Ras is a central regulator of cellular signaling pathways. It is mutated in 20-30% of human tumors. To perform its function, Ras has to be bound to a membrane by a posttranslationally attached lipid anchor. Surprisingly, we identified here dimerization of membrane anchored Ras by combining attenuated total reflectance Fourier transform infrared spectroscopy, biomolecular simulations, and Förster resonance energy transfer experiments. By analyzing x-ray structural models and molecular-dynamics simulations, we propose a dimerization interface between α-helices 4 and 5 and the loop between β2 and β3. This seems to explain why the residues D47, E49, R135, R161, and R164 of this interface are influencing Ras signaling in cellular physiological experiments, although they are not positioned in the catalytic site. Dimerization could catalyze nanoclustering, which is well accepted for membrane-bound Ras. The interface could provide a new target for a seemingly novel type of small molecule interfering with signal transduction in oncogenic Ras mutants.

Heterotrimeric G proteins are crucial molecular switches that maintain a large number of physiological processes in cells. The signal is encoded into surface alterations of the Gα subunit that carries GTP in its active state and GDP in its inactive state. The ability of the Gα subunit to hydrolyze GTP is essential for signal termination. Regulator of G protein signaling (RGS) proteins accelerates this process. A key player in this catalyzed reaction is an arginine residue, Arg178 in Gα i1 , which is already an intrinsic part of the catalytic center in Gα in contrast to small GTPases, at which the corresponding GTPase-activating protein (GAP) provides the arginine "finger." We applied time-resolved FTIR spectroscopy in combination with isotopic labeling and site-directed mutagenesis to reveal the molecular mechanism, especially of the role of Arg178 in the intrinsic Gα i1 mechanism and the RGS4-catalyzed mechanism. Complementary biomolecular simulations (molecular mechanics with molecular dynamics and coupled quantum mechanics/molecular mechanics) were performed. Our findings show that Arg178 is bound to γ-GTP for the intrinsic Gα i1 mechanism and pushed toward a bidentate α-γ-GTP coordination for the Gα i1 ·RGS4 mechanism. This movement induces a charge shift toward β-GTP, increases the planarity of γ-GTP, and thereby catalyzes the hydrolysis.GTPase | FTIR spectroscopy | QM/MM calculations | arginine finger | reaction mechanism

Background: Multiple turnover GTPase assays of G␣ are dominated by nucleotide exchange. Results: FTIR elucidates single turnover rates and individual phosphate vibrations. Conclusion: G␣ i1 -R178S is slowed down in single turnover hydrolysis by 2 orders of magnitude, G␣ i1 -Asp 229 and -Asp 231 are key players in Ras-like/all-␣ domain coordination. Significance: With FTIR on G␣ established, detailed information on the reaction mechanism can be obtained.

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Multi-resistant bacteria are a major threat in modern medicine. The gram-negative coccobacillus Acinetobacter baumannii currently leads the WHO list of pathogens in critical need for new therapeutic development. The maintenance of lipid asymmetry (MLA) protein complex is one of the core machineries that transport lipids from/to the outer membrane in gram-negative bacteria. It also contributes to broad-range antibiotic resistance in several pathogens, most prominently in A. baumannii. Nonetheless, the molecular details of its role in lipid transport has remained largely elusive. Here, we report the cryo-EM maps of the core MLA complex, MlaBDEF, from the pathogen A. baumannii, in the apo-, ATP- and ADP-bound states, revealing multiple lipid binding sites in the cytosolic and periplasmic side of the complex. Molecular dynamics simulations suggest their potential trajectory across the membrane. Collectively with the recently-reported structures of the E. coli orthologue, this data also allows us to propose a molecular mechanism of lipid transport by the MLA system.

GTPases are central switches in cells. Their dysfunctions are involved in severe diseases. The small GTPase Ras regulates cell growth, differentiation and apoptosis by transmitting external signals to the nucleus. In one group of oncogenic mutations, the 'switch-off' reaction is inhibited, leading to persistent activation of the signaling pathway. The switch reaction is regulated by GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis in Ras, and by guanine nucleotide exchange factors, which catalyze the exchange of GDP for GTP. Heterotrimeric G-proteins are activated by G-protein coupled receptors and are inactivated by GTP hydrolysis in the Gα subunit. Their GAPs are called regulators of G-protein signaling. In the same way that Ras serves as a prototype for small GTPases, Gα i1 is the most well-studied Gα subunit. By utilizing X-ray structural models, time-resolved infrared-difference spectroscopy, and biomolecular simulations, we elucidated the detailed molecular reaction mechanism of the GTP hydrolysis in Ras and Gα i1 . In both proteins, the charge distribution of GTP is driven towards the transition state, and an arginine is precisely positioned to facilitate nucleophilic attack of water. In addition to these mechanistic details of GTP hydrolysis, Ras dimerization as an emerging factor in signal transduction is discussed in this review.

The phage shock proteins (Psp) system is activated in bacteria in response to various membrane stress conditions. PspA, the main Psp effector, preserves the integrity and functions of the bacterial inner membrane. Here, we present the 3.6 Å resolution cryo-EM structure of PspA assembled in helical rods. The structure reveals that PspA adopts a canonical ESCRT-III fold in an extended open conformation with the C-terminal helix facing the outside of the helical tube. In addition to the structural homology, we visualized how PspA fuses small vesicles into μm-sized vesicles when reconstituted with bacterial membrane lipids. This membrane remodeling activity is in line with the functional properties of ESCRT-III family proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

24The maintenance of lipid asymmetry (MLA) system is involved in lipid transport from/to 25 the outer membrane in gram-negative bacteria, and contributes to broad-range 26 antibiotic resistance. Here, we report the cryo-EM structure of the A. baumannii 27 MlaBDEF core complex, in the apo, ADP-and AppNHp-bound states. This reveals 28 multiple lipid binding sites, and suggests a mechanism for their transport. 29 30 31Gram-negative bacteria are enveloped by two lipid bilayers, separated by the periplasmic 32 space containing the peptidoglycan cell wall. The two membranes have distinct lipid 33 composition: The inner membrane (IM) consists of glycerophospholipids, with both leaflets 34 having similar compositions, while the outer membrane (OM) is asymmetric, with an outer 35 leaflet of lipopolysaccharides (LPS) and an inner leaflet of glycerophospholipids (1). This lipid 36 gradient is maintained by several machineries, including YebT, PqiB, and the multicomponent 37MLA system (2, 3), which consists of MlaA present in the OM (4, 5), the shuttle MlaC in the 38 periplasmic space, and the MlaBDEF ABC transporter system in the IM (6). The directionality 39 of lipid transport by the MLA system has been the subject of debate, with initial reports 40 suggesting that it recycles lipids from the OM to the IM (7), but recent results (6, 8) indicated 41 that it might exports glycerophospholipids to the outer membrane. Low-resolution cryo-EM 42 maps of the MlaBDEF core complex, from Escherichia coli (2) and Acinetobacter baumannii 43 (6) have revealed the overall architecture of the complex, but did not allow to elucidate the 44 molecular details of lipid binding and transport. 45To address the mechanism of MLA functioning, we have determined the cryo-EM structure of 46 the A. baumannii MlaBDEF complex, bound to the non-hydrolizable ATP analogue AppNHp, 47