Molecular determinants of Ras-mTORC2 signaling.

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.

Protein glutaminylation is a yeast-specific posttranslational modification of elongation factor 1A.

Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the α amino group of mono-l-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A. The MS analysis suggested that all eEF1A molecules are modified by this glutaminylation and that this posttranslational modification occurs at all stages of yeast growth. The mutational studies revealed that this glutaminylation is not essential for the normal functions of eEF1A in S. cerevisiae However, eEF1A glutaminylation slightly reduced growth under antibiotic-induced translational stress conditions. Moreover, we identified the same posttranslational modification in eEF1A from Schizosaccharomyces pombe but not in various other eukaryotic organisms tested despite strict conservation of the Glu45 residue among these organisms. We therefore conclude that eEF1A glutaminylation is a yeast-specific posttranslational modification that appears to influence protein translation.

Atypical Switch-I Arginine plays a catalytic role in GTP hydrolysis by Rab21 from Entamoeba histolytica.

Entamoeba histolytica, the causative agent of amoebic dysentery, liver abscess and colitis, exploits its vesicular trafficking machinery for survival and virulence. Rab family of small GTPases play a key role in the vesicular transport by undergoing the GTP/GDP cycle which is central to the biological processes. Amoebic genome encodes several atypical Rab GTPases which are unique due to absence of conserved sequence motif(s) or atypical residues in their catalytic site [Saito-Nakano et al., 2005 ]. Previously, EhRab21 has been reported to involve in amoebic invasion and migration [Emmanuel et al., 2015 ]. The conserved Glutamine of switch-II region is universally accepted to be crucial for GTP hydrolysis. Mutations that reduce the sidechain polarity of Glutamine render the protein GTPase activity deficient [Krengel et al., 1990]. Here, we report a catalytic role of atypical switch-I Arginine (R36) in intrinsic GTP hydrolysis catalysed by EhRab21. Unlike the GTPase activity deficient QL mutants, the GTPase activity of EhRab21Q64L was found to be marginally enhanced compared to the wild-type protein. Although EhRab21R36L mutant showed normal GTPase activity, the double mutant (R36L/Q64L) was found to be GTPase deficient. Thus, EhRab21 is a unique member of small GTPase family in which an atypical switch-I Arginine is capable of driving GTP hydrolysis independent of the conserved switch-II Glutamine.

Review: Ras GTPases and myosin: Qualitative conservation and quantitative diversification in signal and energy transduction.

Most GTPases and many ATPases belong to the P-loop class of proteins with significant structural and mechanistic similarities. Here we compare and contrast the basic properties of the Ras family GTPases and myosin, and conclude that there are fundamental similarities but also distinct differences related to their specific roles. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 422-430, 2016.

Linking receptor activation to changes in Sw I and II of Gα proteins.

G-protein coupled receptors catalyze nucleotide exchange on G proteins, which results in subunit dissociation and effector activation. In the recent ß2AR-Gs structure, portions of Switch I and II of Gα are not fully elucidated. We paired fluorescence studies of receptor-Gαi interactions with the ß2AR-Gs and other Gi structures to investigate changes in Switch I and II during receptor activation and GTP binding. The ß2/ß3 loop containing Leu194 of Gαi is located between Switches I and II, in close proximity to IC2 of the receptor and the C-terminus of Gα, thus providing an allosteric connection between these Switches and receptor activation. We compared the environment of residues in myristoylated Gαi proteins in the heterotrimer to that upon receptor activation and subsequent GTP binding. Upon receptor activation, residues in both Switch regions are less solvent-exposed, as compared to the heterotrimer. Upon GTPγS binding, the environment of several residues in Switch I resemble the receptor-bound state, while Switch II residues display effects on their environment which are consistent with their role in GTP binding and Gßγ dissociation. The ability to merge available crystal structures with solution studies is a powerful tool to gain insight into conformational changes associated with receptor-mediated Gi protein activation.

Inside the cracked kernel: establishing the molecular basis of AMG510 and MRTX849 in destabilising KRASG12C mutant switch I and II in cancer treatment.

The Kirsten rat sarcoma oncoprotein (KRAS) has been punctuated by drug development failures for decades due to frequent mutations that occur mostly at codon 12 and the seemingly intractable targeting of the protein. However, with advances in covalent targeting, the oncoprotein is being expunged from the 'undruggable' list of proteins. This feat has seen some covalent drugs at different stages of clinical trials. The advancement of AMG510 and MRTX849 as inhibitors of cysteine mutated KRAS (KRASG12C) to phase-III clinical trials informed the biased selection of AMG510 and MRTX849 for this study. Despite this advance, the molecular and atomistic modus operandi of these drugs is yet to come to light. In this study, we employed computational tools to unravel the atomistic interactions and subsequent conformational effects of AMG510 and MRTX849 on the mutant KRASG12C. It was revealed that AMG510 and MRTX849 complexes presented similar total free binding energies, (ΔGbind), of -88.15 ± 5.96 kcal/mol and -88.71 ± 7.70 kcal/mol, respectively. Gly10, Lys16, Thr58, Gly60, Glu62, Glu63, Arg68, Asp69, Met72, His95, Tyr96, Gln99, Arg102 and Val103 interacted prominently with AMG510 and MRTX849. These residues interacted with the pharmacophoric moieties of AMG510 and MRTX849 via hydrogen bonds with decreasing bond lengths at various stages of the simulation. These interactions together with pi-pi stacking, pi-sigma and pi-alkyl interactions induced unfolding of switch I whiles compacting switch II, which could interrupt the binding of effector proteins to these interfaces. These insights present useful atomistic perspectives into the success of AMG510 and MRTX849 which could guide the design of more selective and potent KRAS inhibitors.Communicated by Ramaswamy H. Sarma.

A New Dawn for Targeted Cancer Therapy: Small Molecule Covalent Binding Inhibitor Targeting K-Ras (G12C).

K-Ras is a frequently mutated oncogene in human malignancies, and the development of inhibitors targeting various oncogenic K-Ras mutant proteins is a major challenge in targeted cancer therapy, especially K-Ras(G12C) is the most common mutant, which occurs in pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and other highly prevalent malignancies. In recent years, significant progress has been made in developing small molecule covalent inhibitors targeting K-Ras(G12C), thanks to the production of nucleophilic cysteine by the G12C mutant, breaking the "spell" that K-Ras protein cannot be used as a drug target. With the successful launch of sotorasib and adagrasib, the development of small molecule inhibitors targeting various K-Ras mutants has continued to gain momentum. In recent years, with the popularization of highly sensitive surface plasmon resonance (SPR) technology, fragment-based drug design strategies have shown great potential in the development of small molecule inhibitors targeting K-Ras(G12C), but with the increasing number of clinically reported acquired drug resistance, addressing inhibitor resistance has gradually become the focus of this field, indirectly indicating that such small molecule inhibitors still the potential for the development of these small molecule inhibitors are also indirectly indicated. This paper traces the development of small molecule covalent inhibitors targeting K-Ras(G12C), highlighting and analyzing the structural evolution and optimization process of each series of inhibitors and the previous inhibitor design methods and strategies, as well as their common problems and general solutions, in order to provide inspiration and help to the subsequent researchers.

Drug Target Exploitable Structural Features of Adenylyl Cyclase Activity in Schistosoma mansoni.

The draft genome sequence of the parasitic flatworm Schistosoma mansoni (S. mansoni), a cause of schistosomiasis, encodes a predicted guanosine triphosphate (GTP) binding protein tagged Smp_059340.1. Smp_059340.1 is predicted to be a member of the G protein alpha-s subunit responsible for regulating adenylyl cyclase activity in S. mansoni and a possible drug target against the parasite. Our structural bioinformatics analyses identified key amino acid residues (Ser53, Thr188, Asp207 and Gly210) in the two molecular switches responsible for cycling the protein between active (GTP bound) and inactive (GDP bound) states. Residue Thr188 is located on Switch I region while Gly210 is located on Switch II region with Switch II longer than Switch I. The Asp207 is located on the G3 box motif and Ser53 is the binding residue for magnesium ion. These findings offer new insights into the dynamic and functional determinants of the Smp_059340.1 protein in regulating the S. mansoni life cycle. The binding interfaces and their residues could be used as starting points for selective modulations of interactions within the pathway using small molecules, peptides or mutagenesis.