RESUMEN
A Type 4b secretion system (T4bSS) is required for Legionella growth in alveolar macrophages. IcmQ associates with IcmR, binds to membranes, and has a critical role in the T4bSS. We have now solved a crystal structure of IcmR-IcmQ to further our understanding of this complex. This structure revealed an amphipathic four-helix bundle, formed by IcmR and the N-terminal domain of IcmQ, which is linked to a novel C-terminal domain of IcmQ (Qc) by a linker helix. The Qc domain has structural homology with ADP ribosyltransferase domains in certain bacterial toxins and binds NAD(+) with a dissociation constant in the physiological range. Structural homology and molecular dynamics were used to identify an extended NAD(+) binding site on Qc, and the resulting model was tested by mutagenesis and binding assays. Based on the data, we suggest that IcmR-IcmQ binds to membranes, where it may interact with, or perhaps modify, a protein in the T4bSS when NAD(+) is bound.
Asunto(s)
Proteínas Bacterianas/química , Legionella pneumophila , NAD/química , Secuencia de Aminoácidos , Sistemas de Secreción Bacterianos , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Membrana Dobles de Lípidos/química , Modelos Moleculares , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Transporte de Proteínas/genética , Animales , Línea Celular , Línea Celular Tumoral , Citosol/metabolismo , Citosol/microbiología , Femenino , Células HEK293 , Humanos , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Mutación , Pliegue de Proteína , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Células U937 , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
Legionella pneumophila promotes intracellular growth by moving bacterial proteins across membranes via the Icm/Dot system. A strategy was devised to identify large numbers of Icm/Dot translocated proteins, and the resulting pool was used to identify common motifs that operate as recognition signals. The 3' end of the sidC gene, which encodes a known translocated substrate, was replaced with DNA encoding 200 codons from the 3' end of 442 potential substrate-encoding genes. The resulting hybrid proteins were then tested in a high throughput assay, in which translocated SidC antigen was detected by indirect immunofluorescence. Among translocated substrates, regions of 6-8 residues called E Blocks were identified that were rich in glutamates. Analysis of SidM/DrrA revealed that loss of three Glu residues, arrayed in a triangle on an α-helical surface, totally eliminated translocation of a reporter protein. Based on this result, a second strategy was employed to identify Icm/Dot substrates having carboxyl terminal glutamates. From the fusion assay and the bioinformatic queries, carboxyl terminal sequences from 49 previously unidentified proteins were shown to promote translocation into target cells. These studies indicate that by analysing subsets of translocated substrates, patterns can be found that allow predictions of important motifs recognized by Icm/Dot.
Asunto(s)
Proteínas Bacterianas/metabolismo , Legionella pneumophila/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Bacterianas/genética , Genes Reporteros , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
RGS14 is a multifunctional protein that contains an RGS domain, which binds active Gi/o alpha-GTP, a GoLoco/GPR domain, which binds inactive Gi alpha-GDP, and a tandem Rap1/2 binding domain (RBD). Studies were initiated to determine the roles of these domains and their interactions with Gi alpha on RGS14 subcellular localization. We report that RGS14 dynamic subcellular localization in HeLa cells depends on distinct domains and selective interactions with preferred Gi alpha isoforms. RGS14 shuttles rapidly between the nucleus and cytoplasm, and associates with centrosomes during interphase and mitosis. RGS14 localization to the nucleus depends on the RGS and RBD domains, its translocation out of the nucleus depends on the GoLoco/GPR domain, and its localization to centrosomes depends on the RBD domain. Gi alpha subunits (Gi alpha1, 2 and 3) localize predominantly at the plasma membrane. RGS14 binds directly to inactive and active forms of Gi alpha1 and Gi alpha3, but not Gi alpha2, both as a purified protein and when recovered from cells. RGS14 localizes predominantly at the plasma membrane in cells with inactive Gi alpha1 and Gi alpha3, but not Gi alpha2, whereas less RGS14 associates with active Gi alpha1/3 at the plasma membrane. RGS14 binding to inactive, but not active Gi alpha1/3 also prevents association with centrosomes or nuclear localization. Removal or functional inactivation of the GoLoco/GPR domain causes RGS14 to accumulate at centrosomes and in the nucleus, but renders it insensitive to recruitment to the plasma membrane by Gi alpha1/3. These findings highlight the importance of the GoLoco/GPR domain and its interactions with Gi alpha1/3 in determining RGS14 subcellular localization and linked functions.