RESUMO
The kinetic and thermodynamic consequences of intrinsic disorder in protein-protein recognition are controversial. We address this by inducing one partner of the high-affinity colicin E3 rRNase domain-Im3 complex (K(d) ≈ 10(-12) M) to become an intrinsically disordered protein (IDP). Through a variety of biophysical measurements, we show that a single alanine mutation at Tyr507 within the hydrophobic core of the isolated colicin E3 rRNase domain causes the enzyme to become an IDP (E3 rRNase(IDP)). E3 rRNase(IDP) binds stoichiometrically to Im3 and forms a structure that is essentially identical to the wild-type complex. However, binding of E3 rRNase(IDP) to Im3 is 4 orders of magnitude weaker than that of the folded rRNase, with thermodynamic parameters reflecting the disorder-to-order transition on forming the complex. Critically, pre-steady-state kinetic analysis of the E3 rRNase(IDP)-Im3 complex demonstrates that the decrease in affinity is mostly accounted for by a drop in the electrostatically steered association rate. Our study shows that, notwithstanding the advantages intrinsic disorder brings to biological systems, this can come at severe kinetic and thermodynamic cost.
Assuntos
Colicinas/metabolismo , Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Mapas de Interação de Proteínas , Ribonucleases/metabolismo , Colicinas/química , Colicinas/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Modelos Moleculares , Mutação Puntual , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Ribonucleases/química , Ribonucleases/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , TermodinâmicaRESUMO
Strains of Clostridium difficile produce toxins A and B that can cause diarrhoea and pseudomembranous colitis. Currently, there is no preventative therapy for this infection but antibodies to the toxins provide protection, therefore a toxoid-based vaccine is needed. To evaluate thermal stability, a lyophilized and liquid formulation of toxoids A and B were stored at a range of temperatures for 5 weeks. Changes in toxoid structures and immune responses in an animal model before and after the incubation period were assessed. The structural integrity and the immune responses to liquid formulations were affected when stored at 56°C but the lyophilized formulation was thermally stable and same treatment did not result in significant loss of immunological responses when immunized in an animal model.
Assuntos
Clostridioides difficile/imunologia , Toxoides/química , Animais , Chlorocebus aethiops , Cromatografia em Gel , Cricetinae , Estabilidade de Medicamentos , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulina A/sangue , Mesocricetus , Testes de Neutralização , Toxoides/efeitos adversos , Toxoides/imunologia , Células VeroRESUMO
The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.
Assuntos
Colicinas/toxicidade , Escherichia coli/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Ribossômico 16S/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Eletroforese em Gel de Poliacrilamida , Modelos Biológicos , RNA Bacteriano/metabolismoRESUMO
Colicins are a family of antibacterial cytotoxins produced by Escherichia coli and released into the environment to reduce competition from other bacterial strains. Colicins kill the target cell by a variety of effects that include depolarisation of the cytoplasmic membrane, a non-specific DNase activity, a highly specific RNase activity or by inhibition of murein synthesis. This review summarises some important findings that implicate colicins as potential anti-tumor agents. Colicins appear to inhibit proliferation of tumor cell lines in a colicin-type--and cell line-dependent fashion and are more toxic to tumor cells than to normal cells within the body. This opens a potential for using bacterial colicins in combating cancer and raises a number of questions concerning the mechanism of action of colicins in targeting tumor cells, their specificity and applicability as anti-tumor therapeutics.
