Conformational Switch and Structural Basis for Oncogenic Mutations of Ras Proteins

Oncogenic mutations in RAS genes underlie the pathogenesis of many human tumours, and there has been intense effort for over 30 years to develop effective and tolerated targeted therapeutics for patients with Ras-driven cancers. This review summarises the progress made in Ras drug discovery, highlighting some of the recent developments in directly targeting Ras through advances in small molecule drug design and novel therapeutic strategies.

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The structural basis of the oncogenic mutant K-Ras4B homodimers

10.1101/2020.09.07.285783 ◽

2020 ◽

Author(s):

Kayra Kosoglu ◽

Meltem Eda Omur ◽

Hyunbum Jang ◽

Ruth Nussinov ◽

Ozlem Keskin ◽

...

Keyword(s):

Plasma Membrane ◽

Structural Basis ◽

Ras Proteins ◽

Oncogenic Ras ◽

Extracellular Stimuli ◽

Prediction Server ◽

Physical Interactions ◽

Multiple Conformations ◽

Dynamics Simulations ◽

Membrane Signaling

AbstractRas proteins activate their effectors through physical interactions in response to the various extracellular stimuli at the plasma membrane. Oncogenic Ras forms dimer and nanoclusters at the plasma membrane, boosting the downstream MAPK signal. It was reported that K-Ras4B can dimerize through two major interfaces: (i) the effector lobe interface, mapped to Switch I and effector binding regions; (ii) the allosteric lobe interface involving α3 and α4 helices. Recent experiments showed that constitutively active, oncogenic mutant K-Ras4BG12D dimers are enriched in the plasma membrane. Here, we perform molecular dynamics simulations of K-Ras4BG12D homodimers aiming to quantify the two major interfaces in atomic level. To examine the effect of mutations on dimerization, two double mutations, K101D/R102E on the allosteric lobe and R41E/K42D on the effector lobe interfaces were added to the K-Ras4BG12D dimer simulations. We observed that the effector lobe K-Ras4BG12D dimer is stable, while the allosteric lobe dimer alters its helical interface during the simulations, presenting multiple conformations. The K101D/R102E mutations slightly weakens the allosteric lobe interface. However, the R41E/K42D mutations disrupt the effector lobe interface. Using the homo-oligomers prediction server, we obtained trimeric, tetrameric, and pentameric complexes with the allosteric lobe K-Ras4BG12D dimers. However, the allosteric lobe dimer with the K101D/R102E mutations is not capable of generating multiple higher order structures. Our detailed interface analysis may help to develop inhibitor design targeting functional Ras dimerization and high order oligomerization at the membrane signaling platform.

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Structural Basis for Innate Immune Sensing by M-ficolin and Its Control by a pH-dependent Conformational Switch

Journal of Biological Chemistry ◽

10.1074/jbc.m705741200 ◽

2007 ◽

Vol 282 (49) ◽

pp. 35814-35820 ◽

Cited By ~ 51

Author(s):

Virginie Garlatti ◽

Lydie Martin ◽

Evelyne Gout ◽

Jean-Baptiste Reiser ◽

Teizo Fujita ◽

...

Keyword(s):

Molecular Mechanisms ◽

Physiological Role ◽

Innate Immune ◽

Structural Basis ◽

Structural Determinants ◽

Conformational Switch ◽

Binding Function ◽

Monocytes And Macrophages ◽

Immune Sensing ◽

The One

Ficolins are soluble oligomeric proteins with lectin-like activity, assembled from collagen fibers prolonged by fibrinogen-like recognition domains. They act as innate immune sensors by recognizing conserved molecular markers exposed on microbial surfaces and thereby triggering effector mechanisms such as enhanced phagocytosis and inflammation. In humans, L- and H-ficolins have been characterized in plasma, whereas a third species, M-ficolin, is secreted by monocytes and macrophages. To decipher the molecular mechanisms underlying their recognition properties, we previously solved the structures of the recognition domains of L- and H-ficolins, in complex with various model ligands (Garlatti, V., Belloy, N., Martin, L., Lacroix, M., Matsushita, M., Endo, Y., Fujita, T., Fontecilla-Camps, J. C., Arlaud, G. J., Thielens, N. M., and Gaboriaud, C. (2007) EMBO J. 24, 623–633). We now report the ligand-bound crystal structures of the recognition domain of M-ficolin, determined at high resolution (1.75–1.8 Å), which provides the first structural insights into its binding properties. Interaction with acetylated carbohydrates differs from the one previously described for L-ficolin. This study also reveals the structural determinants for binding to sialylated compounds, a property restricted to human M-ficolin and its mouse counterpart, ficolin B. Finally, comparison between the ligand-bound structures obtained at neutral pH and nonbinding conformations observed at pH 5.6 reveals how the ligand binding site is dislocated at acidic pH. This means that the binding function of M-ficolin is subject to a pH-sensitive conformational switch. Considering that the homologous ficolin B is found in the lysosomes of activated macrophages (Runza, V. L., Hehlgans, T., Echtenacher, B., Zahringer, U., Schwaeble, W. J., and Mannel, D. N. (2006) J. Endotoxin Res. 12, 120–126), we propose that this switch could play a physiological role in such acidic compartments.

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Antibodies to synthetic peptide from the residue 33 to 42 domain of c-Ha-ras p21 block reconstitution of the protein with different effectors.

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3904 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3904-3910 ◽

Cited By ~ 15

Author(s):

I Rey ◽

P Soubigou ◽

L Debussche ◽

C David ◽

A Morgat ◽

...

Keyword(s):

Synthetic Peptide ◽

Binding Pocket ◽

Inhibitory Effect ◽

Effector Proteins ◽

Structural Basis ◽

Ras Proteins ◽

Ras P21 ◽

Reduced Rate ◽

Additional Support

Residues 32 to 40, which are conserved among ras proteins from different species, are likely to participate in interactions with the p21 effector system. With the goal of understanding the structural basis of the regulatory functions of c-Ha-ras p21, we produced rabbit antisera against a synthetic peptide corresponding to amino acids 33 to 42 of the protein. The affinity-purified antibodies interacted specifically with p21 and with the antigenic peptide. The epitope recognized by the antibodies appeared to be centered on threonine 35. The antibodies inhibited both in vitro p21-induced production of cyclic AMP in detergent extracts of RAS-defective yeast membranes and GAP-stimulated GTPase activity. However, monoclonal anti-ras antibodies Y13-259 and Y13-238 were not capable of specifically inhibiting interactions of p21 with these two putative effector proteins. The apparent inhibitory effect of Y13-259 on stimulation of p21 by GAP was due to a greatly reduced rate of exchange of nucleotides in the binding pocket of the protein. These findings provide additional support for the essential role of the residue 32 to 40 domain as the true effector site and further evidence of the involvement of GAP as a cellular effector of ras proteins.

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Small molecule inhibitors of RAS proteins with oncogenic mutations

Cancer and Metastasis Reviews ◽

10.1007/s10555-020-09911-9 ◽

2020 ◽

Vol 39 (4) ◽

pp. 1107-1126 ◽

Cited By ~ 1

Author(s):

Zoltán Orgován ◽

György M. Keserű

Keyword(s):

Clinical Trials ◽

Small Molecules ◽

Molecular Switches ◽

Membrane Association ◽

Ras Proteins ◽

Direct Inhibition ◽

Cellular Processes ◽

Oncogenic Mutations ◽

Covalent Inhibitors ◽

Ras Signalling

AbstractRAS proteins control a number of essential cellular processes as molecular switches in the human body. Presumably due to their important signalling role, RAS proteins are among the most frequently mutated oncogenes in human cancers. Hence, numerous efforts were done to develop appropriate therapies for RAS-mutant cancers in the last three decades. This review aimed to collect all of the reported small molecules that affect RAS signalling. These molecules can be divided in four main branches. First, we address approaches blocking RAS membrane association. Second, we focus on the stabilization efforts of non-productive RAS complexes. Third, we examine the approach to block RAS downstream signalling through disturbance of RAS-effector complex formation. Finally, we discuss direct inhibition; particularly the most recently reported covalent inhibitors, which are already advanced to human clinical trials.

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RAS Nanoclusters Selectively Sort Distinct Lipid Headgroups and Acyl Chains

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.686338 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yong Zhou ◽

Alemayehu A. Gorfe ◽

John F. Hancock

Keyword(s):

Splice Variants ◽

Small Gtpases ◽

Ras Signaling ◽

Ras Proteins ◽

Macromolecular Assembly ◽

Oncogenic Mutations ◽

Constitutively Active Mutants ◽

Primary Location ◽

Acyl Chains ◽

Ras Isoforms

RAS proteins are lipid-anchored small GTPases that switch between the GTP-bound active and GDP-bound inactive states. RAS isoforms, including HRAS, NRAS and splice variants KRAS4A and KRAS4B, are some of the most frequently mutated proteins in cancer. In particular, constitutively active mutants of KRAS comprise ∼80% of all RAS oncogenic mutations and are found in 98% of pancreatic, 45% of colorectal and 31% of lung tumors. Plasma membrane (PM) is the primary location of RAS signaling in biology and pathology. Thus, a better understanding of how RAS proteins localize to and distribute on the PM is critical to better comprehend RAS biology and to develop new strategies to treat RAS pathology. In this review, we discuss recent findings on how RAS proteins sort lipids as they undergo macromolecular assembly on the PM. We also discuss how RAS/lipid nanoclusters serve as signaling platforms for the efficient recruitment of effectors and signal transduction, and how perturbing the PM biophysical properties affect the spatial distribution of RAS isoforms and their functions.

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Structural basis of the regulation of normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

10.1101/2020.12.05.413278 ◽

2020 ◽

Author(s):

Ko Sato ◽

Amarjeet Kumar ◽

Keisuke Hamada ◽

Chikako Okada ◽

Asako Oguni ◽

...

Keyword(s):

Histone H3 ◽

Point Mutations ◽

Polycomb Group ◽

Structural Basis ◽

Nucleosomal Dna ◽

Oncogenic Mutations ◽

Repressive Effect ◽

Binding Cleft ◽

Dynamics Simulations ◽

Nucleosome Binding

SummaryDimethylated histone H3 Lys36 (H3K36me2) regulates gene expression by antagonizing the repressive effect of polycomb-group proteins. Aberrant upregulation of H3K36me2, either by overexpression or point mutations of NSD2/MMSET, an H3K36 dimethyltransferase, is found in various cancers, including multiple myeloma. To understand the mechanism underlying its regulation, here we report the cryo-electron microscopy structure of the catalytic fragment of NSD2 bound to the nucleosome at 2.8 Å resolution. The nucleosomal DNA is partially unwrapped at superhelix location +5.5, facilitating the access of NSD2 to H3K36. NSD2 interacts with DNA and H2A along with H3. The autoinhibitory loop of NSD2 changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed two oncogenic mutations, E1099K and T1150A, to aberrantly activate NSD2 by increasing its catalytic turnover but not the nucleosome affinity. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing the catalytic turnover. Our analyses would guide the development of specific inhibitors of NSD2 for the treatment of various cancers.

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Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

Nature Communications ◽

10.1038/s41467-021-26913-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ko Sato ◽

Amarjeet Kumar ◽

Keisuke Hamada ◽

Chikako Okada ◽

Asako Oguni ◽

...

Keyword(s):

Histone H3 ◽

Structural Basis ◽

Wild Type ◽

Cryo Electron Microscopy ◽

Nucleosomal Dna ◽

Oncogenic Mutations ◽

Binding Cleft ◽

Dynamics Simulations ◽

Nucleosome Binding ◽

Nucleosomal Histone

AbstractDimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

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Bispecific antibodies targeting mutantRASneoantigens

Science Immunology ◽

10.1126/sciimmunol.abd5515 ◽

2021 ◽

Vol 6 (57) ◽

pp. eabd5515 ◽

Cited By ~ 7

Author(s):

Jacqueline Douglass ◽

Emily Han-Chung Hsiue ◽

Brian J. Mog ◽

Michael S. Hwang ◽

Sarah R. DiNapoli ◽

...

Keyword(s):

Cancer Cells ◽

T Cell Activation ◽

Cell Activation ◽

Human Leukocyte ◽

Bispecific Antibodies ◽

Ras Proteins ◽

Single Chain ◽

Hla Alleles ◽

Oncogenic Mutations ◽

Low Levels

Mutations in theRASoncogenes occur in multiple cancers, and ways to target these mutations has been the subject of intense research for decades. Most of these efforts are focused on conventional small-molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular. Peptides derived from recurrentRASmutations, G12V and Q61H/L/R, are presented on cancer cells in the context of two common human leukocyte antigen (HLA) alleles, HLA-A3 and HLA-A1, respectively. Using phage display, we isolated single-chain variable fragments (scFvs) specific for each of these mutant peptide-HLA complexes. The scFvs did not recognize the peptides derived from the wild-type form of RAS proteins or other related peptides. We then sought to develop an immunotherapeutic agent that was capable of killing cells presenting very low levels of theseRAS-derived peptide-HLA complexes. Among many variations of bispecific antibodies tested, one particular format, the single-chain diabody (scDb), exhibited superior reactivity to cells expressing low levels of neoantigens. We converted the scFvs to this scDb format and demonstrated that they were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles. CRISPR-mediated alterations of theHLAandRASgenes provided strong genetic evidence for the specificity of the scDbs. Thus, this approach could be applied to other common oncogenic mutations that are difficult to target by conventional means, allowing for more specific anticancer therapeutics.

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Decoding RAS isoform and codon-specific signalling

Biochemical Society Transactions ◽

10.1042/bst20140057 ◽

2014 ◽

Vol 42 (4) ◽

pp. 742-746 ◽

Cited By ~ 11

Author(s):

Anna U. Newlaczyl ◽

Fiona E. Hood ◽

Judy M. Coulson ◽

Ian A. Prior

Keyword(s):

Present Article ◽

Ras Proteins ◽

Oncogenic Mutations ◽

Underlying Mechanisms ◽

Ras Isoforms

RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms.

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