Michael Spoerner

Dynamic properties of the Ras switch I region and its importance for binding to effectors

We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)⋅GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. 31P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. 31P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.

Conformational states of human rat sarcoma (Ras) protein complexed with its natural ligand GTP and their role for effector interaction and GTP hydrolysis.

The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH2p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer 31P NMR experiments and effector binding studies we show that Ras(wt)·Mg2+·GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2. At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1–166) K12 is 11.3. K12 of full-length Ras is >20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (kex) for Ras(wt)·Mg2+·GTP is 7 s−1 and thus 18-fold lower compared with that found for the Ras·GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.

Growth and Thallus Morphogenesis of Ulva mutabilis (Chlorophyta) Depends on A Combination of Two Bacterial Species Excreting Regulatory Factors.

Axenic Ulva mutabilis gametes develop parthenogenetically into callus‐like colonies consisting of undifferentiated cells without normal cell walls. From the accompanying microbial flora of established laboratory strains of U. mutabilis with normal morphology, a Roseobacter, a Sulfitobacter, and a Halomonas species were isolated. Each of these microbe species alone induced the development of the Ulva gametes into thalli composed of differentiated cells with characteristic deficiencies. Typical traits of these thalli were: an enhanced rate of cell division not followed by cell expansion, the presence of unusual cell wall protrusions, and the absence of differentiated rhizoid cells. The addition of a Cytophaga species, also derived from the same microbial flora, to either one of the three other strains resulted in the development of normal fast growing thalli with the typical morphology of the algal strain used. These effects are mediated by specific regulatory factors that are excreted into the environment by the bacteria and could be also isolated from the bacterial cell extracts. In contrast with the Cytophaga‐factor, the regulatory factor of the three other bacterial species was also found intracellularly in other bacterial strains not associated with Ulva, but in this case it was not excreted. Functionally, the Roseobacter‐, Sulfitobacter‐, and Halomonas‐factors resemble a cytokinin, while the Cytophaga‐factor acts similar to auxin. Neither factor could be replaced by known phytohormones. The Roseobacter species exhibits a specific chemotactic affinity to the rhizoid cells of U. mutabilis and seems to cooperate with the Cytophaga strain and the alga by chemical communication forming a symbiotic tripartite community.

Perturbation of the conformational equilibria in Ras by selective mutations as studied by 31P NMR spectroscopy.

Ras regulates a variety of different signal transduction pathways acting as molecular switch. It was shown by liquid and solid‐state 31P NMR spectroscopy that Ras exists in the guanosine‐5′‐(β,γ‐imido)triphosphate bound form in at least two conformational states interconverting in millisecond time scale. The relative population between the two conformational states affects drastically the affinity of Ras to its effectors. 31P NMR spectroscopy shows that the conformational equilibrium can be shifted specifically by point mutations, including mutations with oncogenic potential, thus modifying the effector interactions and their coupling to dynamic properties of the protein.

Metal–Bis(2-picolyl)amine Complexes as State 1(T) Inhibitors of Activated Ras Protein

Allosteric interactions: Metal(II) cyclens inhibit Ras-effector interactions by stabilizing a weak effector-binding state of Ras, state 1(T), and binding directly in the active site. The novel state (1T) inhibitor Zn(2+)-BPA (BPA = bis(2-picolyl)amine) binds outside the nucleotide binding pocket but nevertheless allosterically stabilizes state 1(T) and thus inhibits the Ras-Raf interaction.

Slow conformational dynamics of the guanine nucleotide‐binding protein Ras complexed with the GTP analogue GTPγS

The guanine nucleotide‐binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K12 of 2.0, when the GTP analogue guanosine‐5′‐(β,γ‐imido)triphosphate or guanosine‐5′‐(β,γ‐methyleno)triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state (state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) leads to 31P NMR spectra that indicate the prevalence of only one conformational state with K12 > 10. Titration with the Ras‐binding domain of Raf‐kinase (Raf‐RBD) shows that this state corresponds to effector binding state 2. In the GTPγS complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf‐RBD suggest that both mutants exist mainly in low‐affinity states 1a and 1b. From line‐shape analysis of the spectra measured at various temperatures an activation energy ΔH|1a1b of 61 kJ·mol−1 and an activation entropy ΔS|1a1b of 65 J·K−1·mol−1 are derived. Isothermal titration calorimetry on Ras bound to the different GTP‐analogues shows that the effective affinity KA for the Raf‐RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild‐type with the strongest reduction observed for the GTPγS complex.

A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins

Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP–N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. 1H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

Crystal structure of a Schistosoma mansoni septin reveals the phenomenon of strand slippage in septins dependent on the nature of the bound nucleotide.

Background: Septins are filament-forming proteins involved in membrane-remodeling events. Results: Two crystal structures of a septin with the highest resolution to date reveal the phenomenon of β-strand slippage. Conclusion: A novel mechanistic framework for the influence of the nature of the bound nucleotide and the presence of Mg2+ in septins is proposed. Significance: Identification of strand slippage might contribute to elucidating the mechanism of septin association with membranes. Septins are filament-forming GTP-binding proteins involved in important cellular events, such as cytokinesis, barrier formation, and membrane remodeling. Here, we present two crystal structures of the GTPase domain of a Schistosoma mansoni septin (SmSEPT10), one bound to GDP and the other to GTP. The structures have been solved at an unprecedented resolution for septins (1.93 and 2.1 Å, respectively), which has allowed for unambiguous structural assignment of regions previously poorly defined. Consequently, we provide a reliable model for functional interpretation and a solid foundation for future structural studies. Upon comparing the two complexes, we observe for the first time the phenomenon of a strand slippage in septins. Such slippage generates a front-back communication mechanism between the G and NC interfaces. These data provide a novel mechanistic framework for the influence of nucleotide binding to the GTPase domain, opening new possibilities for the study of the dynamics of septin filaments.

Structural insights into the small G-protein Arl13B and implications for Joubert syndrome

Ciliopathies are human diseases arising from defects in primary or motile cilia. The small G-protein Arl13B (ADP-ribosylation factor-like 13B) localizes to microtubule doublets of the ciliary axoneme and is mutated in Joubert syndrome. Its GDP/GTP mechanistic cycle and the effect of its mutations in patients with Joubert syndrome remain elusive. In the present study we applied high resolution structural and biochemical approaches to study Arl13B. The crystal structure of Chlamydomonas rheinhardtii Arl13B, comprising the G-domain and part of its unique C-terminus, revealed an incomplete active site, and together with biochemical data the present study accounts for the absence of intrinsic GTP hydrolysis by this protein. The structure shows that the residues representing patient mutations R79Q and R200C are involved in stabilizing important intramolecular interactions. Our studies suggest that Arg79 is crucial for the GDP/GTP conformational change by stabilizing the large two-residue register shift typical for Arf (ADP-ribosylation factor) and Arl subfamily proteins. A corresponding mutation in Arl3 induces considerable defects in effector and GAP (GTPase-activating protein) binding, suggesting a loss of Arl13B function in patients with Joubert syndrome.

Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity

The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras·Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras·GTP versus Ras·GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.