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1.
Biochim Biophys Acta Proteins Proteom ; 1872(1): 140967, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-37757925

RESUMO

CBL1 is an EF hand Ca2+ binding protein from A. thaliana that is involved in the detection of cellular Ca2+ signals and the downstream signal transmission by interaction with the protein kinase CIPK23. So far, the structure and calcium ion binding affinities of CBL1 remain elusive. In this study it was observed that CBL1 tends to form higher oligomeric states due to an intrinsic hydrophobicity and the presence of the detergent BriJ35 was required for the purification of monomeric and functional protein. Functional insights into the in vitro Ca2+ binding capabilities of CBL1 were obtained by isothermal titration calorimetry (ITC) of the wildtype protein as well as single site EF hand mutants. Based on our results, a binding model of CBL1 for Ca2+in vivo is proposed. Additionally, upon both, ITC measurements and the analysis of an AlphaFold2 model of CBL1, we could gain first insights into the formation of the dimer interface. We could identify an area around EF hand 4 to be relevant for the structural and functional integrity of monomeric CBL1 and likely EF hand 1 to be involved in the dimer interface.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ligação ao Cálcio , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Ligação Proteica
2.
Sci Rep ; 14(1): 8994, 2024 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-38637678

RESUMO

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Domínios Proteicos
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