Extent of N-Terminus Folding of Semenogelin 1 Cleavage Product Determines Tendency to Amyloid Formation.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into ß-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.

Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus.

Ribosome biogenesis is a complex and highly accurate conservative process of ribosomal subunit maturation followed by association. Subunit maturation comprises sequential stages of ribosomal RNA and proteins' folding, modification and binding, with the involvement of numerous RNAses, helicases, GTPases, chaperones, RNA, protein-modifying enzymes, and assembly factors. One such assembly factor involved in bacterial 30S subunit maturation is ribosomal binding factor A (RbfA). In this study, we present the crystal (determined at 2.2 Å resolution) and NMR structures of RbfA as well as the 2.9 Å resolution cryo-EM reconstruction of the 30S-RbfA complex from Staphylococcus aureus (S. aureus). Additionally, we show that the manner of RbfA action on the small ribosomal subunit during its maturation is shared between bacteria and mitochondria. The obtained results clarify the function of RbfA in the 30S maturation process and its role in ribosome functioning in general. Furthermore, given that S. aureus is a serious human pathogen, this study provides an additional prospect to develop antimicrobials targeting bacterial pathogens.

The data of heterologous expression protocol for synthesis of 15N, 13C-labeled SEM1(68-107) peptide fragment of homo sapiens semenogelin 1.

The semenogelin 1 protein is secreted in the seminal vesicles. After ejaculation it is split into small peptide fragments using internal proteases. It was shown that the fragments SEM1(45-107), SEM1(49-107), SEM1(68-107) (SEM1(86-107) form amyloid fibrils, which increase the possibility of HIV infection. The article presents a protocol for the synthesis and purification of a 15N, 13C-labeled SEM1(68-107) peptide for further structural studies by high-resolution NMR spectroscopy. The work describes cloning, expression of fusion protein GB1-SEM1(68-107) in E.coli, its purification, removal of GB1 and purification of SEM1(68-107). The purity of SEM1(68-107) samples on each purification steps was evaluated by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) and tricine-SDS-PAGE. The developed protocol allows to obtain SEM1(68-107) peptide for NMR studies (using 3D experiments), instead of costly solid-phase synthesis.

NMR and crystallographic structural studies of the Elongation factor P from Staphylococcus aureus.

Elongation factor P (EF-P) is a translation protein factor that plays an important role in specialized translation of consecutive proline amino acid motifs. EF-P is an essential protein for cell fitness in native environmental conditions. It regulates synthesis of proteins involved in bacterial motility, environmental adaptation and bacterial virulence, thus making EF-P a potential drug target. In the present study, we determined the solution and crystal structure of EF-P from the pathogenic bacteria Staphylococcus aureus at 1.48 Å resolution. The structure can serve as a platform for structure-based drug design of novel antibiotics to combat the growing antibiotic resistance of S. aureus.

Dimerization of long hibernation promoting factor from Staphylococcus aureus: Structural analysis and biochemical characterization.

Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6 Šresolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.

Oligomerization of the antimicrobial peptide Protegrin-5 in a membrane-mimicking environment. Structural studies by high-resolution NMR spectroscopy.

Protegrin pore formation is believed to occur in a stepwise fashion that begins with a nonspecific peptide interaction with the negatively charged bacterial cell walls via hydrophobic and positively charged amphipathic surfaces. There are five known nature protegrins (PG1-PG5), and early studies of PG-1 (PDB ID:1PG1) shown that it could form antiparallel dimer in membrane mimicking environment which could be a first step for further oligomeric membrane pore formation. Later, we solved PG-2 (PDB ID:2MUH) and PG-3 (PDB ID:2MZ6) structures in the same environment and for PG-3 observed a strong dαα NOE effects between residues R18 and F12, V14, and V16. These "inconsistent" with monomer structure NOEs appears due to formation of an additional antiparallel ß-sheet between two monomers. It was also suggested that there is a possible association of protegrins dimers to form octameric or decameric ß-barrels in an oligomer state. In order to investigate a more detailed oligomerization process of protegrins, in the present article we report the monomer (PDB ID: 2NC7) and octamer pore structures of the protegrin-5 (PG-5) in the presence of DPC micelles studied by solution NMR spectroscopy. In contrast to PG-1, PG-2, and PG-3 studies, for PG-5 we observed not only dimer NOEs but also several additional NOEs between side chains, which allows us to calculate an octamer pore structure of PG-5 that was in good agreement with previous AFM and PMF data.