Linker Dependence of Avidity in Multivalent Interactions Between Disordered Proteins

Sørensen, Charlotte Mehlin; Jendroszek, Agnieszka; Kjærgaard, Magnus

doi:10.1016/j.jmb.2019.09.001

Cited by 40 publications

(27 citation statements)

References 54 publications

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“…Back of the envelope calculations, using available structural data and assuming disordered regions are fully extended peptide chains, reveal that the proximity of adjacent MST2 kinase domains in either a MST2 homodimer, complex with SAV1, or a FKBP mediated homodimer are all roughly within 1000 Angstroms. Recent measurements of the relationship between linker length and effective concentrations suggest, however, the relationship may not be linear (83). Precise measurements of the effective concentration of MST2 in cells for each signaling event will be required to fully understand the differences among each scenario.…”

Section: Discussionmentioning

confidence: 99%

Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

Tran

Mitra

et al. 2020

Journal of Biological Chemistry

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The Hippo pathway plays an important role in developmental biology, mediating organ size by controlling cell proliferation through the activity of a core kinase cassette. Multiple upstream events activate the pathway, but how each controls this core kinase cassette is not fully understood. Activation of the core kinase cassette begins with phosphorylation of the kinase MST1/2 (also known as STK3/4). Here, using a combination of in vitro biochemistry, and cell-based assays including chemically induced dimerization and single-molecule pull down we revealed that increasing the proximity of adjacent kinase domains, rather than formation of a specific protein assembly, is sufficient to trigger autophosphorylation. We validate this mechanism in cells and demonstrate that multiple events associated with the active pathway, including SARAH-domain mediated homodimerization, membrane recruitment, and complex formation with the effector protein SAV1, each increase the kinase domain proximity and autophosphorylation of MST2. Together, our results reveal that multiple and distinct upstream signals each utilize the same common molecular mechanism to stimulate MST2 autophosphorylation. This mechanism is likely conserved among MST2 homologs. Our works also highlights potential differences in Hippo signal propagation between each activating event owing to differences in the dynamics and regulation of each protein ensemble that triggers MST2 autophosphorylation and possible redundancy in activation.

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Section: Discussionmentioning

confidence: 99%

Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

Tran

Mitra

et al. 2020

Journal of Biological Chemistry

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“…A similar conceptual analysis was recently described for tethered protein-ligand interactions. 25 To construct a non-covalently tethered kinase substrate system, we used heterodimeric coiled-coil interactions to recruit the substrate to the kinase via a flexible tether ( Figure 5A). We used the heterodimeric coiled-coils SYNZIP 5 and SYNZIP 6 because this pair is wellcharacterized structurally and biophysically and has been used for many applications in synthetic biology and protein design.…”

Section: A Non-covalent Tether Can Accelerate the Kinase Reaction At mentioning

confidence: 99%

The relationship between effective molarity and affinity governs rate enhancements in tethered kinase-substrate reactions

Speltz

Zalatan

2020

Preprint

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Scaffold proteins are thought to accelerate protein phosphorylation reactions by tethering kinases and substrates together, but there is little quantitative data on their functional effects. To assess the contribution of tethering to kinase reactivity, we compared intramolecular and intermolecular kinase reactions in a minimal model system. We find that tethering can enhance reaction rates in a flexible tethered kinase system, and the magnitude of the effect is sensitive to the structure of the tether. The largest effective molarity we obtained was ~0.08 µM, which is much lower than the effects observed in small molecule model systems and tethered protein-ligand interactions. We further demonstrate that the tethered, intramolecular reaction only makes a significant contribution to observed rates when the scaffolded complex assembles at concentrations below the effective molarity. These findings provide a quantitative framework that can be applied to understand endogenous protein scaffolds and to engineer synthetic networks.

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“…Previous effective concentration measurements suggest a similar scaling with linker length for such a system, so we disregard the contribution of the coiled-coil domain although it potentially introduces a systematic error. 19 The phosphorylation rate saturated at high effective concentration for all variants. The WT substrate was close to saturation already at the concentrations enforced by the longest linkers.…”

Section: Resultsmentioning

confidence: 95%

“…18 The contact frequency can be expressed as an effective concentration, which in our case corresponds to the concentration of free substrate that would encounter the enzyme as often as the tethered substrate. Such effective concentration can occasionally be measured by competition experiments, 12,19,20 although in most proteins has to be estimated theoretically. [21][22][23] Our group previously measured the scaling of effective concentrations with the length of disordered linkers including the sequences used here.…”

Section: Resultsmentioning

confidence: 99%

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Intrinsically disordered linkers control tethered kinases via effective concentration

Dyla

Kjærgaard

2020

Preprint

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Kinase specificity is crucial to the fidelity of signalling pathways, yet many pathways use the same kinases to achieve widely different effects. Specificity arises in part from the enzymatic domain, but also from the physical tethering of kinases to their substrates.Such tethering can occur via protein interaction domains in the kinase or via anchoring and scaffolding proteins, and can drastically increase the kinetics of phosphorylation.However, we do not know how such intra-complex reactions depend on the link between enzyme and substrate. Here we show that the kinetics of tethered kinases follow a Michaelis-Menten like dependence on effective concentration. We find that phosphorylation kinetics scale with the length of the intrinsically disordered linkers that join the enzyme and substrate, but that the scaling differs between substrates. Steadystate kinetics can only partially predict rates of tethered reactions as product release may obscure the rate of phospho-transfer. Our results suggest that changes in signalling complex architecture not only enhance the rates of phosphorylation reactions, but may also alter the relative substrate usage. This suggests a mechanism for how scaffolding proteins can allosterically modify the output from a signalling pathway. Preparation of DNA constructs:The plasmid containing the catalytic domain of PKA (PKAc) was a gift from Susan Taylor via Addgene (#14921). 13 The other plasmids were synthesized by Genscript and codon optimized for expression in E. coli. The coding regions were cloned into pET15b vectors using the NdeI/XhoI sites. The disordered GS linkers from the linker library generated previously 12 were cloned into the protein constructs using unique NheI/KpnI sites. The sequences of protein constructs are given in the SI.Protein expression and purification: Protein constructs containing the catalytic domain of PKA linked by variable length linkers to the MBD2 coiled-coil domain (MBD2-(GS)n-

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Linker Dependence of Avidity in Multivalent Interactions Between Disordered Proteins

Abstract: Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affect avidity by modulating the

Cited by 40 publications

References 54 publications

Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

Increasing kinase domain proximity promotes MST2 autophosphorylation during Hippo signaling

The relationship between effective molarity and affinity governs rate enhancements in tethered kinase-substrate reactions

Intrinsically disordered linkers control tethered kinases via effective concentration

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