Mutations in the Hydrophobic Core of Ubiquitin Differentially Affect Its Recognition by Receptor Proteins

Haririnia, Aydin; Verma, Rati; Purohit, Nisha; Twarog, Michael Z.; Deshaies, Raymond J.; Bolon, Daniel N.; Fushman, David

doi:10.1016/j.jmb.2007.11.016

Cited by 46 publications

(60 citation statements)

References 49 publications

(62 reference statements)

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“…2f). These structural analyses are consistent with the chemical intuition that the functional sensitivity of a position to mutation is primarily determined by direct binding interfaces 39 as well as structural integrity 40 and dynamics 41 .…”

Section: Resultssupporting

confidence: 80%

Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast

Roscoe

Bolon

2014

Journal of Molecular Biology

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The complexity of biological interaction networks poses a challenge to understanding the function of individual connections in the overall network. To address this challenge, we developed a high throughput reverse engineering strategy to analyze how thousands of specific perturbations (encompassing all point mutations in a central gene) impact both a specific edge (interaction to a directly connected node) as well as overall network function. We analyzed the effects of ubiquitin mutations on activation by the E1 enzyme and compared these to effects on yeast growth rate. Using this approach, we delineated ubiquitin mutations that selectively impacted the ubiquitin-E1 edge. We find that the elasticity function relating the efficiency of ubiquitin-E1 interaction to growth rate is non-linear and that a greater than 50-fold decrease in E1 activation efficiency is required to reduce growth rate by two fold. Despite the robustness of fitness to decreases in E1 activation efficiency, the effects of most ubiquitin mutations on E1 activation paralleled the effects on growth rate. Our observations indicate that most ubiquitin mutations that disrupt E1 activation also disrupt other functions. The structurally characterized ubiquitin-E1 interface encompasses the interfaces of ubiquitin with most other known binding partners, and we propose that this enables E1 in wild-type cells to selectively activate ubiquitin protein molecules capable of binding to other partners from the cytoplasmic pool of ubiquitin protein that will include molecules with chemical damage and/or errors from transcription and translation.

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Section: Resultssupporting

confidence: 80%

Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast

Roscoe

Bolon

2014

Journal of Molecular Biology

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“…Populations of excited states have recently been implicated in function (12,34,35), and our data and others' indicate that the rate at which ubiquitin moves among states also appears to be critical for its biological function (16). Although our data do not definitively assign the conformations between which the U14Ub variants slowly transition, we postulate that their motions may correspond to the same states observed for WT ubiquitin's β1-β2 loop.…”

Section: Discussioncontrasting

confidence: 44%

“…Precisely how the slowing of motion in the U14Ubs has increased their affinity for USP14 remains unclear. Point mutations that decrease ubiquitin's overall stability and increase its flexibility over slow, second-minute timescales have been shown to abrogate interactions with ubiquitin interacting motif domains (16), but the U14Ubs appear to be gain-of-function dynamic mutants in that they have enhanced affinities for human USP14. We speculate that an increase in the energy barrier connecting ground and excited states restricts the rate at which the apo U14Ubs access a possible high-affinity excited state, but this energetic barrier can be overcome through binding to the DUB, resulting in the observed shift away from conformational selection and toward an induced-fit binding mode (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Other reports suggest that at least some ubiquitin-partner interactions cannot be solely attributed to a conformational selection binding mechanism and that induced fit plays some role in ubiquitin binding (13)(14)(15). Intriguingly, single point mutations within ubiquitin's hydrophobic core that decrease overall stability and increase the flexibility of the β-sheet binding interface abrogate recognition by some partner proteins without dramatically altering the ubiquitin fold (16). Although these studies have hinted at the importance of conformational dynamics in ubiquitin recognition, it is currently unknown whether the rates of ubiquitin's motions are functionally important.…”

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confidence: 99%

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Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling

Phillips¹,

Zhang²,

Cunningham³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.computational design | phage display | protein dynamics | protein-protein interactions | ubiquitin signaling

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“…Several recent studies have designed mutations in and around the core of ubiquitin to shift its conformational equilibrium toward or away from binding-competent states (27)(28)(29)(30). In one case, the mutations introduced widespread millisecond timescale motions that were not present in WT (29).…”

Section: Significancementioning

confidence: 99%

Allosteric switch regulates protein–protein binding through collective motion

Smith

Ban

Pratihar

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

105

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Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.allostery | protein dynamics | concerted motion | relaxation dispersion | nuclear magnetic resonance I ntermolecular interactions are one of the key mechanisms by which proteins mediate their biological functions. For many proteins, these interactions are enhanced or suppressed by allosteric networks that couple distant regions together (1). The mechanisms by which these networks function are just starting to be understood (2-4), and many of the important details have yet to be uncovered. In particular, the role of intrinsic protein motion and kinetics remains particularly poorly characterized. A number of structural ensembles representing ubiquitin motion have been recently proposed (5-9). Additionally, it has been suggested that through motion at the binding interface, its free state visits the same conformations found in complex with its many binding partners (5, 10). However, it remains an unanswered question if the dynamics that enable this multispecificity are only clustered around the canonical binding interface or whether this motion is allosterically coupled to the rest of the protein, especially given the presence of motion at distal sites (11). ResultsTo answer this question and to provide a detailed structural picture of the underlying mechanism, we applied recently developed high-power relaxation dispersion (RD) experiments (12, 13) to both the backbone amide proton ( 1 H N ) and nitrogen ( 15 N) nuclei of ubiquitin. This survey yielded a nearly twofold increase in the number of nuclei where RD had been previously observed (11)(12)(13)(14) (from 17 to 31; Fig. 1A and Fig. S1). When fit individually, the full set of backbone and side-chain nuclei shows a consistent time scale of motion [exchange lifetime (τ ex ) = 55 μs; Fig. 1B]. Furthermore, the nuclei showing exchange are spread throughout the structure (Fig. 1C). Put together, these data suggest that the motions are not independent but share a common molecular mechanism.To determine whether the RD data could be modeled using a single collective motion, we developed a computational method to take a set of molecu...

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Mutations in the Hydrophobic Core of Ubiquitin Differentially Affect Its Recognition by Receptor Proteins

Cited by 46 publications

References 49 publications

Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast

Systematic Exploration of Ubiquitin Sequence, E1 Activation Efficiency, and Experimental Fitness in Yeast

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling

Allosteric switch regulates protein–protein binding through collective motion

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