Lothar Gremer

The C2 domain of SynGAP is essential for stimulation of the Rap GTPase reaction

The brain‐specific synaptic guanosine triphosphatase (GTPase)‐activating protein (SynGAP) is important in synaptic plasticity. It shows dual specificity for the small guanine nucleotide‐binding proteins Rap and Ras. Here, we show that RapGAP activity of SynGAP requires its C2 domain. In contrast to the isolated GAP domain, which does not show any detectable RapGAP activity, a fragment comprising the C2 and GAP domains (C2–GAP) stimulates the intrinsic GTPase reaction of Rap by approximately 1 × 104. The C2–GAP crystal structure, complemented by modelling and biochemical analyses, favours a concerted movement of the C2 domain towards the switch II region of Rap to assist in GTPase stimulation. Our data support a catalytic mechanism similar to that of canonical RasGAPs and distinct from the canonical RapGAPs. SynGAP presents the first example, to our knowledge, of a GAP that uses a second domain for catalytic activity, thus pointing to a new function of C2 domains.

Germline KRAS mutations cause aberrant biochemical and physical properties leading to developmental disorders.

The KRAS gene is the most common locus for somatic gain‐of‐function mutations in human cancer. Germline KRAS mutations were shown recently to be associated with developmental disorders, including Noonan syndrome (NS), cardio‐facio‐cutaneous syndrome (CFCS), and Costello syndrome (CS). The molecular basis of this broad phenotypic variability has in part remained elusive so far. Here, we comprehensively analyzed the biochemical and structural features of ten germline KRAS mutations using physical and cellular biochemistry. According to their distinct biochemical and structural alterations, the mutants can be grouped into five distinct classes, four of which markedly differ from RAS oncoproteins. Investigated functional alterations comprise the enhancement of intrinsic and guanine nucleotide exchange factor (GEF) catalyzed nucleotide exchange, which is alternatively accompanied by an impaired GTPase‐activating protein (GAP) stimulated GTP hydrolysis, an overall loss of functional properties, and a deficiency in effector interaction. In conclusion, our data underscore the important role of RAS in the pathogenesis of the group of related disorders including NS, CFCS, and CS, and provide clues to the high phenotypic variability of patients with germline KRAS mutations. Hum Mutat 31:1–11, 2010.

Synthesis of the Rheb and K-Ras4B GTPases

K-Ras4B is the mostimportant isoform of the Ras proteins, which hold a centralposition in the transduction of growth-promoting signalsacross the plasma membrane to regulate cell growth anddifferentiation. Mutations in Ras that lead to misregulatedsignaling are found in approximately 30% of all humancancers.

Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans. Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli.

The carbon monoxide (CO) dehydrogenase ofOligotropha carboxidovorans is composed of anS-selanylcysteine-containing 88.7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)2 subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)2- or (LS)2-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced inEscherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)2 species occurred with second-order kinetics (k +1 = 1350m −1 s−1) and high affinity (K d = 1.0 × 10−9 m). The structure of the resulting flavo (LMS)2 species at a 2.8-Å resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas theS-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein.

Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction.

Abstract Down-regulation of Ras signalling is mediated by specific GTPase-activating proteins (GAPs), which stimulate the very slow GTPase reaction of Ras by 105-fold. The basic features of the GAP activity involve the stabilisation of both switch regions of Ras in the transition state, and the insertion of an arginine finger. In the case of oncogenic Ras mutations, the features of the active site are disturbed. To understand these features in more detail, we investigated the effects of oncogenic mutations of Ras and compared the GAP-stimulated GTPase reaction with the ability to form GAP-mediated aluminium or beryllium fluoride complexes. In general, we found a correlation between the size of the amino acid at position 12, the GTPase activity and ability to form aluminium fluoride complexes. While Gly12 is very sensitive to even the smallest possible structural change, Gly13 is much less sensitive to steric hindrance, but is sensitive to charge. Oncogenic mutants of Ras defective in the GTPase activity can however form ground-state GppNHp complexes with GAP, which can be mimicked by beryllium fluoride binding. We show that beryllium fluoride complexes are less sensitive to structural changes and report on a state close to but different from the ground state of the GAP-stimulated GTPase reaction.

Arl3 regulates a transport system for farnesylated cargo

Arl3 is a small G-protein that is found exclusively in ciliated organisms. In addition, knocking out of Arl3 results in a plethora of ciliopathies. Arl3 is known to bind the photoreceptor (specialized cilia) specific PDE delta subunit (PDE6D), which in turn bind to prenylated proteins. The significance of this interaction and the function of Arl3 in cilia are poorly understood. Here in this study, by solving the crystal structure of a fully modified prenylated (farnesylated) Rheb in complex with PDE6D and comparing it to a structure of PDE6D in complex with the Arl3 homologue Arl2, we show that Arl3 is an allosteric regulator of PDE6D. Arl3, in a nucleotide dependent manner, releases the farnesylated cargo bound to PDE6D. We explain the molecular mechanism of this release and we further verify the mechanism in vitro and by live cell imaging. Based on this study we hypothesize that Arl3 regulate the targeting of prenylated cargo in and out the cilia.