A Redox-Sensitive Cysteine Is Required for PIN1At Function

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

In-solution buffer-free digestion for the analysis of SARS-CoV-2 RBD proteins allows a full sequence coverage and detection of post-translational modifications in a single ESI-MS spectrum

Subunit vaccines based on the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2, are among the most promising strategies to fight the COVID-19 pandemic. The detailed characterization of the protein primary structure by mass spectrometry (MS) is mandatory, as described in ICHQ6B guidelines. In this work, several recombinant RBD proteins produced in five expression systems were characterized using a non-conventional protocol known as in-solution buffer-free digestion (BFD). In a single ESI-MS spectrum, BFD allowed very high sequence coverage (≥ 99 %) and the detection of highly hydrophilic regions, including very short and hydrophilic peptides (2-8 amino acids), the His6-tagged C-terminal peptide carrying several post-translational modifications at Cys538 such as cysteinylation, glutathionylation, cyanilation, among others. The analysis using the conventional digestion protocol allowed lower sequence coverage (80-90 %) and did not detect peptides carrying some of the above-mentioned post-translational modifications. The two C-terminal peptides of a dimer [RBD(319-541)-(His)6]2 linked by an intermolecular disulfide bond (Cys538-Cys538) with twelve histidine residues were only detected by BFD. This protocol allows the detection of the four disulfide bonds present in the native RBD and the low-abundance scrambling variants, free cysteine residues, O-glycoforms and incomplete processing of the N-terminal end, if present. Artifacts that might be generated by the in-solution BFD protocol were also characterized. BFD can be easily implemented and we foresee that it can be also helpful to the characterization of mutated RBD.

A Comprehensive Analysis of Novel Disulfide Bond Introduction Site into the Constant Domain of Human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bonds can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the H: F130C-L: Q124C, H: F174C-L: S176C, H: V177C-L: Q160C, H: F174C-L: S162C, H: F130C-L: S121C, and H: A145C-L: F116C mutants mostly formed intermolecular disulfide bonds. All these mutants showed increased thermal stabilities compared to those without intermolecular disulfide bonds. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C-F118C mutant showed only a slight decrease in binding activity and β-helix content, owing to adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals the introduction of intermolecular disulfide bonds in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

Tetragonal crystal form of the cyanobacterial bicarbonate-transporter regulator SbtB from Synechocystis sp. PCC 6803

The PII-like protein SbtB has been identified as a regulator of SbtA, which is one of the key bicarbonate transporters in cyanobacteria. While SbtB from Synechocystis sp. PCC 6803 has previously been shown to be a trimer, a new crystal form is reported here which crystallizes in what is thought to be a non-native tetramer in the crystal, with the C-terminus in an extended conformation. The crystal structure shows the formation of an intermolecular disulfide bond at Cys94 between SbtB monomers, which may stabilize this conformation in the crystal. This motivates the need for future studies to investigate the potential role that the oxidation and reduction of these cysteines may play in the activation and/or function of SbtB.

Conjugative Transfer of IncP-9 Catabolic Plasmids Requires a Previously Uncharacterized Gene, mpfK, Whose Homologs Are Conserved in Various MPFT-Type Plasmids

ABSTRACT Conjugative transfer of bacterial plasmids to recipient cells is often mediated by type IV secretion machinery. Experimental investigations into the minimal gene sets required for efficient conjugative transfer suggest that such gene sets are variable, depending on plasmids. We have been analyzing the conjugative transfer of Pseudomonas-derived and IncP-9 plasmids, NAH7 and pWW0, whose conjugation systems belong to the MPFT type. Our deletion analysis and synthetic biology analysis in this study showed that these plasmids require previously uncharacterized genes, mpfK (formerly orf34) and its functional homolog, kikA, respectively, for their efficient conjugative transfer. MpfK was localized in periplasm and had four cysteine residues whose intramolecular or intermolecular disulfide bond formation was suggested to be important for efficient conjugative transfer. The mpfK homologs were specifically carried by many MPFT-type plasmids, including non-IncP-9 plasmids, such as R388 and R751. Intriguingly, the mpfK homologs from the two non-IncP-9 plasmids were not required for conjugation of their plasmids, but were able to complement efficiently the transfer defect of the NAH7 mpfK mutant. Our results suggested the importance of the mpfK homologs for conjugative transfer of MPFT-type plasmids. IMPORTANCE IncP-9 plasmids are important mobile genetic elements for the degradation of various aromatic hydrocarbons. Elucidation of conjugative transfer of such plasmids is expected to greatly contribute to our understanding of its role in the bioremediation of polluted environments. The present study mainly focused on the conjugation system of NAH7, a well-studied and naphthalene-catabolic IncP-9 plasmid. Our analysis showed that the NAH7 conjugation system uniquely requires, in addition to the conserved components of the type IV secretion system (T4SS), a previously uncharacterized periplasmic protein, MpfK, for successful conjugation. Our findings collectively revealed a unique type of T4SS-associated conjugation system in the IncP-9 plasmids.