Preprotein mature domains contain translocase targeting signals that are essential for secretion

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly “catch and release” trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted

Exploring the binding interactions of NOP receptor with designed natural phytochemical-neuropeptide conjugates: an in silico and SPR study

Abstract The Nociceptin/orphanin FQ peptide (NOP) receptor is considered a member of the opioid receptor subfamily of G-protein coupled receptors (GPCRs) which has been shown to be present in many parts of the central nervous system (CNS). It plays biologically diverse roles in pain modulation, immune response and in neurodegenerative diseases. In this work, phytochemical conjugates of two known neuropeptides, melanocyte inhibition factor (MiF-1) and mammalian amidated neuropeptide NPFF with pain modulating ability were developed. The binding interactions of those conjugates with NOP receptor was examined as an approach to develop novel natural compounds that can modulate NOP receptor activity. The selected phytochemicals are well-known for their antioxidant abilities and are derived either from natural alkaloids (betanin), polyphenols (gallic acid and sinapic acid) or terpenes (pomolic acid). Each of the phytochemicals selected are antioxidants which may play a role in mitigating diseases. Three conjugates of betanin were designed with each peptide by conjugating each of the three carboxylic acid groups of betanin with the peptides, while all others were mono-conjugates. Our results indicated that the betanin conjugates with both peptides showed strong binding interactions while the pomolate-peptide conjugates showed moderate binding. In general, NPFF and its conjugates showed stronger binding with the receptor. Docking and molecular dynamics studies revealed that binding interactions occurred at the binding pocket encompassing the transmembrane helices TM1, TM3 and TM7 in most cases, with the ligands binding deep within the hydrophobic core. The binding interactions were further confirmed experimentally through SPR analysis, which also showed higher binding with the betanin conjugates. MMGBSA studies indicated that the binding energies of MiF-1 conjugates were higher compared to neat MiF-1. However, in the case of NPFF, while the betanin conjugates showed enhancement, in some cases the binding energies were found to be slightly reduced compared to neat NPFF. Overall our studies reveal that such natural phytochemical derivatives that can bind to the NOP receptor when conjugated to the mammalian amidated neuropeptide NPFF and the short sequence of melanocyte inhibiting factor MiF-1 may be potentially developed for further laboratory studies for potential pharmaceutical applications

Developing New Peptides and Peptide–Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids

In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism

SummaryProtein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated and secretes &gt;500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptide promotes clamp closing; their mature domain overcomes the rate limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly ‘catch and release’ trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics and become secreted.</jats:p

Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome

Secretory preproteins carry signal peptides fused amino-terminally to mature domains. They are post-translationally targeted to cross the plasma membrane in non-folded states with the help of translocases, and fold only at their final destinations. The mechanism of this process of postponed folding is unknown, but is generally attributed to signal peptides and chaperones. We herein demonstrate that, during targeting, most mature domains maintain loosely packed folding intermediates. These largely soluble states are signal peptide independent and essential for translocase recognition. These intermediates are promoted by mature domain features: residue composition, elevated disorder, and reduced hydrophobicity. Consequently, a mature domain folds slower than its cytoplasmic structural homolog. Some mature domains could not evolve stable, loose intermediates, and hence depend on signal peptides for slow folding to the detriment of solubility. These unique features of secretory proteins impact our understanding of protein trafficking, folding, and aggregation, and thus place them in a distinct class.status: publishe