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

Mitochondrial homeostasis: How do dimers of mitofusins mediate mitochondrial fusion?

Mitochondria have high fusion and fission rates to maintain their size and number throughout the cell cycle. How is fusion mediated? New structural studies propose mechanisms by which the dynamin-like mitofusin proteins promote fusion of mitochondria

Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily

Dynamin superfamily proteins are multi-domain mechano-chemical GTPases which are implicated in nucleotide-dependent membrane remodeling events. A prominent feature of these proteins is their assembly-stimulated mechanism of GTP hydrolysis. The molecular basis for this reaction has been initially clarified for the dynamin-related guanylate binding protein 1 (GBP1) and involves the transient dimerization of the GTPase domains in a parallel head-to-head fashion. A catalytic arginine finger from the phosphate binding (P-) loop is repositioned towards the nucleotide of the same molecule to stabilize the transition state of GTP hydrolysis. Dynamin uses a related dimerization-dependent mechanism, but instead of the catalytic arginine, a monovalent cation is involved in catalysis. Still another variation of the GTP hydrolysis mechanism has been revealed for the dynamin-like Irga6 which bears a glycine at the corresponding position in the P-loop. Here, we highlight conserved and divergent features of GTP hydrolysis in dynamin superfamily proteins and show how nucleotide binding and hydrolysis are converted into mechano-chemical movements. We also describe models how the energy of GTP hydrolysis can be harnessed for diverse membrane remodeling events, such as membrane fission or fusion

A complex water network contributes to high-affinity binding in an antibody-antigen interface

This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment), J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA). The 1.89 {Angstrom} X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: "Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma", Mol. Oncol. 9 (7) (2015) pp. 1348–58

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

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