Phosphorylation by kinases governs many key cellular and extracellular processes, such as transcription, cell cycle progression, differentiation, secretion and apoptosis. Unsurprisingly, tight and precise kinase regulation is a prerequisite for normal cell functioning, whereas kinase dysregulation often leads to disease. Moreover, the functions of many kinases are regulated through protein-protein interactions, which in turn are mediated by phosphorylated motifs and often involve associations with the scaffolding and chaperon protein 14-3-3. Therefore, the aim of this review article is to provide an overview of the state of the art on 14-3-3-mediated kinase regulation, focusing on the most recent mechanistic insights into these important protein-protein interactions and discussing in detail both their structural aspects and functional consequences.
- MeSH
- alosterická regulace genetika MeSH
- apoptóza genetika MeSH
- fosforylace genetika MeSH
- lidé MeSH
- mitogenem aktivované proteinkinasy p38 genetika MeSH
- proteinkinasy genetika MeSH
- proteiny 14-3-3 genetika MeSH
- signální transdukce genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: The Hsp70 proteins maintain proteome integrity through the capacity of their nucleotide- and substrate-binding domains (NBD and SBD) to allosterically regulate substrate affinity in a nucleotide-dependent manner. Crystallographic studies showed that Hsp70 allostery relies on formation of contacts between ATP-bound NBD and an interdomain linker, accompanied by SBD subdomains docking onto distinct sites of the NBD leading to substrate release. However, the mechanics of ATP-induced SBD subdomains detachment is largely unknown. METHODS: Here, we investigated the structural and allosteric properties of human HSPA1A using hydrogen/deuterium exchange mass spectrometry, ATPase assays, surface plasmon resonance and fluorescence polarization-based substrate binding assays. RESULTS: Analysis of HSPA1A proteins bearing mutations at the interface of SBD subdomains close to the interdomain linker (amino acids L399, L510, I515, and D529) revealed that this region forms a folding unit stabilizing the structure of both SBD subdomains in the nucleotide-free state. The introduced mutations modulate HSPA1A allostery as they localize to the NBD-SBD interfaces in the ATP-bound protein. CONCLUSIONS: These findings show that residues forming the hydrophobic structural unit stabilizing the SBD structure are relocated during ATP-activated detachment of the SBD subdomains to different NBD-SBD docking interfaces enabling HSPA1A allostery. GENERAL SIGNIFICANCE: Mutation-induced perturbations tuned HSPA1A sensitivity to peptide/protein substrates and to Hsp40 in a way that is common for other Hsp70 proteins. Our results provide an insight into structural rearrangements in the SBD of Hsp70 proteins and highlight HSPA1A-specific allostery features, which is a prerequisite for selective targeting in Hsp-related pathologies.
- MeSH
- adenosintrifosfát chemie genetika MeSH
- alosterická regulace genetika MeSH
- konformace proteinů * MeSH
- lidé MeSH
- mutace genetika MeSH
- proteinové domény genetika MeSH
- proteiny tepelného šoku HSP70 chemie genetika MeSH
- vazba proteinů genetika MeSH
- vazebná místa genetika MeSH
- vodík-deuteriová výměna MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many Gram-positive pathogenic bacteria employ ribosomal protection proteins (RPPs) to confer resistance to clinically important antibiotics. In Bacillus subtilis, the RPP VmlR confers resistance to lincomycin (Lnc) and the streptogramin A (SA) antibiotic virginiamycin M (VgM). VmlR is an ATP-binding cassette (ABC) protein of the F type, which, like other antibiotic resistance (ARE) ABCF proteins, is thought to bind to antibiotic-stalled ribosomes and promote dissociation of the drug from its binding site. To investigate the molecular mechanism by which VmlR confers antibiotic resistance, we have determined a cryo-electron microscopy (cryo-EM) structure of an ATPase-deficient B. subtilis VmlR-EQ2 mutant in complex with a B. subtilis ErmDL-stalled ribosomal complex (SRC). The structure reveals that VmlR binds within the E site of the ribosome, with the antibiotic resistance domain (ARD) reaching into the peptidyltransferase center (PTC) of the ribosome and a C-terminal extension (CTE) making contact with the small subunit (SSU). To access the PTC, VmlR induces a conformational change in the P-site tRNA, shifting the acceptor arm out of the PTC and relocating the CCA end of the P-site tRNA toward the A site. Together with microbiological analyses, our study indicates that VmlR allosterically dissociates the drug from its ribosomal binding site and exhibits specificity to dislodge VgM, Lnc, and the pleuromutilin tiamulin (Tia), but not chloramphenicol (Cam), linezolid (Lnz), nor the macrolide erythromycin (Ery).
- MeSH
- ABC transportéry chemie genetika metabolismus MeSH
- alosterická regulace účinky léků genetika MeSH
- antibakteriální látky chemie farmakologie MeSH
- Bacillus subtilis enzymologie genetika MeSH
- bakteriální léková rezistence * MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- ribozomy chemie genetika metabolismus MeSH
- RNA transferová chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- alosterická regulace genetika MeSH
- receptory metabotropního glutamátu chemie MeSH
- Publikační typ
- kongresy MeSH
