RESUMO
This work presents a protein structure that has been designed purely for aesthetic reasons, symbolizing decades of coiled-coil research and praising its most fundamental model system, the GCN4 leucine zipper. The GCN4 leucine zipper is a highly stable coiled coil which can be tuned to adopt different oligomeric states via mutation of its core residues. For these reasons it is used in structural studies as a stabilizing fusion adaptor. On the occasion of the 50th birthday of Andrei N. Lupas, we used it to create the first personalized protein structure: we fused the sequence ANDREI-N-LVPAS in heptad register to trimeric GCN4 adaptors and determined its structure by X-ray crystallography. The structure demonstrates the robustness and versatility of GCN4 as a fusion adaptor. We learn how proline can be accommodated in trimeric coiled coils, and put the structure into the context of the other GCN4-fusion structures known to date.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Prolina , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Bacterial cell division requires the coordinated action of cell division proteins and murein (peptidoglycan) synthases. Interactions involving the essential cell division protein FtsN and murein synthases were studied by affinity chromatography with membrane fraction. The murein synthases PBP1A, PBP1B, and PBP3 had an affinity to immobilized FtsN. FtsN and PBP3, but not PBP1A, showed an affinity to immobilized PBP1B. The direct interaction between FtsN and PBP1B was confirmed by pulldown experiments and surface plasmon resonance. The interaction was also detected by bacterial two-hybrid analysis. FtsN and PBP1B could be cross-linked in intact cells of the wild type and in cells depleted of PBP3 or FtsW. FtsN stimulated the in vitro murein synthesis activities of PBP1B. Thus, FtsN could have a role in controlling or modulating the activity of PBP1B during cell division in Escherichia coli.
Assuntos
Divisão Celular/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo , Cromatografia de Afinidade , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Proteínas de Ligação às Penicilinas/química , Peptidoglicano Glicosiltransferase/química , Ligação Proteica/fisiologia , D-Ala-D-Ala Carboxipeptidase Tipo Serina/química , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-HíbridoRESUMO
The translocon at the inner envelope membrane of chloroplasts (Tic) plays a central role in plastid biogenesis by coordinating the sorting of nucleus-encoded preproteins across the inner membrane and coordinating the interactions of preproteins with the processing and folding machineries of the stroma. Despite these activities, the precise roles of known Tic proteins in translocation, sorting, and preprotein maturation have not been defined. In this report, we examine the in vivo function of a major Tic component, Tic110. We demonstrate that Arabidopsis thaliana Tic110 (atTic110) is essential for plastid biogenesis and plant viability. The downregulation of atTic110 expression results in the reduced accumulation of a wide variety of plastid proteins. The expression of dominant negative mutants of atTic110 disrupts assembly of Tic complexes and the translocation of preproteins across the inner envelope membrane. Together, these data suggest that Tic110 plays a general role in the import of nuclear-encoded preproteins as a common component of Tic complexes.