The Structure of RalF, an ADP-ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

Amor, Juan Carlos; Swails, Jennifer L; Zhu, Xinjun; Roy, Craig R.; Nagai, Hiroki; Ingmundson, Alyssa; Cheng, Xiaodong; Kahn, Richard

doi:10.1074/jbc.m410820200

Cited by 94 publications

(104 citation statements)

References 52 publications

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“…In the absence of chaperones, the prediction would be that these C-terminal translocation domains should be exposed and available for binding to components of the type IV secretion system. The RalF crystal structure has recently been solved (36), and, consistent with this prediction, these data show that the RalF C-terminal domain is located at the end of a long alpha helix comprised of amino acid residues 331-348 (Fig. 6, which is published as supporting information on the PNAS web site).…”

Section: Discussionsupporting

confidence: 58%

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

Nagai

Cambronne

Kagan

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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264

308

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The Legionella pneumophila Dot͞Icm system is a type IV secretion apparatus that transfers bacterial proteins into eukaryotic host cells. The RalF protein is a substrate engaged and translocated into host cells by the Dot͞Icm system. In this study, the mechanism of Dot͞Icm-mediated translocation of RalF has been investigated. It was determined that RalF translocation into host cells occurs before bacterial internalization. Sequences essential for RalF translocation were located at the C terminus of the RalF protein. A fusion protein consisting of a 20-aa C-terminal RalF peptide appended to the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis adenylate cyclase protein was translocated into host cells by the Dot͞Icm system. A leucine (L372) residue at the ؊3 position in relation to the RalF C terminus was critical for translocation. Consistent with RalF L372 playing an important role in substrate recognition by the Dot͞Icm system, most other Dot͞Icm substrates were found to have amino acid residues with similar physical properties at their ؊3 or ؊4 C-terminal positions. These data demonstrate that the Dot͞Icm system can transfer bacterial proteins that modulate host cellular functions before uptake and indicate that substrate recognition involves a C-terminal translocation signal. Thus, Legionella has the ability to engage synthesized substrate proteins and transfer them into host cells on contact, enabling Legionella to rapidly alter transport of the vacuole in which it resides.

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Section: Discussionsupporting

confidence: 58%

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

Nagai

Cambronne

Kagan

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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264

308

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“…The C termini of both RalF and VirE2 are disordered and solvent exposed, as first indicated by secondary structure prediction algorithms and more recently by X-ray crystallography (5,88). These C-terminal domains (CTDs) are thus likely accessible to bind cognate T4SS receptors.…”

Section: C-terminal Secretion Signalsmentioning

confidence: 99%

Biological Diversity of Prokaryotic Type IV Secretion Systems

Martı́nez

Christie

2009

Microbiol Mol Biol Rev

621

780

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SUMMARY Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

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“…The genomes of Legionella and of Rickettsia encode a type IV effector with a Sec7 domain homologous to that of eukaryotic ArfGEFs which functions as a GEF to activate host Arf GTPases. 65 The structure of Legionella RalF showed that the Sec7 domain is strongly autoinhibited by a C-terminal domain that blocks access to the Arf-binding site, 66 and a similar autoinhibited conformation was observed in the Rickettsia RalF homolog. 67 Both bacterial ArfGEFs are strongly activated by membranes, and the membrane-binding site is identical to the elements in the autoinhibitory domain that blocks the Arf-binding site.…”

Section: Regulation Of Bacterial Arfgefs By Membranesmentioning

confidence: 99%

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

2016

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Arf GTPases assemble protein complexes on membranes to carry out major functions in cellular traffic. An essential step is their activation by guanine nucleotide exchange factors (GEFs), whose Sec7 domain stimulates GDP/GTP exchange. ArfGEFs form 2 major families: ArfGEFs with DCB, HUS and HDS domains (GBF1 and BIG1/BIG2 in humans), which act at the Golgi; and ArfGEFs with a Cterminal PH domain (cytohesin, EFA6 and BRAG), which function at the plasma membrane and endosomes. In addition, pathogenic bacteria encode an ArfGEF with a unique membrane-binding domain. Here we review the allosteric regulation of Arf GTPases and their GEFs at the membrane interface. Membranes contribute several regulatory layers: at the GTPase level, where activation by GTP is coupled to membrane recruitment by a built-in structural device; at the Sec7 domain, which manipulates this device to ensure that Arf-GTP is attached to membranes; and at the level of noncatalytic ArfGEF domains, which form direct or GTPase-mediated interactions with membranes that enable a spectacular diversity of regulatory regimes. Notably, we show here that membranes increase the efficiency of a large ArfGEF (human BIG1) by 32-fold by interacting directly with its Nterminal DCB and HUS domains. The diversity of allosteric regulatory regimes suggests that ArfGEFs can function in cascades and circuits to modulate the shape, amplitude and duration of Arf signals in cells. Because Arf-like GTPases feature autoinhibitory elements similar to those of Arf GTPases, we propose that their activation also requires allosteric interactions of these elements with membranes or other proteins.

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The Structure of RalF, an ADP-ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

Cited by 94 publications

References 52 publications

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

A C-terminal translocation signal required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells

Biological Diversity of Prokaryotic Type IV Secretion Systems

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

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