KRAS Ubiquitination at Lysine 104 Retains Exchange Factor Regulation by Dynamically Modulating the Conformation of the Interface

Yin, Guowei; Zhang, Jerry; Nair, Vinay; Truong, Vinh H.; Chaia, Angelo; Petela, Johnny; Harrison, Joseph S.; Gorfe, Alemayehu A.; Campbell, Sharon L.

doi:10.1016/j.isci.2020.101448

Cited by 16 publications

(17 citation statements)

References 35 publications

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“…To generate the ensemble of conjugated E2 structures, we used the E2 thioester protocol in Rosetta (Saha et al, 2011). This protocol is a tethered docking protocol which we have previously used to construct and analyze the conformational ensemble of conjugated Ub (Yin et al, 2020). We have previously observed that this conjugated docking quickly settles in a local minimum and then remains in that relative orientation.…”

Section: Rosetta Modelingmentioning

confidence: 99%

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

et al. 2021

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Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the Nterminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turnhelix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2 HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2S HTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2 HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

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Section: Rosetta Modelingmentioning

confidence: 99%

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

et al. 2021

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“…Clearly, K104 and K147 are solvent exposed and amenable for ubiquitin ligation. We then used a structure of wild-type KRAS described in previous studies , to build four structural models of KRAS monoubiquitinated at K104 (mUbKRAS 104 ) and four models of KRAS monoubiquitinated at K147 (mUbKRAS 147 ). Ubiquitin (Ub) was linked to K104 or K147 via an isopeptide bond as described previously and placed in different orientations relative to KRAS in the four different starting configurations of both mUbKRAS 104 and mUbKRAS 147 .…”

Section: Methodsmentioning

confidence: 99%

“…Similarly, the full-length ubiquitin G76C mutant was expressed in E. coli BL21 (pLysS). Both the proteins were purified as described with protein purity validated using SDS-PAGE. Ubiquitin G76C was ligated to KRAS K104C using a chemical ligation strategy adapted from Baker et al NMR sample preparation and data collection were conducted as described previously .…”

Section: Methodsmentioning

confidence: 99%

Monoubiquitination of KRAS at Lysine104 and Lysine147 Modulates Its Dynamics and Interaction with Partner Proteins

et al. 2021

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KRAS, a 21 kDa guanine nucleotide-binding protein that functions as a molecular switch, plays a key role in regulating cellular growth. Dysregulation of this key signaling node leads to uncontrolled cell growth, a hallmark of cancer cells. KRAS undergoes post-translational modification by monoubiquitination at various locations, including at lysine104 (K104) and lysine147 (K147). Previous studies have suggested that K104 stabilizes helix-2/helix-3 interactions and K147 is involved in nucleotide binding. However, the impact of monoubiquitination at these residues on the overall structure, dynamics, or function of KRAS is not fully understood. In this study, we examined KRAS monoubiquitination at these sites using data from extensive (12 μs aggregate time) molecular dynamics simulations complemented by nuclear magnetic resonance spectroscopy data. We found that ubiquitin forms dynamic nonspecific interactions with various regions of KRAS and that ubiquitination at both sites modulates conformational fluctuations. In both cases, ubiquitin samples a broad range of conformational space and does not form long-lasting noncovalent contacts with KRAS but it adopts several preferred orientations relative to KRAS. To examine the functional impact of these preferred orientations, we performed a systematic comparison of the dominant configurations of the ubiquitin/KRAS simulated complex with experimental structures of KRAS bound to regulatory and effector proteins as well as a model membrane. Results from these analyses suggest that conformational selection and population shift may minimize the deleterious effects of KRAS ubiquitination at K104 and K147 on binding to some but not all interaction partners. Our findings thus provide new insights into the steric effects of ubiquitin and suggest a potential avenue for therapeutic targeting.

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“…Despite the differences, previous work in the Ras ubiquitylation field allows us to think more critically about our Rap2 findings and future directions (Baker et al 2012(Baker et al , 2013Yin et al 2020;Thurman, Siraliev-Perez, and Campbell 2020). Specifically, mono-ubiquitylation of KRas-K147 impedes GAP-mediated GTP hydrolysis and promotes effector binding (Sasaki et al 2011;Baker et al 2012Baker et al , 2013.…”

Section: Differential Regulation Between Rap and Ras Family Membersmentioning

confidence: 99%

Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration

Duncan

Han

Trout

et al. 2021

Preprint

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Cell migration is a complex process that involves coordinated changes in membrane transport, actin cytoskeleton dynamics, and extracellular matrix remodeling. Ras-like small monomeric GTPases, such as Rap2, play a key role in regulating actin cytoskeleton dynamics and cell adhesions. However, how Rap2 function, localization, and activation are regulated during cell migration is not fully understood. We previously identified the small GTPase Rab40b as a regulator of breast cancer cell migration. Rab40b contains a Suppressor of Cytokine Signaling (SOCS) box, which facilitates binding to Cullin5, a known E3 Ubiquitin Ligase component responsible for protein ubiquitylation. In this study, we show that the Rab40b/Cullin5 complex ubiquitylates Rap2. Importantly, we demonstrate that ubiquitylation regulates Rap2 activation, as well as recycling of Rap2 from the endolysosomal compartment to the lamellipodia of migrating breast cancer cells. Based on these data, we propose that Rab40b/Cullin5 ubiquitylates and regulates Rap2-dependent actin dynamics at the leading-edge, a process that is required for breast cancer cell migration and invasion.

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KRAS Ubiquitination at Lysine 104 Retains Exchange Factor Regulation by Dynamically Modulating the Conformation of the Interface

Cited by 16 publications

References 35 publications

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes

Monoubiquitination of KRAS at Lysine104 and Lysine147 Modulates Its Dynamics and Interaction with Partner Proteins

Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration

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