RESUMO
Protein transport into the nucleus is mediated by transport receptors. Import of highly charged proteins, such as histone H1 and ribosomal proteins, requires a dimer of two transport receptors. In this study, we determined the cryo-EM structure of the Imp7:Impß:H1.0 complex, showing that the two importins form a cradle that accommodates the linker histone. The H1.0 globular domain is bound to Impß, whereas the acidic loops of Impß and Imp7 chaperone the positively charged C-terminal tail. Although it remains disordered, the H1 tail serves as a zipper that closes and stabilizes the structure through transient non-specific interactions with importins. Moreover, we found that the GGxxF and FxFG motifs in the Imp7 C-terminal tail are essential for Imp7:Impß dimerization and H1 import, resembling importin interaction with nucleoporins, which, in turn, promote complex disassembly. The architecture of many other complexes might be similarly defined by rapidly exchanging electrostatic interactions mediated by disordered regions.
Assuntos
Núcleo Celular/metabolismo , Histonas/metabolismo , Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Sítios de Ligação , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica , Humanos , Carioferinas/genética , Carioferinas/ultraestrutura , Modelos Moleculares , Complexos Multiproteicos , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Relação Estrutura-Atividade , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
We investigated the presence of carbapenemases in carbapenem-resistant Pseudomonas aeruginosa isolates, which were collected over a 14-month period in a Turkish hospital, with in-depth molecular characterization of carbapenemase-producing isolates. Among 45 study isolates, 2 isolates were identified as carbapenemase producers by both Carba NP and Carbapenem Inactivation Method tests, and only 1 of them gave a positive result in polymerase chain reaction tests for a carbapenemase gene (blaVIM). Whole genome sequencing of the 2 isolates revealed the presence of blaVIM-5 gene in an ST308 isolate, while the other one expressed IMP-7 in an ST357 isolate; both STs are considered high-risk clones. The 2 carbapenemase-producing isolates were multidrug resistant, as they harbored other resistance determinants, including a variant of the recently described plasmid-encoded fluoroquinolone resistance determinant crpP gene, crpP-2. We report for the first time P. aeruginosa high-risk clones carrying VIM-5- and IMP-7-type carbapenemases with multiple resistance determinants in Turkey.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , DNA Bacteriano/genética , Fluoroquinolonas/farmacologia , Genoma Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Plasmídeos/genética , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Centros de Atenção Terciária , Turquia , Sequenciamento Completo do Genoma , beta-Lactamases/biossíntese , beta-Lactamases/metabolismoRESUMO
Metallo-ß-lactamases (MBLs) are a growing threat to the continued efficacy of ß-lactam antibiotics. Recently, aspergillomarasmine A (AMA) was identified as an MBL inhibitor, but the mode of inhibition was not fully characterized. Equilibrium dialysis and metal analysis studies revealed that 2 equiv of AMA effectively removes 1 equiv of Zn(II) from MBLs NDM-1, VIM-2, and IMP-7 when the MBL is at micromolar concentrations. Conversely, 1H NMR studies revealed that 2 equiv of AMA remove 2 equiv of Co(II) from Co(II)-substituted NDM-1, VIM-2, and IMP-7 when the MBL/AMA are at millimolar concentrations. Our findings reveal that AMA inhibits the MBLs by removal of the active site metal ions required for ß-lactam hydrolysis among the most clinically significant MBLs.