedGoogle Scholar
- Hyyryläinen H, Marciniak BC, Dahncke K, Pietiäinen M, Courtin P, Vitikainen M, et al. Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis. Mol Microbiol. 2010;77:108–27. doi:10.1111/j.1365-2958.2010.07188.x.View ArticlePubMedGoogle Scholar
- Dartigalongue C, Raina S. A new heat-shock gene, ppiD, encodes a peptidyl-prolyl isomerase required for folding of outer membrane proteins in Escherichia coli. EMBO J. 1998;17:3968–80. doi:10.1093/emboj/17.14.3968.View ArticlePubMedPubMed CentralGoogle Scholar
- Cahoon LA, Freitag NE, Prehna G. A structural comparison of Listeria monocytogenes protein chaperones PrsA1 and PrsA2 reveals molecular features required for virulence. Mol Microbiol. 2016. doi:10.1111/mmi.13367.PubMedGoogle Scholar
- Walker CB, de la Torre JR, Klotz MG, Urakawa H, Pinel N, Arp DJ, et al. Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. Proc Natl Acad Sci U S A. 2010;107:8818–23. doi:10.1073/pnas.0913533107.View ArticlePubMedPubMed CentralGoogle Scholar
- Lederer C, Heider D, van den Boom J, Hoffmann D, Mueller JW, Bayer P. Single-domain parvulins constitute a specific marker for recently proposed deep-branching archaeal subgroups. Evol Bioinform Online. 2011;7:135–48. doi:10.4137/EBO.S7683.PubMedPubMed CentralGoogle Scholar
- Könneke M, Schubert DM, Brown PC, Hügler M, Standfest S, Schwander T, et al. Ammonia-oxidizing archaea use the most energy-efficient aerobic pathway for CO2 fixation. Proc Natl Acad Sci U S A. 2014;111:8239–44. doi:10.1073/pnas.1402028111.View ArticlePubMedPubMed CentralGoogle Scholar
- Stahl DA, de la Torre JR. Physiology and diversity of ammonia-oxidizing archaea. Annu Rev Microbiol. 2012;66:83–101. doi:10.1146/annurev-micro-092611-150128.View ArticlePubMedGoogle Scholar
- Yaffe MB, Schutkowski M, Shen M, Zhou XZ, Stukenberg PT, Rahfeld JU, et al. Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science. 1997;278:1957–60.View ArticlePubMedGoogle Scholar
- Verdecia MA, Bowman ME, Lu KP, Hunter T, Noel JP. Structural basis for phosphoserine-proline recognition by group IV WW domains. Nat Struct Biol. 2000;7:639–43. doi:10.1038/77929.View ArticlePubMedGoogle Scholar
- Sun L, Wu X, Peng Y, Goh JY, Liou Y, Lin D, et al. Solution structural analysis of the single-domain parvulin TbPin1. PLoS One. 2012;7:e43017. doi:10.1371/journal.pone.0043017.View ArticlePubMedPubMed CentralGoogle Scholar
- Konagurthu AS, Whisstock JC, Stuckey PJ, Lesk AM. MUSTANG: a multiple structural alignment algorithm. Proteins. 2006;64:559–74. doi:10.1002/prot.20921.View ArticlePubMedGoogle Scholar
- Fanghänel J, Fischer G. Insights into the catalytic mechanism of peptidyl prolyl cis/trans isomerases. Front Biosci. 2004;9:3453–78.View ArticlePubMedGoogle Scholar
- Schouten S, Hopmans EC, Baas M, Boumann H, Standfest S, Könneke M, et al. Intact membrane lipids of “Candidatus Nitrosopumilus maritimus,” a cultivated representative of the cosmopolitan mesophilic group I Crenarchaeota. Appl Environ Microbiol. 2008;74:2433–40. doi:10.1128/AEM.01709-07.View ArticlePubMedPubMed CentralGoogle Scholar
- Pitcher A, Hopmans EC, Mosier AC, Park S, Rhee S, Francis CA, et al. Core and intact polar glycerol dibiphytanyl glycerol tetraether lipids of ammonia-oxidizing archaea enriched from marine and estuarine sediments. Appl Environ Microbiol. 2011;77:3468–77. doi:10.1128/AEM.02758-10.View ArticlePubMedPubMed CentralGoogle Scholar
- Elling FJ, Könneke M, Lipp JS, Becker KW, Gagen EJ, Hinrichs K. Effects of growth phase on the membrane lipid composition of the thaumarchaeon Nitrosopumilus maritimus and their implications for archaeal lipid distributions in the marine environment. Geochim Cosmochim Acta. 2014;141:579–97. doi:10.1016/j.gca.2014.07.005.View ArticleGoogle Scholar
- de la Torre JR, Walker CB, Ingalls AE, Könneke M, Stahl DA. Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol. Environ Microbiol. 2008;10:810–8. doi:10.1111/j.1462-2920.2007.01506.x.View ArticlePubMedGoogle Scholar
- Könneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl DA. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature. 2005;437:543–6. doi:10.1038/nature03911.View ArticlePubMedGoogle Scholar
- Pelve EA, Lindås A, Martens-Habbena W, de la Torre JR, Stahl DA, Bernander R. Cdv-based cell division and cell cycle organization in the thaumarchaeon Nitrosopumilus maritimus. Mol Microbiol. 2011;82:555–66. doi:10.1111/j.1365-2958.2011.07834.x.View ArticlePubMedGoogle Scholar
- Albers S, Meyer BH. The archaeal cell envelope. Nat Rev Microbiol. 2011;9:414–26. doi:10.1038/nrmicro2576.View ArticlePubMedGoogle Scholar
- Scholz C, Rahfeld J, Fischer G, Schmid FX. Catalysis of protein folding by parvulin. J Mol Biol. 1997;273:752–62. doi:10.1006/jmbi.1997.1301.View ArticlePubMedGoogle Scholar
- Rouvière PE, Gross CA. SurA, a periplasmic protein with peptidyl-prolyl isomerase activity, participates in the assembly of outer membrane porins. Genes Dev. 1996;10:3170–82.View ArticlePubMedGoogle Scholar
- Kennelly PJ. Protein Ser/Thr/Tyr phosphorylation in the Archaea. J Biol Chem. 2014;289:9480–7. doi:10.1074/jbc.R113.529412.View ArticlePubMedPubMed CentralGoogle Scholar
- Lu PJ, Zhou XZ, Shen M, Lu KP. Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science. 1999;283:1325–8.View ArticlePubMedGoogle Scholar
- Li Z, Li H, Devasahayam G, Gemmill T, Chaturvedi V, Hanes SD, et al. The structure of the Candida albicans Ess1 prolyl isomerase reveals a well-ordered linker that restricts domain mobility. Biochemistry. 2005;44:6180–9. doi:10.1021/bi050115l.View ArticlePubMedPubMed CentralGoogle Scholar
- Matena A, Sinnen C, van den Boom J, Wilms C, Dybowski JN, Maltaner R, et al. Transient domain interactions enhance the affinity of the mitotic regulator Pin1 toward phosphorylated peptide ligands. Structure. 2013;21:1769–77. doi:10.1016/j.str.2013.07.016.View ArticlePubMedGoogle Scholar
- Yao JL, Kops O, Lu PJ, Lu KP. Functional conservation of phosphorylation-specific prolyl isomerases in plants. J Biol Chem. 2001;276:13517–23. doi:10.1074/jbc.M007006200.PubMedGoogle Scholar
- Uchida T, Fujimori F, Tradler T, Fischer G, Rahfeld JU. Identification and characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase. FEBS Lett. 1999;446:278–82.View ArticlePubMedGoogle Scholar
- Kelly S, Wickstead B, Gull K. Archaeal phylogenomics provides evidence in support of a methanogenic origin of the Archaea and a thaumarchaeal origin for the eukaryotes. Proc Biol Sci. 2011;278:1009–18. doi:10.1098/rspb.2010.1427.View ArticlePubMedGoogle Scholar
- Guy L, Ettema TJG. The archaeal ‘TACK’ superphylum and the origin of eukaryotes. Trends Microbiol. 2011;19:580–7. doi:10.1016/j.tim.2011.09.002.View ArticlePubMedGoogle Scholar
- Williams TA, Foster PG, Cox CJ, Embley TM. An archaeal origin of eukaryotes supports only two primary domains of life. Nature. 2013;504:231–6. doi:10.1038/nature12779.View ArticlePubMedGoogle Scholar
- Raymann K, Brochier-Armanet C, Gribaldo S. The two-domain tree of life is linked to a new root for the Archaea. Proc Natl Acad Sci U S A. 2015;112:6670–5. doi:10.1073/pnas.1420858112.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang J, Nakatsu Y, Shinjo T, Guo Y, Sakoda H, Yamamotoya T, et al. Par14 protein associates with insulin receptor substrate 1 (IRS-1), thereby enhancing insulin-induced IRS-1 phosphorylation and metabolic actions. J Biol Chem. 2013;288(28):20692–701. doi:10.1074/jbc.M113.485730. Epub 2013 May 29.View ArticlePubMedPubMed CentralGoogle Scholar
- Fujiyama-Nakamura S, Yoshikawa H, Homma K, Hayano T, Tsujimura-Takahashi T, Izumikawa K, et al. Parvulin (Par14), a peptidyl-prolyl cis-trans isomerase, is a novel rRNA processing factor that evolved in the metazoan lineage. Mol Cell Proteomics. 2009;8(7):1552–65. doi:10.1074/mcp.M900147-MCP200.View ArticlePubMedPubMed CentralGoogle Scholar
- Mulgrew-Nesbitt A, Diraviyam K, Wang J, Singh S, Murray P, Li Z, et al. The role of electrostatics in protein-membrane interactions. Biochim Biophys Acta. 2006;1761:812–26. doi:10.1016/j.bbalip.2006.07.002.View ArticlePubMedGoogle Scholar
- Honig B, Nicholls A. Classical electrostatics in biology and chemistry. Science. 1995;268:1144–9.View ArticlePubMedGoogle Scholar
- Boddey JA, O’Neill MT, Lopaticki S, Carvalho TG, Hodder AN, Nebl T, et al. Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding. Nat Commun. 2016;7:10470.View ArticlePubMedPubMed CentralGoogle Scholar
- Messner P, Pum D, Sára M, Stetter KO, Sleytr UB. Ultrastructure of the cell envelope of the archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus. J Bacteriol. 1986;166:1046–54.PubMedPubMed CentralGoogle Scholar
- Baumeister W, Lembcke G. Structural features of archaebacterial cell envelopes. J Bioenerg Biomembr. 1992;24:567–75.View ArticlePubMedGoogle Scholar
- Albers S, Szabo Z, Driessen AJM. Protein secretion in the Archaea: multiple paths towards a unique cell surface. Nat Rev Microbiol. 2006;4:537–47. doi:10.1038/nrmicro1440.View ArticlePubMedGoogle Scholar
- Ellen AF, Zolghadr B, Driessen AMJ, Albers S. Shaping the archaeal cell envelope. Archaea. 2010;2010:608243. doi:10.1155/2010/608243.View ArticlePubMedPubMed CentralGoogle Scholar
- Peters J, Nitsch M, Kühlmorgen B, Golbik R, Lupas A, Kellermann J, et al. Tetrabrachion: a filamentous archaebacterial surface protein assembly of unusual structure and extreme stability. J Mol Biol. 1995;245:385–401.View ArticlePubMedGoogle Scholar
- Peters J, Baumeister W, Lupas A. Hyperthermostable surface layer protein tetrabrachion from the archaebacterium Staphylothermus marinus: evidence for the presence of a right-handed coiled coil derived from the primary structure. J Mol Biol. 1996;257:1031–41. doi:10.1006/jmbi.1996.0221.View ArticlePubMedGoogle Scholar
- Kessler D, Papatheodorou P, Stratmann T, Dian EA, Hartmann-Fatu C, Rassow J, et al. The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae. BMC Biol. 2007;5:37. doi:10.1186/1741-7007-5-37.View ArticlePubMedPubMed CentralGoogle Scholar
- Grum D, Franke S, Kraff O, Heider D, Schramm A, Hoffmann D, et al. Design of a modular protein-based MRI contrast agent for targeted application. PLoS One. 2013;8:e65346. doi:10.1371/journal.pone.0065346.View ArticlePubMedPubMed CentralGoogle Scholar
- Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA. Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria. Nature. 2009;461:976–9. doi:10.1038/nature08465.View ArticlePubMedGoogle Scholar
- Kofron JL, Kuzmic P, Kishore V, Colón-Bonilla E, Rich DH. Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay. Biochemistry. 1991;30:6127–34.View ArticlePubMedGoogle Scholar
- Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins. 2005;59:687–96. doi:10.1002/prot.20449.View ArticlePubMedGoogle Scholar
- López-Méndez B, Güntert P. Automated protein structure determination from NMR spectra. J Am Chem Soc. 2006;128:13112–22. doi:10.1021/ja061136l.View ArticlePubMedGoogle Scholar
- Krieger E, Koraimann G, Vriend G. Increasing the precision of comparative models with YASARA NOVA--a self-parameterizing force field. Proteins. 2002;47:393–402.View ArticlePubMedGoogle Scholar
- Yan J, Wen W, Xu W, Long J, Adams ME, Froehner SC, et al. Structure of the split PH domain and distinct lipid-binding properties of the PH-PDZ supramodule of alpha-syntrophin. EMBO J. 2005;24:3985–95. doi:10.1038/sj.emboj.7600858.View ArticlePubMedPubMed CentralGoogle Scholar
- Krojer T, Sawa J, Schäfer E, Saibil HR, Ehrmann M, Clausen T. Structural basis for the regulated protease and chaperone function of DegP. Nature. 2008;453:885–90. doi:10.1038/nature07004.View ArticlePubMedGoogle Scholar
- Trent JD, Kagawa HK, Yaoi T, Olle E, Zaluzec NJ. Chaperonin filaments: the archaeal cytoskeleton? Proc Natl Acad Sci U S A. 1997;94:5383–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Krieger E, Nielsen JE, Spronk CA, Vriend G. Fast empirical pKa prediction by Ewald summation. J Mol Graph Model. 2006;25:481–6. doi:10.1016/j.jmgm.2006.02.009.View ArticlePubMedGoogle Scholar
- Bayer E, Goettsch S, Mueller JW, Griewel B, Guiberman E, Mayr LM, et al. Structural analysis of the mitotic regulator hPin1 in solution: insights into domain architecture and substrate binding. J Biol Chem. 2003;278:26183–93. doi:10.1074/jbc.M300721200.View ArticlePubMedGoogle Scholar
- Mueller JW, Link NM, Matena A, Hoppstock L, Rüppel A, Bayer P, et al. Crystallographic proof for an extended hydrogen-bonding network in small prolyl isomerases. J Am Chem Soc. 2011;133:20096–9. doi:10.1021/ja2086195.View ArticlePubMedGoogle Scholar
- Landrieu I, Wieruszeski J, Wintjens R, Inzé D, Lippens G. Solution structure of the single-domain prolyl cis/trans isomerase PIN1At from Arabidopsis thaliana. J Mol Biol. 2002;320:321–32. doi:10.1016/S0022-2836(02)00429-1.View ArticlePubMedGoogle Scholar
- Bailey ML, Shilton BH, Brandl CJ, Litchfield DW. The dual histidine motif in the active site of Pin1 has a structural rather than catalytic role. Biochemistry. 2008;47:11481–9. doi:10.1021/bi800964q.View ArticlePubMedGoogle Scholar