Structural insights into Cullin4-RING ubiquitin ligase remodelling by Vpr from simian immunodeficiency viruses

Viruses have evolved means to manipulate the host's ubiquitin-proteasome system, in order to down-regulate antiviral host factors. The Vpx/Vpr family of lentiviral accessory proteins usurp the substrate receptor DCAF1 of host Cullin4-RING ligases (CRL4), a family of modular ubiquitin ligases involved in DNA replication, DNA repair and cell cycle regulation. CRL4DCAF1 specificity modulation by Vpx and Vpr from certain simian immunodeficiency viruses (SIV) leads to recruitment, poly-ubiquitylation and subsequent proteasomal degradation of the host restriction factor SAMHD1, resulting in enhanced virus replication in differentiated cells. To unravel the mechanism of SIV Vpr-induced SAMHD1 ubiquitylation, we conducted integrative biochemical and structural analyses of the Vpr protein from SIVs infecting Cercopithecus cephus (SIVmus). X-ray crystallography reveals commonalities between SIVmus Vpr and other members of the Vpx/Vpr family with regard to DCAF1 interaction, while cryo-electron microscopy and cross-linking mass spectrometry highlight a divergent molecular mechanism of SAMHD1 recruitment. In addition, these studies demonstrate how SIVmus Vpr exploits the dynamic architecture of the multi-subunit CRL4DCAF1 assembly to optimise SAMHD1 ubiquitylation. Together, the present work provides detailed molecular insight into variability and species-specificity of the evolutionary arms race between host SAMHD1 restriction and lentiviral counteraction through Vpx/Vpr proteins

Phospho-dependent Regulation of SAMHD1 Oligomerisation Couples Catalysis and Restriction.

SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells

Struktur- und Funktionsuntersuchung von immunassoziierten GTPasen

GTPases of Immunity-Associated Proteins (GIMAPs) are a distinctive family of GTPases, which control apoptosis in lymphocytes and play a central role in lymphocyte maturation and lymphocyte-associated diseases. To explore their function and mechanism, we determined crystal structures of several GIMAP family members in different nucleotide-loading and oligomerization states. Nucleotide-free and GDP-bound GIMAP2 were monomeric and revealed a guanine nucleotide binding domain (G domain) of the TRAFAC (Translation Factor associated) GTPase superclass with a unique amphipathic helix alpha 7 packing against the switch II region of the G domain. In the absence of alpha 7 and presence of GTP, GIMAP2 oligomerized via two distinct interfaces in the crystal. GTP-induced stabilization of switch I mediates dimerization across the nucleotide binding site which also involves the GIMAP conserved box, which is a sequence stretch downstream of the G3 motif, and the nucleotide base. Structural rearrangements in switch II appear to induce the release of alpha 7 allowing oligomerization to proceed via a second interface. The unique architecture of the linear oligomer was confirmed by mutagenesis. Furthermore, we showed a function for the GIMAP2 oligomer at the surface of lipid droplets. The structure of the GDP-bound GIMAP5 monomer was very similar to GDP-bound GIMAP2, suggesting the conservation of the fold within the GIMAPs. The protein was shown to partially co-localize with the lysosomal compartment in a T cell line. In the crystal structure of GMPPNP-bound GIMAP7, a dimeric arrangement of the protein was observed. The dimer interface proved to be identical to one of the GIMAP2 oligomerization interfaces. We termed this binding interface G-interface, since it involves the guanine-nucleotide binding site and the bound nucleotide. Using site-directed mutagenesis, we identified a conserved arginine residue within the G-interface, which stimulates the GTPase reaction in the opposing protomer within the GIMAP7 dimer. In GIMAP2, the corresponding arginine plays a structural role in the dimerization process and is not involved in catalysis, since GIMAP2 does not show any GTP-hydrolytic activity. While earlier studies indicated that GIMAPs are related to the septins, the current structure also revealed a strikingly similar nucleotide coordination and dimerization mode as in the dynamin GTPase. Based on this, we re-examined the relationships of the septin- and dynamin-like GTPases and demonstrate that these are likely to have emerged from a common membrane-associated dimerizing ancestor. This ancestral property appears to be critical for the role of GIMAPs as nucleotide-regulated scaffolds on intracellular membranes.GTPasen der immun-assoziierten Proteine (GIMAPs) sind eine spezielle GTPase- Familie, die Apoptosevorgänge in Lymphozyten kontrollieren und eine zentrale Rolle in der Lymphozytenentwicklung und in Lymphozyten-Erkrankungen spielen. Um ihre Funktion und ihre Mechanismen zu verstehen, wurden mehrere GIMAP- Kristallstrukturen gelöst, in verschiedenen Nukleotidbeladungs- und Oligomerisierungsstadien. Nukleotidfreies, monomeres GIMAP2 besteht aus einer Guaninnukleotidbindedomäne (G-Domäne) der translationsfaktor-assoziierten (TRAFAC) Klasse und einer amphipatischen Helix alpha 7, die gegen die switch II Region der G-Domäne faltet. Ohne diese Helix alpha 7 und in GTP-gebundenem Zustand oligomerisiert GIMAP2 über zwei verschiedende Kontaktflächen. GTP- induzierte Stabilisierung der switch I Region ermöglicht die GTP-spezifische Dimerisierung über die GTP-Bindestelle, wobei auch die GIMAP-spezifische konservierte Box beteiligt ist, ein Sequenzabschnitt direkt nach dem G3-Motiv. Die Nukleotidbase selbst spielt auch eine Rolle in der Dimerisierung. GTP- induzierte Strukturänderungen in der switch II Region könnten zur Ablösung von alpha 7 führen und eine weitere Oligomerisierung über eine zweite Kontaktfläche hervorrufen. Die Architektur des Oligomers wurde durch Mutagenese einzelner Aminosäuren und weiterfürende Studien bestätigt. Weiterhin konnten wir in einer T-Zellline die Lokalisierung von GIMAP2 an der Hülle von intrazellulären Lipidtröpfchen zeigen. Die Struktur des GDP- gebundenen, monomeren GIMAP5-Proteins erwies sich der Struktur des GDP- gebundenen GIMAP2 als sehr ähnlich, daher könnte die festgestellte Faltung stellvertretend für die ganze Proteinfamilie gelten. In einer T-Zelllinie wurde eine teilweise Kolokalisierung des Proteins mit Lysosomen festgestellt. In der Kristallstruktur von GMP-PNP-gebundenem GIMAP7 lag das Protein als Dimer vor. Die Dimer-Kontaktfläche ist äquivalent zu der, die in GTP- gebundenem GIMAP2 beobachtet wurde. Durch Mutagenesestudien konnte ein konservierter Arginin-Aminosäurerest identifiziert werden, der die GTP Hydrolyse im gegenüberliegenden Protomer des GIMAP7 Dimers stimuliert. Der entsprechende Argininrest im GTP-abhängigen GIMAP2 Dimer ist hingegen nur ein struktureller Rest, der an der Dimerisierung beteiligt ist. GIMAP2 besitzt auch keinerlei GTP-Hydrolyseaktivität. Frühere Studien zeigten, dass die GIMAP-Familie mit den Septin-Proteinen verwandt ist. Der GTP-abhängige Dimer, der in dieser Arbeit gefunden wurde, zeigt jedoch einen Dimerisierungsmodus, der auch im Dynamin G-Domänendimer vorliegt, dessen Struktur kürzlich ermittelt wurde. Unter diesem Gesichtspunkt wurden die phylogenetischen Beziehungen zwischen Septin- und Dynamin-ähnlichen GTPasen erneut untersucht, und es wurde gezeigt, dass diese einen gemeinsamen dimeren membran- assoziierten Vorfahren besitzen. Diese Ureigenschaft scheint auch wichtig zu sein für die Funktion von GIMAPs als nukleotid-gesteuerte Gerüstproteine an intrazellulären Membranen

GTP-dependent scaffold formation in the GTPase of Immunity Associated Protein family

GTP ases of Immunity-Associated Proteins (GIMAPs) are a family of guanine nucleotide binding (G) proteins which are implicated in the regulation of apoptosis in lymphocytes. GIMAPs are composed of an amino-terminal G domain and carboxy-terminal extensions of varying size. Our recent biochemical and structural analysis of a representative GIMAP family member, GIMAP2, revealed the molecular basis of GTP-dependent oligomerization which involves two interfaces in the G domain. Whereas the amphipathic helix α7 in the C-terminal extension closely folds against the G domain in the GDP-bound state, it might be released in the GTP-bound state to assemble interaction partners. We also showed that the GIMAP2 oligomer functions at the surface of lipid droplets in a Jurkat T cell line. Here, we review our recent work and discuss the GIMAP2 oligomer as a GTP-dependent protein scaffold at the surface of lipid droplets controlling apoptosis