Konstantin S. Usachev

Structures and dynamics of hibernating ribosomes from Staphylococcus aureus mediated by intermolecular interactions of HPF

In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress‐induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation‐promoting factor (SaHPF) that we solved using cryo‐electron microscopy. Our reconstructions reveal that the N‐terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C‐terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD‐dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA. We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.

High-resolution NMR structure of the antimicrobial peptide protegrin-2 in the presence of DPC micelles

AbstractPG-1 adopts a dimeric structure in dodecylphosphocholine (DPC) micelles, and a channel is formed by the association of several dimers but the molecular mechanisms of the membrane damage by non-α-helical peptides are still unknown. The formation of the PG-1 dimer is important for pore formation in the lipid bilayer, since the dimer can be regarded as the primary unit for assembly into the ordered aggregates. It was supposed that only 12 residues (RGGRL-CYCRR-RFCVC-V) are needed to endow protegrin molecules with strong antibacterial activity and that at least four additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin (PG-2) represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi. As the peptide conformation and peptide-to-membrane binding properties are very sensitive to single amino acid substitutions, the solution structure of PG-2 in solution and in a membrane mimicking environment are crucial. In order to find evidence if the oligomerization state of PG-1 in a lipid environment will be the same or not for another protegrins, we investigate in the present work the PG-2 NMR solution structure in the presence of perdeuterated DPC micelles. The NMR study reported in the present work indicates that PG-2 form a well-defined structure (PDB: 2MUH) composed of a two-stranded antiparallel β-sheet when it binds to DPC micelles.

Antimicrobial peptide protegrin-3 adopt an antiparallel dimer in the presence of DPC micelles: a high-resolution NMR study.

A tendency to dimerize in the presence of lipids was found for the protegrin. The dimer formation by the protegrin-1 (PG-1) is the first step for further oligomeric membrane pore formation. Generally there are two distinct model of PG-1 dimerization in either a parallel or antiparallel β-sheet. But despite the wealth of data available today, protegrin dimer structure and pore formation is still not completely understood. In order to investigate a more detailed dimerization process of PG-1 and if it will be the same for another type of protegrins, in this work we used a high-resolution NMR spectroscopy for structure determination of protegrin-3 (RGGGL-CYCRR-RFCVC-VGR) in the presence of perdeuterated DPC micelles and demonstrate that PG-3 forms an antiparallel NCCN dimer with a possible association of these dimers. This structural study complements previously published solution, solid state and computational studies of PG-1 in various environments and validate the potential of mean force simulations of PG-1 dimers and association of dimers to form octameric or decameric β-barrels.

Spatial structure of heptapeptide Aβ16–22 (beta-amyloid Aβ1–40 active fragment) in solution and in complex with a biological membrane model

The spatial structure of an active fragment of beta‐amyloid Aβ1–40 heptapeptide Aβ16–22 (Lys‐Leu‐Val‐Phe‐Phe‐Ala‐Glu) in aqueous buffer solution and in complex with sodium dodecyl sulfate micelles as a model membrane system was investigated by 1H NMR spectroscopy and two‐dimensional NMR (TOCSY, HSQC‐HECADE (Heteronuclear Couplings from ASSCI‐domain experiments with E.COSY‐type crosspeaks), NOESY) spectroscopy. Complex formation was confirmed by the chemical shift changes of the heptapeptides 1H NMR spectra, as well as by the signs and values of the NOE effects in different environments. We compared the spatial structure of the heptapeptide in borate buffer solution and in complex with a model of the cell surface membrane. Copyright

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 β-sheet between two monomers. It was also suggested that there is a possible association of protegrins dimers to form octameric or decameric β-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.