RESUMEN
Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Proteínas de Ciclo Celular/química , Proteínas del Citoesqueleto/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.
Asunto(s)
Proteínas ADAM/metabolismo , Tiorredoxinas/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Reactivos de Enlaces Cruzados/farmacología , Activación Enzimática/efectos de los fármacos , Células HEK293 , Células HeLa , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Immunoblotting , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Acetato de Tetradecanoilforbol/farmacología , Tiorredoxinas/químicaRESUMEN
Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.