Development of Antibacterial Peptides with Membrane Disruption and Folate Pathway Inhibitory Activities against Methicillin-Resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) offer an opportunity to overcome multidrug resistance. Here, novel peptides were designed based on AMP fragments derived from sea cucumber hemolytic lectin to enhance anti-methicillin-resistant Staphylococcus aureus (MRSA) activity with less side effects. Two designed peptides, CGS19 (LARVARRVIRFIRRAW-NH2) and CGS20 (RRRLARRLIFFIRRAW-NH2), exhibited strong antibacterial activities against clinically isolated MRSA with MICs of 3-6 µM, but no obvious cytotoxicity was observed. Consistently, CGS19 and CGS20 exerted rapid bactericidal activity and effectively induced 5.9 and 5.8 log reduction of MRSA counts in mouse subeschar, respectively. Further, CGS19 and CGS20 kill bacteria not only through disturbing membrane integrity but also by binding formate-tetrahydrofolate ligase, a key enzyme in the folate metabolism pathway, thereby inhibiting the folate pathway of MRSA. CGS19 and CGS20 are promising lead candidates for drug development against MRSA infection. The dual mechanisms on the identical peptide sequence or scaffold might be an underappreciated manner of treating life-threatening pathogens.

Advances in the structural characterization of complexes of therapeutic antibodies with PD-1 or PD-L1.

Antibody-based immune checkpoint blockade (ICB)-based therapeutics have become effective clinical applications for cancers. Applications of monoclonal antibodies (mAbs) to de-activate the PD-1-PD-L1 pathway could effectively reverse the phenotype of depleted activated thymocytes (T cells) to recover their anti-tumoral activities. High-resolution structures of the complexes of the therapeutic monoclonal antibodies with PD-1 or PD-L1 have revealed the key inter-molecular interactions and provided valuable insights into the fundamental mechanisms by which these antibodies inhibit PD-L1-PD-1 binding. Each anti-PD-1 mAb exhibits a unique blockade mechanism, such as interference with large PD-1-PD-L1 contacting interfaces, steric hindrance by overlapping a small area of this site, or binding to an N-glycosylated site. In contrast, all therapeutic anti-PD-L1 mAbs bind to a similar area of PD-L1. Here, we summarized advances in the structural characterization of the complexes of commercial mAbs that target PD-1 or PD-L1. In particular, we focus on the unique characteristics of those mAb structures, epitopes, and blockade mechanisms. It is well known that the use of antibodies as anti-tumor drugs has increased recently and both PD-1 and PD-L1 have attracted substantial attention as target for antibodies derived from new technologies. By focusing on structural characterization, this review aims to aid the development of novel antibodies targeting PD-1 or PD-L1 in the future.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

NMR Spectroscopic Studies Reveal the Critical Role of the Isopeptide Bond in Forming the Otherwise Unstable SpyTag-SpyCatcher Mutant Complexes.

The interplay between protein folding and chemical reaction has been an intriguing subject. In this contribution, we report the study of SpyTag and SpyCatcher reactive mutants using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, liquid chromatography and mass spectrometry, circular dichroism, and NMR spectroscopy. It was found that the wild-type SpyCatcher is well-folded in solution and docks with SpyTag to form an intermediate that promotes isopeptide bond formation. By contrast, the double mutant SpyCatcherVA is disordered in solution yet remains reactive toward SpyTag, forming a well-folded covalent complex. Control experiments using the catalytically inactive mutants further reveal the critical role of the isopeptide bond in stabilizing the otherwise loose SpyTag-SpyCatcherVA complex, amplifying the effect of the minute sequence disparity. We believe that the synergy between protein folding and isopeptide bonding is an effective way to enhance protein stability and engineer protein-protein interactions.

Application of nuclear magnetic resonance spectroscopy in food adulteration determination: the example of Sudan dye I in paprika powder.

Carcinogenic Sudan I has been added illegally into spices for an apparent freshness. 1H solution and solid-state (SS) nuclear magnetic resonance (NMR) spectroscopies were applied and compared for determination of Sudan I in paprika powders (PPs). For solution NMR, PPs spiked with Sudan I were extracted with acetonitrile, centrifuged, rotor-evaporated, and re-dissolved in DMSO-d6 for spectral collection. For SSNMR, Sudan I contaminated PPs were mixed with DMSO-d6 solution and used for spectral collection. Linear regression models constructed for quantitative analyses resulted in the average accuracies for unknown samples as 98% and 105%, respectively. Limits of detection for the solution NMR and SSNMR spectrometers were 6.7 and 128.6 mg kg-1, while the limits of quantification were 22.5 and 313.7 mg kg-1. The overall analysis time required by both methods was similar (35 and 32 min). Both NMR techniques are feasible for rapid and accurate determination of Sudan I adulteration in PPs.

Mechanism of Cu(A) assembly.

Copper is essential for proper functioning of cytochrome c oxidases, and therefore for cellular respiration in eukaryotes and many bacteria. Here we show that a new periplasmic protein (PCu(A)C) selectively inserts Cu(I) ions into subunit II of Thermus thermophilus ba(3) oxidase to generate a native Cu(A) site. The purported metallochaperone Sco1 is unable to deliver copper ions; instead, it works as a thiol-disulfide reductase to maintain the correct oxidation state of the Cu(A) cysteine ligands.

An atomic-level investigation of the disease-causing A629P mutant of the Menkes protein, ATP7A.

Menkes disease is a fatal disease that can be induced by various mutations in the ATP7A gene, leading to unpaired uptake of dietary copper. The ATP7A gene encodes a copper(I)-translocating ATPase. Here the disease-causing A629P mutation, which occurs in the last of the six copper(I)-binding soluble domains of the ATPase (hereafter MNK6), was investigated. To understand why this apparently minor amino acid replacement is pathogenic, the solution structures and dynamics on various time-scales of wild-type and A629P-MNK6 were determined both in the apo- and copper(I)-loaded forms. The interaction in vitro with the physiological ATP7A copper(I)-donor (HAH1) was additionally studied. The A629P mutation makes the protein beta-sheet more solvent accessible, possibly resulting in an enhanced susceptibility of ATP7A to proteolytic cleavage and/or in reduced capability of copper(I)-translocation. A small reduction of the affinity for copper(I) is also observed. Both effects could concur to pathogenicity.