Interaction Of Signal Peptide Research Articles

Conserved SecA Signal Peptide-Binding Site Revealed by Engineered Protein Chimeras and Förster Resonance Energy Transfer.

Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study, we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger subdomain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger subdomain of SecA, and they are also consistent with the proposed role of this subdomain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of SecYEG protein. In addition, our work highlights the utility of this system to conveniently engineer and study the interaction of SecA with any signal peptide of interest as well as its potential use for X-ray crystallographic studies given issues with exogenous signal peptide solubility.

Influence of GTP on system specific chaperone – Twin arginine signal peptide interaction

Many bacterial respiratory redox enzymes depend on the twin-arginine translocase (Tat) system for translocation and membrane insertion. Tat substrates contain an N-terminal twin-arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone – redox enzyme maturation protein (REMP) – for final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin-arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase. In this study, we have utilized the in vitro protein–protein interaction technique of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF) to investigate the interaction of guanosine nucleotides (GNPs) with DmsD. Here we have shown highly cooperative binding of DmsD with GTP. A dissociative ligand-binding style isotherm was generated upon GTP titration into the DmsD:DmsAL interaction, yielding sigmoidal release of DmsD with a Hill coefficient of 2.09 and a dissociation constant of 0.99 mM. DSF further illustrated the change in thermal stability upon DmsD interaction with DmsAL and GTP. These results imply the possibility of DmsD detection and binding of GTP during the DMSO protein maturation mechanism, from ribosomal translation to membrane targeting and final assembly. Conceivably, GTP is shown to act as a molecular regulator in the biochemical pathway.

Changes to signal peptide and the level of transforming growth factor- β1 due to T869C polymorphism of TGF β1 associated with lupus renal fibrosis.

Lupus Nephritis (LN) is a serious manifestation of lupus that can lead to End Stage Renal Disease (ESRD). Fibrosis is the main feature of ESRD, and it is likely influenced by Transforming Growth Factor Beta1 (TGFβ1). The T869C gene polymorphism of TGFβ1 is assumed to change the signal peptide, that has potential to interfere the urine production and renal protein expression of TGFβ1. The influence of T869C gene polymorphism on TGFβ1 production and renal fibrosis was evaluated in this study. Subjects were 45 patients LN with renal fibrosis and 45 participants without renal fibrosis as control, that were recruited from 2011 to 2013.Their urinary TGFβ1 levels and TGFβ1 gene polymorphisms were examined. All lupus patients underwent renal biopsy to assess their protein expression of TGFβ1 in the renal tissue by immunohistochemistry and their renal fibrosis by morphometry and chronicity index. Changes in the signal peptide interaction with Signal Recognition Particle (SRP) and translocon of endoplasmic reticulum were analyzed by Bioinformatics. Levels of urinary and protein expression of TGFβ1 increased in the LN with renal fibrosis group. There were significant differences in levels of urinary TGFβ1 in T, C allele and TT, TC, CC genotypes between case and control groups. Furthermore, patients with C allele are 3.86 times more at risk of renal fibrosis than T allele. The C allele encodes proline, which stabilizes the interaction of the TGFβ1 signal peptide with SRP and translocon, resulting in elevation of TGFβ1 secretion. Our results indicated that T869C gene polymorphism of TGFβ1 changes the signal peptide, that contributes to the production of urinary TGFβ1 and affects renal fibrosis in lupus nephritis.

Characterization of the Escherichia coli SecA Signal Peptide-Binding Site

SecA signal peptide interaction is critical for initiating protein translocation in the bacterial Sec-dependent pathway. Here, we have utilized the recent nuclear magnetic resonance (NMR) and Förster resonance energy transfer studies that mapped the location of the SecA signal peptide-binding site to design and isolate signal peptide-binding-defective secA mutants. Biochemical characterization of the mutant SecA proteins showed that Ser226, Val310, Ile789, Glu806, and Phe808 are important for signal peptide binding. A genetic system utilizing alkaline phosphatase secretion driven by different signal peptides was employed to demonstrate that both the PhoA and LamB signal peptides appear to recognize a common set of residues at the SecA signal peptide-binding site. A similar system containing either SecA-dependent or signal recognition particle (SRP)-dependent signal peptides along with the prlA suppressor mutation that is defective in signal peptide proofreading activity were employed to distinguish between SecA residues that are utilized more exclusively for signal peptide recognition or those that also participate in the proofreading and translocation functions of SecA. Collectively, our data allowed us to propose a model for the location of the SecA signal peptide-binding site that is more consistent with recent structural insights into this protein translocation system.

Another Factor Besides Hydrophobicity Can Affect Signal Peptide Interaction with Signal Recognition Particle

Translocation of alkaline extracellular protease (AEP) into the endoplasmic reticulum of Yarrowia lipolytica is cotranslational and signal recognition particle (SRP)-dependent, whereas translocation of P17M AEP (proline to methionine at position 17, second amino acid in the pro-region) is posttranslational and SRP-independent. P17M signal peptide mutations that resulted in more rapid SRP-dependent translocation of AEP precursor were isolated. Most of these mutations significantly increased hydrophobicity, but the A12P/P17M mutation did not. The switch from SRP-dependent to SRP-independent translocation without a decrease in hydrophobicity (wild type to P17M) and restoration of SRP-dependent translocation without an increase in hydrophobicity (P17M to A12P/P17M) indicate that some factor(s) in addition to hydrophobicity determines selection of targeting pathway. Models of extended forms of wild type and A12P/P17M signal peptides are kinked, whereas the P17M signal peptide is relatively straight. Possibly the conformation/orientation of signal peptides at the ribosomal surface affects SRP binding and consequently the targeting route to the endoplasmic reticulum. Kinked signal peptides might approach SRP more closely more often. Most likely, these effects were only detectable because of the short length and low average hydrophobicity of the AEP signal peptide.

Anionic phospholipids are essential for alpha-helix formation of the signal peptide of prePhoE upon interaction with phospholipid vesicles.

The conformational consequences of the interaction of the PhoE signal peptide with bilayers of different types of phospholipids was investigated using circular dichroism. It was found that interaction of the signal peptide with anionic phospholipid vesicles of dioleoylphosphatidylglycerol and dioleoylphosphatidylserine results in induction of high amounts of alpha-helical structure of 70% and 57%, respectively. Upon addition of the signal peptide to cardiolipin vesicles, less but still significant alpha-helical structure was induced (29%). In contrast, no alpha-helix formation was observed upon the interaction of the signal peptide with zwitterionic dioleoylphosphatidylcholine vesicles. In bilayers of dioleoylphosphatidylcholine with dioleoylphosphatidylglycerol, it was shown that in the presence of 100 mM NaCl a minimum amount of 50% of negatively charged lipid was required for induction of the maximal percentage of alpha-helix, whereas in the absence of salt a minimum amount of 35% of negatively charged lipid was necessary. Induction of alpha-helix structure appeared to be correlated with functionality, since, in a less functional analogue of the PhoE signal peptide, the PhoE-[Asp-19,20] signal peptide, less alpha-helix was induced than in the wild-type PhoE signal peptide. It is proposed that the interaction with anionic phospholipids is essential for a functional conformation of the PhoE signal sequence during protein translocation.

Stereochemical Analysis of Interaction of Signal Peptide with Phospholipids at the Initiation of Protein Translocation Across the Membrane

Stereochemical analysis of signal peptide interaction with E. coli membrane phospholipids revealed the structural complementarity of N-terminus of signal peptide alpha-helix and acid phospholipids. The formation of their complex leads to neutralization of charges and decrease in hydrophilicity of both components, and promotes insertion of peptide and phospholipid into the membrane, not separately but as a complex. Interaction of acid phospholipids with the E. coli alkaline phosphatase (AP) signal peptide was thoroughly analyzed, and it was shown that in this case a complex of signal peptide alpha-helix with phosphatidylglycerol is inserted into the membrane with the lowest energy expense. On the basis of the results of stereochemical analysis and the available experimental data, a molecular mechanism of protein translocation initiation across the membrane has been proposed, in which the key events are the formation of the complex "signal peptide alpha-helix-acid phospholipid", the coupled insertion of hydrophobic peptide-lipid complex into a nonpolar membrane interior and translocation across the membranes.