Scaffold proteins as dynamic integrators of biological processes

Scaffold proteins act as molecular hubs for the docking of multiple proteins to organize efficient functional units for signaling cascades. Over 300 human proteins have been characterized as scaffolds, acting in a variety of signaling pathways. While the term scaffold implies a static, supportive platform, it is now clear that scaffolds are not simply inert docking stations but can undergo conformational changes that affect their dependent signaling pathways. In this review, we catalog scaffold proteins that have been shown to undergo actionable conformational changes, with a focus on the role that conformational change plays in the activity of the classic yeast scaffold STE5, as well as three human scaffold proteins (KSR, NEMO, SHANK3) that are integral to well-known signaling pathways (RAS, NF-κB, postsynaptic density). We also discuss scaffold protein conformational changes vis-à-vis liquid–liquid phase separation. Changes in scaffold structure have also been implicated in human disease, and we discuss how aberrant conformational changes may be involved in disease-related dysregulation of scaffold and signaling functions. Finally, we discuss how understanding these conformational dynamics will provide insight into the flexibility of signaling cascades and may enhance our ability to treat scaffold-associated diseases. Scaffold proteins act as molecular hubs for the docking of multiple proteins to organize efficient functional units for signaling cascades. Over 300 human proteins have been characterized as scaffolds, acting in a variety of signaling pathways. While the term scaffold implies a static, supportive platform, it is now clear that scaffolds are not simply inert docking stations but can undergo conformational changes that affect their dependent signaling pathways. In this review, we catalog scaffold proteins that have been shown to undergo actionable conformational changes, with a focus on the role that conformational change plays in the activity of the classic yeast scaffold STE5, as well as three human scaffold proteins (KSR, NEMO, SHANK3) that are integral to well-known signaling pathways (RAS, NF-κB, postsynaptic density). We also discuss scaffold protein conformational changes vis-à-vis liquid–liquid phase separation. Changes in scaffold structure have also been implicated in human disease, and we discuss how aberrant conformational changes may be involved in disease-related dysregulation of scaffold and signaling functions. Finally, we discuss how understanding these conformational dynamics will provide insight into the flexibility of signaling cascades and may enhance our ability to treat scaffold-associated diseases. Intracellular signaling cascades are complex processes that require precise spatiotemporal control (1Good M.C. Zalatan J.G. Lim W.A. Scaffold proteins: hubs for controlling the flow of cellular information.Science. 2011; 332: 680-686Crossref PubMed Scopus (624) Google Scholar). From the initial binding of a ligand to a cell surface receptor to the downstream gene expression changes required for many biological responses, the cell often coordinates complex molecular events via control boards that organize the timing and strength of such signals and the array of proteins involved. The term scaffold has been used in several ways, but the general consensus is that a scaffold is a protein that binds two or more proteins to increase the efficiency of a molecular event, often one that is involved in signal transduction (2Shaw A.S. Filbert E.L. Scaffold proteins and immune-cell signalling.Nat. Rev. Immunol. 2009; 9: 47-56Crossref PubMed Scopus (158) Google Scholar). As such, scaffolds serve as docking platforms, which act as switchboards to reduce the chaos of the cellular "soup." Thus, the primary function of scaffolds is to overcome the difficulty of organizing complex signal transduction and feedback mechanisms within a cell by regulating these molecular events in time and space. As such, scaffolds provide the advantage of modular regulation in that they are responsible for rapid mobilization of signaling components based on external signals. By organizing a set of master controlling molecules such as scaffolds, instead of the larger pool of signaling enzymes, a cell can increase the flexibility of signaling while minimizing energy expenditure. In this way, scaffolds ensure that proper responses occur in either one or multiple signaling pathways. Overall, scaffolds comprise a class of proteins that are central to enhancing signaling cascades, have interaction domains such that they can interact with multiple binding partners, facilitate higher order complex formation, and are often highly conserved (3Buday L. Tompa P. 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As such, the term scaffold has recently taken on new connotations, especially in the field of conformational change and liquid–liquid phase separation (LLPS). Therefore, it is important to rethink how scaffolds function in a dynamic cellular context and how conformational changes in scaffolds are central to the regulation of signaling pathways and molecular processes. In this review, we present a critical analysis of scaffolds as plastic regulators of signaling cascades and provide examples of how conformational changes within scaffolds can impact their dynamic roles in signal transduction. We catalog conformational changes in scaffold proteins and describe examples of how conformational flexibility imparts a regulatory property onto several well studied scaffolds. We also describe what is known about scaffold dysfunction in human disease and how the understanding of scaffold dynamics may be applied to the modulation of scaffold function and new disease therapies. To comprehensively search for scaffolds that have been reported to undergo conformational changes, we used a human scaffold database (ScaPD) (8Han X. Wang J. Wang J. Liu S. Hu J. Zhu H. et al.ScaPD: a database for human scaffold proteins.BMC Bioinform. 2017; 18: 386Crossref PubMed Scopus (0) Google Scholar), as well as an ad hoc literature search. From this, we identified 35 human scaffolds (20 out of 291 from ScaPD and 15 from our manual literature search) that have been documented to undergo conformational changes during signaling (Table 1). These 35 scaffolds are part of many signaling pathways, including ones involved in cell proliferation, cell survival and cell death, cell adhesion and motility, neural function, immunity, and differentiation. For most scaffolds in Table 1, conformational changes have been documented by structural approaches, including X-ray crystallography (XRC), NMR, small angle X-ray scattering (SAXS), and cryo-EM, or by computational modeling based on existing crystal structures. For others, biochemical or biophysical methods strongly suggest conformational changes, such as for the exposure of protein binding domains or altered mobility during electrophoresis. It is likely that the number of scaffolds with validated conformational changes will continue to increase.Table 1Scaffold proteins that undergo conformational change during signaling and diseases associated with scaffold protein mutationsaList was curated from ScaPD database as well as a manual literature search. Proteins are organized by best fit biological process.ScaffoldbAbbreviations: AKAP, A-kinase anchoring protein 18; AKAP-Lbc, A-kinase-anchoring protein-Lbc; AP-1/AP-2, Adaptor Protein Complex 1/2; ATM, ataxia-telangiectasia mutated; BCL10, B cell lymphoma 10; BCR, B-cell receptor; CARMA1, Caspase recruitment domain-containing MAGUK protein 1; CRK, Chicken tumor virus number 10 regulator of kinase; DISK1, Disrupted-in-schizophrenia-1; eIF4G, Eukaryotic initiation factor 4 gamma; FAK, Focal adhesion kinase; FLNA, Filamin A; GPCR, G-protein coupled receptor; Grb2, Growth factor receptor bound protein 2; IGF-1, Insulin growth factor 1; IQGAP1, IQ motif containing GTPase activating protein 1; IRS-1, Insulin receptor substrate-1; ITK, IL2 inducible T cell kinase; JIP1, JNK-interacting protein; JNK, c-Jun N-terminal kinase; KSR2; kinase suppressor of RAS; MAPK, mitogen-activated protein kinase; NBS1, Nijmegen breakage syndrome 1; NEMO, NF-kappa-B essential modulator; NHERF1, Na+/H+ exchanger regulatory factor 1; NHERF3, Na+/H+ exchanger regulatory factor 3; p130Cas, Crk-associated substrate; NMDA, N-methyl-D-aspartate; PAK1, P21-activated protein kinase 1; PKA, protein kinase A; PKC, Protein kinase C; PSD-95, Postsynaptic density protein-95; RACK1, receptor for activated C kinases; RGS4, Regulator of G protein signaling 4;; RTK, receptor tyrosine kinase; SH3RF1, SH3 domains and ring finger; SHANK3, SH3 and multiple Ankyrin repeat domains 3; SHC1, SHC-transforming protein 1; SHOC-2, Leucine-rich repeat protein SHOC-2; SQSTM1, Sequestosome 1; TCR, T-cell receptor; VAV, Vav guanine nucleotide exchange factor 1; WASP, Wiskott–Aldrich syndrome protein; ZO1, zonula occludens-1.Signaling pathwayScaffold-associated diseases (mutation effect)cGOF, gain-of-function mutation; LOF, loss-of-function mutation.GeneCards identifierConformational change validation methoddAMF, atomic force microscopy; BiFC, bimolecular fluorescence complementation; BRET, bioluminescence resonance energy transfer; EM, electron microscopy; ITC, isothermal titration calorimetry; MS, mass spectroscopy; PPI, protein–protein interaction; SANS, small angle neutron scattering; SAXS, small angle X-ray scattering; SIM, structured illumination microscopy; TRFS, time-resolved fluorescence spectroscopy.Cell cycleAKAP18cAMP, PKA, and Ca+2 signalingFebrile seizures, Familial, 7; gestational diabetes insipidus (LOF)GC06P131126XRC, EM (81Bjerregaard-Andersen K. 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Proteins are organized by best fit biological process.b Abbreviations: AKAP, A-kinase anchoring protein 18; AKAP-Lbc, A-kinase-anchoring protein-Lbc; AP-1/AP-2, Adaptor Protein Complex 1/2; ATM, ataxia-telangiectasia mutated; BCL10, B cell lymphoma 10; BCR, B-cell receptor; CARMA1, Caspase recruitment domain-containing MAGUK protein 1; CRK, Chicken tumor virus number 10 regulator of kinase; DISK1, Disrupted-in-schizophrenia-1; eIF4G, Eukaryotic initiation factor 4 gamma; FAK, Focal adhesion kinase; FLNA, Filamin A; GPCR, G-protein coupled receptor; Grb2, Growth factor receptor bound protein 2; IGF-1, Insulin growth factor 1; IQGAP1, IQ motif containing GTPase activating protein 1; IRS-1, Insulin receptor substrate-1; ITK, IL2 inducible T cell kinase; JIP1, JNK-interacting protein; JNK, c-Jun N-terminal kinase; KSR2; kinase suppressor of RAS; MAPK, mitogen-activated protein kinase; NBS1, Nijmegen breakage syndrome 1; NEMO, NF-kappa-B essential modulator; NHERF1, Na+/H+ exchanger regulatory factor 1; NHERF3, Na+/H+ exchanger regulatory factor 3; p130Cas, Crk-associated substrate; NMDA, N-methyl-D-aspartate; PAK1, P21-activated protein kinase 1; PKA, protein kinase A; PKC, Protein kinase C; PSD-95, Postsynaptic density protein-95; RACK1, receptor for activated C kinases; RGS4, Regulator of G protein signaling 4;; RTK, receptor tyrosine kinase; SH3RF1, SH3 domains and ring finger; SHANK3, SH3 and multiple Ankyrin repeat domains 3; SHC1, SHC-transforming protein 1; SHOC-2, Leucine-rich repeat protein SHOC-2; SQSTM1, Sequestosome 1; TCR, T-cell receptor; VAV, Vav guanine nucleotide exchange factor 1; WASP, Wiskott–Aldrich syndrome protein; ZO1, zonula occludens-1.c GOF, gain-of-function mutation; LOF, loss-of-function mutation.d AMF, atomic force microscopy; BiFC, bimolecular fluorescence complementation; BRET, bioluminescence resonance energy transfer; EM, electron microscopy; ITC, isothermal titration calorimetry; MS, mass spectroscopy; PPI, protein–protein interaction; SANS, small angle neutron scattering; SAXS, small angle X-ray scattering; SIM, structured illumination microscopy; TRFS, time-resolved fluorescence spectroscopy. Open table in a new tab Mutations in 34 of these 35 scaffolds have been directly linked to human disease. Such mutations affect the ability of the scaffold to bind a partner or render the scaffold unable to undergo proper conformational change upon substrate binding. Indeed, in at least two cases, disease mutations have been shown to affect a key conformational change in the scaffold. Namely, gain-of-function mutations in CARMA1 that disrupt its ability to undergo inhibitory conformational changes occur in some human B-cell lymphomas. Specifically, these mutations induce the stable conversion of CARMA1 to an open, active state, which promotes downstream signaling in the BCL10 pathway to chronically activate NF-κB, which blocks apoptosis and enhances proliferation (9Cheng J. Maurer L.M. Kang H. Lucas P.C. McAllister-Lucas L.M. Critical protein-protein interactions within the CARMA1-BCL10-MALT1 complex: take-home points for the cell