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Disparate Binding Kinetics by an Intrinsically Disordered Domain Enables Temporal Regulation of Transcriptional Complex Formation

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Disparate Binding Kinetics by an Intrinsically Disordered Domain Enables Temporal Regulation of Transcriptional Complex Formation Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation Neil O. Robertsona, Ngaio C. Smitha, Athina Manakasa, Mahiar Mahjouba, Gordon McDonaldb, Ann H. Kwana, and Jacqueline M. Matthewsa,1 aSchool of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia; and bCentre for Translational Data Science, University of Sydney, Sydney, NSW 2006 Edited by G. Marius Clore, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, and approved March 26, 2018 (received for review August 18, 2017) Intrinsically disordered regions are highly represented among C-terminal domain (Fig. 1A). LIM domain binding protein 1 mammalian transcription factors, where they often contribute to (LDB1) interacts with all LIM-HD/LMO proteins through a the formation of multiprotein complexes that regulate gene expres- disordered LIM interaction domain (LID) (Fig. 1A), which folds sion. An example of this occurs with LIM-homeodomain (LIM-HD) on binding to LIM1+2 domains to form extended modular com- proteins in the developing spinal cord. The LIM-HD protein LHX3 and plexes (9–11). Competition for LDB1 by LIM-HD/LMO proteins the LIM-HD cofactor LDB1 form a binary complex that gives rise to contributes to a so-called transcriptional “LIM code” that helps interneurons, whereas in adjacent cell populations, LHX3 and LDB1 determine cell fate in the developing spinal cord (12). A binary form a rearranged ternary complex with the LIM-HD protein ISL1, complex comprising LDB1 bound to the LIM-HD protein resulting in motor neurons. The protein–protein interactions within LHX3 triggers differentiation of V2-interneurons (V2-INs) these complexes are mediated by ordered LIM domains in the LIM- (Fig. 1B) (13). In neighboring cells, a ternary complex is formed HD proteins and intrinsically disordered LIM interaction domains comprising LDB1, LHX3, and a second LIM-HD protein, ISL1. (LIDs) in LDB1 and ISL1; however, little is known about how the Here, LDB1 contacts ISL1 + , forcing LHX3 + to bind a LID LIM1 2 LIM1 2 BIOCHEMISTRY strength or rates of binding contribute to complex assemblies. We LID in the C-terminal domain of ISL1. The ternary complex trig- have measured the interactions of LIM:LID complexes using FRET- gers differentiation of spinal motor neurons (sMNs) (Fig. 1C)(13, based protein–protein interaction studies and EMSAs and used these 14). Paralogues ISL2 and LHX4 can form similar complexes in data to model population distributions of complexes. The protein– developing sMNs (Fig. 1 B and C)(15–17). Despite low sequence protein interactions within the ternary complexes are much weaker identity (Fig. 1D), LDB1LID and ISL1/2LID form very similar than those in the binary complex, yet surprisingly slow LDB1: structures when in complex with their partners (11). sMN progen- ISL1 dissociation kinetics and a substantial increase in DNA binding itors also express LMO4, which has been shown to inhibit the for- affinity promote formation of the ternary complex over the binary mation of the binary complex over the ternary complex by complex in motor neurons. We have used mutational and protein competing with LHX3/4 for LDB1 (18, 19). engineering approaches to show that allostery and modular binding A lack of quantitative protein–protein and protein–DNA by tandem LIM domains contribute to the LDB1LID binding kinetics. binding data means that it is unclear what mechanism governs The data indicate that a single intrinsically disordered region can the regulation of ternary and binary complexes in sMNs. Efforts achieve highly disparate binding kinetics, which may provide a mech- to quantify LID:LIM1+2 interactions were hampered, as the anism to regulate the timing of transcriptional complex assembly. Significance intrinsically disordered proteins | binding kinetics | protein–protein interactions | protein–DNA interactions | transcriptional regulation Different combinations and permutations of transcription fac- tors work together to regulate the expression of target genes. These proteins often contain high levels of intrinsically disor- ntrinsically disordered regions (IDRs) are protein domains that dered regions, which are important mediators of protein–pro- Ilack a well-defined 3D structure. IDRs are frequently involved in tein interactions. We show that unusual binding kinetics – making protein protein interactions. It has been suggested that associated with an intrinsically disordered region in a tran- interactions involving IDRs offer many advantages over those in- scriptional coregulator can regulate the formation of tran- volving solely structured domains, including the combination of scriptional complexes that lead to the specification of neuronal K high specificity with low affinity ( d), increased sensitivity to en- cell subtypes. Notably, a single intrinsically disordered region vironmental conditions and posttranslational modifications, in- shows selective differences in binding kinetics for proteins of creased flexibility, and the ability to provide a hub for multiple the same family, which have implications for how intrinsic interactions (1, 2). Studies of IDR binding kinetics suggest that disorder contributes to regulatory processes and complexity in disorder can increase both association and dissociation rate con- higher organisms. stants (kon and koff, respectively) (3–6). Eukaryotic transcription factors are highly enriched in IDRs compared with other eukary- Author contributions: N.O.R., N.C.S., A.H.K., and J.M.M. designed research; N.O.R., N.C.S., otic proteins and prokaryotic transcription factors (7). Despite and A.M. performed research; N.O.R., N.C.S., A.M., M.M., G.M., A.H.K., and J.M.M. ana- their biological importance, relatively few studies report quanti- lyzed data; and N.O.R., N.C.S., A.M., A.H.K., and J.M.M. wrote the paper. tative data for IDR-mediated transcription factor interactions (8). The authors declare no conflict of interest. The LIM-homeodomain (LIM-HD) and LIM-only (LMO) This article is a PNAS Direct Submission. proteins provide a model for the role of IDRs in transcriptional Published under the PNAS license. complexes. All LIM-HD/LMO proteins contain two LIM domains 1To whom correspondence should be addressed. Email: [email protected]. (LIM1+2) arrayed in tandem that take part in protein–protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. interactions. LIM-HD proteins also contain a central DNA 1073/pnas.1714646115/-/DCSupplemental. binding homeodomain (HD) and an intrinsically disordered www.pnas.org/cgi/doi/10.1073/pnas.1714646115 PNAS Latest Articles | 1of6 Downloaded by guest on September 29, 2021 – A LMO LDB1 Here, we report quantification of the protein protein interac- LIM1+2 SA LID tions involved in the sMN LIM code using solution FRET-based LIM-HD methods. The binding kinetics of the LDB1:ISL1/2 interactions LIM1+2 HD LID are much slower than those of the other LID:LIM1+2 interactions in the system. We combined these data with measurements of B LDB1 NI-2V C LDB1 NMs protein–DNA interactions using EMSAs and modeled the changes in populations of complexes. The formation of sMN LHX3/4 ISL1/2 LHX3/4 LIM1+2 LIM1+2 LIM1+2 ternary complexes is likely to be reliant on both the unusual ki- SA SA netics of LDB1 binding by the different LIM-HD proteins and the LID LID LID higher affinity of the ternary complex for its target DNA sites. HD HD HD Binding data for mutants and single LIM domains suggest that small differences in related ordered LIM domains can modulate Binary complex target genes Ternary complex target genes the LDB1LID binding mechanism to produce highly disparate D LIM2 binding region LIM1 binding region binding kinetics for a single IDR. 339 LDB1LID 291 ISL1LID Results ISL2 301 LID LID:LIM1+2 Interactions Have Disparate Affinities and Kinetics. We K k – – measured d and off values for complex formation between the Fig. 1. Protein protein and protein DNA interactions in the LIM code. (A) + Domain structures of the LIM-HD/LMO proteins and LDB1: tandem LIM do- LIDs from LDB1, ISL1, and ISL2 and the LIM1 2 domains mains (LIM1+2), HD, LID, and self-association (SA) domain. The LID (broken from ISL1, ISL2, LHX3, and LHX4 (Table 1). The homologous box) in LIM-HDs has only been found in ISL1/2. (B) LDB1 and LHX3/4 form a competition approach, in which MBP-LDB1LID was titrated into binary complex to regulate V2-IN development. (C) LDB1 binds ISL1/2, which in cleaved fluorescent complexes, was used to estimate the stronger turn, binds LHX3/4 to form a ternary complex that regulates sMN develop- binding affinities (Kd ≤ 2 nM) (Fig. 2B), and dilution experi- ment. (D) Structure-based sequence alignment of the LDB1 and ISL1/2 LIDs. ments were used to estimate weaker binding affinities (Kd > 10 nM) (Fig. 2C). koff values were determined by titrating with a large excess of MBP-LDB1LID (Fig. 2D). koff values for slowly LIM1+2 domains aggregate when expressed in isolation. How- −5 −1 dissociating complexes (koff < 10 s )werefittedusingafixed ever, engineered “tethered complexes,” comprising the LIM −4 −1 final FRET efficiency taken from the faster (koff ∼ 10 s ) domains fused to LIDs via a flexible linker, are soluble and stable experiments. (10, 20). We recently developed a FRET-based solution method The binding affinities of this network of like interactions span to study these interactions (21). Monomeric FRET protein pairs six orders of magnitude from the weakest intramolecular LID: mYPet and mECFP are fused to the termini of the interacting −4 −5 LIM1+2 interactions in the ISL1/2 proteins (Kd ∼ 10 –10 M) domains within a tethered complex that has a human rhinovirus through the intermediate LDB1LID:ISL1/2LIM1+2 and ISL1/2LID: −7 −8 3C protease (HRV 3C)-cleavable peptide linker. After pro- LHX3/4LIM1+2 (Kd ∼ 10 –10 M) to the strongest LDB1LID: −9 teolysis of the linker, the loss of FRET by competition with a LHX3/4LIM1+2 interactions (Kd ≤ 10 M), which were similar to nonfluorescent LID peptide or by dilution of the complex is that previously reported for LDB1LID:LMO4LIM1+2 (21).
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