Inhibition of RAS function through targeting an allosteric regulatory site

Spencer-Smith, Russell; Koide, Akiko; Zhou, Yong; Eguchi, Raphael R.; Sha, Fern; Gajwani, Priyanka; Santana, Dianicha; Gupta, Ankit; Jacobs, Miranda L.; Herrero-Garcia, Erika; Cobbert, Jacqueline; Lavoie, Hugo; Smith, Matthew J.; Rajakulendran, Thanashan; Dowdell, Evan; Okur, Mustafa Nazir; Dementieva, I.; Sicheri, Frank; Therrien, Marc; Hancock, John F.; Ikura, Mitsuhiko; Koide, Shohei; O’Bryan, John P.

doi:10.1038/nchembio.2231

Cited by 259 publications

(397 citation statements)

References 60 publications

(97 reference statements)

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“…This feature is similar to other artificial binding proteins that can also be expressed in the cytoplasm of mammalian cells (Kummer et al, 2012; Spencer-Smith et al, 2017; Wojcik et al, 2010). This raises the intriguing possibility of generating the necessary reagents, based on Affimers and other artificial binding proteins, to target specific protein domains of the human ‘interactome’.…”

Section: Discussionsupporting

confidence: 62%

Affimer proteins are versatile and renewable affinity reagents

Tiede

Bedford

Heseltine

et al. 2017

eLife

172

162

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Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.DOI: http://dx.doi.org/10.7554/eLife.24903.001

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Section: Discussionsupporting

confidence: 62%

Affimer proteins are versatile and renewable affinity reagents

Tiede

Bedford

Heseltine

et al. 2017

eLife

172

162

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“…These observations underline the notion of previous studies that showed monobody binding to hotspots of protein–protein interactions in structurally diverse targets including other SH2 domains [18], [22], [23], [30], [31].…”

Section: Discussionsupporting

confidence: 86%

Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

Kükenshöner

Schmit

Bouda

et al. 2017

Journal of Molecular Biology

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The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody–SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

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“…Although emerging data indicate that Lys-to-Gln mutations may not fully mimic acetylation, our studies do indicate that Lys 104 , a hot spot for RAS PTMs, plays a key role in maintaining the structural integrity of H3 and H2. Given the proposed role of H3 in RAS-mediated dimerization at the membrane, it is possible that KRAS acetylation may alter RAS dimerization (32). It will be important to evaluate each PTM (acetylation, ubiquitylation) separately to determine how the PTM may directly alter RAS activity as well as protein-protein interactions.…”

Section: Discussionmentioning

confidence: 99%

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

Yin

Kistler

George

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellThe KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cellbased properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.RAS proteins function as molecular switches that cycle between active GTP-and inactive GDP-bound states to regulate signal transduction pathways that modulate cellular growth control. In the unstimulated cell, RAS proteins are populated in their inactive GDP-bound state. However, in response to growth-stimulatory signals, guanine nucleotide exchange factors (GEFs) 2 co-localize and up-regulate RAS by facilitating exchange of GDP for GTP. Inactivation of RAS is achieved through GTPase-activating proteins (GAPs) that bind to GTPbound RAS and promote GTP hydrolysis (1, 2). Several point mutations in RAS have been identified that dysregulate RAS nucleotide exchange or hydrolysis, often leading to hyperactivation and promoting tumorigenesis. The most common RAS mutations identified in cancer occur at residues 12, 13, and 61 and render RAS GAP defective, thereby populating RAS in its active GTP-bound state (3). Constitutive hyperactivation of RAS promotes chronic stimulation of effector-mediated downstream pathways, causing deregulated growth and tumorigenic growth transformation.RAS contains two dynamic regions termed switch I (SWI; residues 30 -37) and switch II (SWII; residues 60 -76 with 66 -74 corresponding to helix 2 (H2)) that populate distinct conformations when the protein is bound to GDP versus GTP. Effectors and GAP proteins recognize specific conformations of the switch regions and bind with preferential affinity to the active GTP-bound state. Activated GTP-bound RAS can interact with multiple effectors (e.g. RAF kinase, RAL exchang...

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Inhibition of RAS function through targeting an allosteric regulatory site

Cited by 259 publications

References 60 publications

Affimer proteins are versatile and renewable affinity reagents

Affimer proteins are versatile and renewable affinity reagents

Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

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