Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation

Abstract

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1 binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

Mutation in a flexible linker modulates binding affinity for modular complexes

“…These assays (Supporting Information Figure S2B) yielded K D values of: ISL2/LHX3 = 160 ± 30 nM (n = 6), ISL2 R282G /LHX3 = 970 ± 20 nM (n = 3), ISL2/LHX4 = 33 ± 5 nM (n = 4), ISL2 R282G /LHX4 = 170 ± 20 nM (n = 4); standard errors are reported. Data for the WT proteins were reported previously . These data are consistent with the Y2H and thermofluor data confirming that WT ISL2 LID binds with a higher affinity than does the R282G mutant to both LHX3 (6.1‐fold higher) and LHX4 (5.2‐fold) and that ISL2 binds to LHX4 more strongly than to LHX3 (4.8‐fold).…”

“…Data for the WT proteins were reported previously. 9 These data are consistent with the Y2H and thermofluor data confirming that WT ISL2 LID binds with a higher affinity than does the R282G mutant to both LHX3 (6.1-fold higher) and LHX4 (5.2-fold) and that ISL2 binds to LHX4 more strongly than to LHX3 (4.8-fold).…”

“…For the ISL2‐R282G mutation we expect that introduction of a glycine residue induces an increased entropic penalty—increased loss of entropy due to binding and folding of a more inherently flexible spacer. This type of penalty likely contributes to the tendency of modular binding to have a binding affinity greater than that of the individual modules but be less than additive …”

“… with samples at 0.6 mg/mL protein. FRET‐based dilution experiments with 3C‐protease treated mYPet–LID–LIM 1 + 2 ‐mECFP fusion proteins were conducted as described previously, over the protein concentration range of 1‐9 μM.…”

“…This type of penalty likely contributes to the tendency of modular binding to have a binding affinity greater than that of the individual modules but be less than additive. 5,9 .…”