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1.
BMC Biol ; 20(1): 176, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945584

RESUMO

BACKGROUND: Calmodulin (CaM) is an evolutionarily conserved eukaryotic multifunctional protein that functions as the major sensor of intracellular calcium signaling. Its calcium-modulated function regulates the activity of numerous effector proteins involved in a variety of physiological processes in diverse organs, from proliferation and apoptosis, to memory and immune responses. Due to the pleiotropic roles of CaM in normal and pathological cell functions, CaM antagonists are needed for fundamental studies as well as for potential therapeutic applications. Calmidazolium (CDZ) is a potent small molecule antagonist of CaM and one the most widely used inhibitors of CaM in cell biology. Yet, CDZ, as all other CaM antagonists described thus far, also affects additional cellular targets and its lack of selectivity hinders its application for dissecting calcium/CaM signaling. A better understanding of CaM:CDZ interaction is key to design analogs with improved selectivity. Here, we report a molecular characterization of CaM:CDZ complexes using an integrative structural biology approach combining SEC-SAXS, X-ray crystallography, HDX-MS, and NMR. RESULTS: We provide evidence that binding of a single molecule of CDZ induces an open-to-closed conformational reorientation of the two domains of CaM and results in a strong stabilization of its structural elements associated with a reduction of protein dynamics over a large time range. These CDZ-triggered CaM changes mimic those induced by CaM-binding peptides derived from physiological protein targets, despite their distinct chemical natures. CaM residues in close contact with CDZ and involved in the stabilization of the CaM:CDZ complex have been identified. CONCLUSION: Our results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists. Calmidazolium is a potent and widely used inhibitor of calmodulin, a major mediator of calcium-signaling in eukaryotic cells. Structural characterization of calmidazolium-binding to calmodulin reveals that it triggers open-to-closed conformational changes similar to those induced by calmodulin-binding peptides derived from enzyme targets. These results provide molecular insights into CDZ-induced dynamics and structural changes of CaM leading to its inhibition and open the way to the rational design of more selective CaM antagonists.


Assuntos
Cálcio , Calmodulina , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Imidazóis , Ligação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
2.
Adv Sci (Weinh) ; 8(9): 2003630, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33977052

RESUMO

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454-484), which exhibits membrane-active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.


Assuntos
Toxina Adenilato Ciclase/metabolismo , Calmodulina/metabolismo , Células Eucarióticas/metabolismo , Domínios Proteicos/fisiologia , Sítios de Ligação/fisiologia , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia
3.
Vaccine ; 28(42): 6930-41, 2010 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-20728521

RESUMO

HIV-Tat based vaccines have been proposed as an attractive option to prevent or treat AIDS. A vaccine to induce optimal anti-Tat neutralizing antibody responses was designed by inserting this protein, or its dominant B-cell epitope, into the CyaA vector, which targets dendritic cells (DC). Tat was inserted into various sites of CyaA, including regions that do not translocate into the cytosol of the targeted DC. The presentation of the Tat CD4(+) T-cell epitope delivered by the CyaA-Tat proteins was observed with a recombinant CyaA in which the entire AC domain was replaced by the entire Tat protein (Tat-Δ373 CyaA) but was abolished with large deletions of the N-terminal region. Moreover, CyaA carrying multiple copies of the dominant Tat: 1-21 B-cell epitope were shown to induce high titers of anti-Tat antibodies, even after a single immunization, that persisted up to 10 weeks post-immunization.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Células Dendríticas/imunologia , Epitopos de Linfócito B/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Toxina Adenilato Ciclase/imunologia , Animais , Apresentação de Antígeno , Células CHO , Ilhas de CpG , Cricetinae , Cricetulus , Citocinas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Anticorpos Anti-HIV/sangue , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Poli I-C/imunologia , Proteínas Recombinantes/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
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