Structural and functional analysis of the GTPase Activating Protein of the small guanine nucleotide binding protein Rap1

Rap1GAP is the founding member of a family of GTPase activating proteins (GAPs) for the small guanine nucleotide binding protein (GNBP) Rap1, which show no sequence homology to GAPs of other small GNBPs. Rap1 does not have a catalytic glutamine residue which is essential for the intrinsic and GAP mediated GTP hydrolysis of all other small GNBPs. In this thesis, the structure and the mechanism of GAP catalysed GTP hydrolysis are examined. Most GAPs provide a catalytic arginine residue to the GNBP to complement the incomplete catalytic machinery. However, site-directed mutagenesis revealed that Rap1GAP does not employ a catalytic arginine. To understand the novel reaction mechanism, the structure of Rap1GAP was determined by X-ray crystallography to a maximal resolution of 2,9 Å. Initial phases were obtained by selenomethionine substituted crystals and a SIRAS phasing protocol. The structure was built and refined to an Rcryst= 23,4% and an Rfree of 27,6%. Two Rap1GAP dimers were observed in the asymmetric unit, consistent with gel filtration experiments in which also dimerisation was observed. A Rap1GAP monomer consists of two domains. Both domains show a mixed alpha-beta fold and were named dimerisation and catalytic domain, respectively. Surprisingly, the catalytic domain has structural similarity to the G domain of GNBPs itself suggesting a common evolutionary origin. No structural similarity to any other GAP was observed. By site-directed mutagenesis, it was shown that dimerisation is not required for GAP function. However, both domains are necessary for full catalytic activity. Mutations around a highly invariant helix, the putative interaction helix, dramatically reduced GAP activity. Using a single-turnover fluorescence reporter assay it could be conclusively proven that Rap1GAP employs a catalytic asparagine from the interaction helix to stimulate GTP hydrolysis in Rap1. In the absence of this asparagine side-chain Rap1GAP was completely inactive but could still bind to Rap1●GTP. In contrast to the wild-type, the Rap1GAP(N290A) mutant can not associate with a transition state mimic of Rap1 GTP hydrolysis. Thus, Rap1GAP is the first example of a GAP which provides a catalytic asparagine for catalysis. Based on the analysis of various mutants, a model for the interaction of Rap1GAP with Rap1 is proposed. The results of this thesis have implications for the disease Tuberous sclerosis. Loss of function mutations in the Rap1GAP homologue Tuberin are associated with this disease and can be rationalised in the view of this work

Pathophysiological Role of Caveolae in Hypertension

Caveolae, flask-shaped cholesterol-, and glycosphingolipid-rich membrane microdomains, contain caveolin 1, 2, 3 and several structural proteins, in particular Cavin 1-4, EHD2, pacsin2, and dynamin 2. Caveolae participate in several physiological processes like lipid uptake, mechanosensitivity, or signaling events and are involved in pathophysiological changes in the cardiovascular system. They serve as a specific membrane platform for a diverse set of signaling molecules like endothelial nitric oxide synthase (eNOS), and further maintain vascular homeostasis. Lack of caveolins causes the complete loss of caveolae; induces vascular disorders, endothelial dysfunction, and impaired myogenic tone; and alters numerous cellular processes, which all contribute to an increased risk for hypertension. This brief review describes our current knowledge on caveolae in vasculature, with special focus on their pathophysiological role in hypertension

Characterization of the CD177 interaction with the ANCA antigen proteinase 3

Proteinase 3 is a serine protease found in neutrophil granules and on the extracellular neutrophil membrane (mPR3). mPR3 is a major antigen for anti- neutrophil cytoplasmic antibodies (PR3-ANCAs), autoantibodies causing fatal autoimmune diseases. In most individuals, a subpopulation of neutrophils also produce CD177, proposed to present additional PR3 on the surface, resulting in CD177neg/mPR3low and CD177pos/mPR3high neutrophil subsets. A positive correlation has been shown between mPR3 abundance, disease incidence, and clinical outcome. We present here a detailed investigation of the PR3:CD177 complex, verifying the interaction, demonstrating the effect of binding on PR3 proteolytic activity and explaining the accessibility of major PR3-ANCA epitopes. We observed high affinity PR3:CD177 complex formation by surface plasmon resonance. Using flow cytometry and a PR3-specific FRET assay, we found that CD177 binding reduced the proteolytic activity of PR3 in vitro using purified proteins, in neutrophil degranulation supernatants containing wtPR3 and directly on mPR3high neutrophils and PR3-loaded HEK cells. Finally, CD177pos/mPR3high neutrophils showed no migration advantage in vitro or in vivo when migrating from the blood into the oral cavity. We illuminate details of the PR3:CD177 interaction explaining mPR3 membrane orientation and proteolytic activity with relevance to ANCA activation of the distinct mPR3 neutrophil populations

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. Results Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands. Conclusion We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.Published versio

The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion

The immunity-related GTPases (IRGs) constitute a powerful cell-autonomous resistance system against several intracellular pathogens. Irga6 is a dynamin-like protein that oligomerizes at the parasitophorous vacuolar membrane (PVM) of Toxoplasma gondii leading to its vesiculation. Based on a previous biochemical analysis, it has been proposed that the GTPase domains of Irga6 dimerize in an antiparallel fashion during oligomerization.Leibniz Graduate School grants: (SFB958, SFB635)

FIP200 Claw Domain Binding to p62 Promotes Autophagosome Formation at Ubiquitin Condensates

The autophagy cargo receptor p62 facilitates the condensation of misfolded, ubiquitin-positive proteins and their degradation by autophagy, but the molecular mechanism of p62 signaling to the core autophagy machinery is unclear. Here, we show that disordered residues 326–380 of p62 directly interact with the C-terminal region (CTR) of FIP200. Crystal structure determination shows that the FIP200 CTR contains a dimeric globular domain that we designated the “Claw” for its shape. The interaction of p62 with FIP200 is mediated by a positively charged pocket in the Claw, enhanced by p62 phosphorylation, mutually exclusive with the binding of p62 to LC3B, and it promotes degradation of ubiquitinated cargo by autophagy. Furthermore, the recruitment of the FIP200 CTR slows the phase separation of ubiquitinated proteins by p62 in a reconstituted system. Our data provide the molecular basis for a crosstalk between cargo condensation and autophagosome formation

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5′ exonuclease-mediated nucleic acid degradation

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3

Divergent architecture of the heterotrimeric NatC complex explains N-terminal acetylation of cognate substrates

The heterotrimeric NatC complex, comprising the catalytic Naa30 and the two auxiliary subunits Naa35 and Naa38, co-translationally acetylates the N-termini of numerous eukaryotic target proteins. Despite its unique subunit composition, its essential role for many aspects of cellular function and its suggested involvement in disease, structure and mechanism of NatC have remained unknown. Here, we present the crystal structure of the Saccharomyces cerevisiae NatC complex, which exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes. Cofactor and ligand-bound structures reveal how the first four amino acids of cognate substrates are recognized at the Naa30–Naa35 interface. A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation. Based on detailed structure–function studies, we suggest a catalytic mechanism and identify a ribosome-binding patch in an elongated tip region of NatC. Our study reveals how NAT machineries have divergently evolved to N-terminally acetylate specific subsets of target proteins

Structural insights into crista junction formation by the Mic60-Mic19 complex

Mitochondrial cristae membranes are the oxidative phosphorylation sites in cells. Crista junctions (CJs) form the highly curved neck regions of cristae and are thought to function as selective entry gates into the cristae space. Little is known about how CJs are generated and maintained. We show that the central coiled-coil (CC) domain of the mitochondrial contact site and cristae organizing system subunit Mic60 forms an elongated, bow tie–shaped tetrameric assembly. Mic19 promotes Mic60 tetramerization via a conserved interface between the Mic60 mitofilin and Mic19 CHCH (CC-helix-CC-helix) domains. Dimerization of mitofilin domains exposes a crescent-shaped membrane-binding site with convex curvature tailored to interact with the curved CJ neck. Our study suggests that the Mic60-Mic19 subcomplex traverses CJs as a molecular strut, thereby controlling CJ architecture and function