Systematic analysis of tropomodulin/tropomyosin interactions uncovers fine‐tuned binding specificity of intrinsically disordered proteins

Abstract

An intriguing regulatory mechanism is the ability of some proteins to recognize their binding partners in an isoform-specific manner. In this study we undertook a systematic analysis of the specificity of the tropomodulin (Tmod) interaction with tropomyosin (TM) to show that affinities of different Tmod isoforms to TM are isoform-dependent. Intrinsic disorder predictions, alignment of sequences, and circular dichroism were utilized to establish a structural basis for these isoform-specific interactions. The affinity of model peptides derived from the N-terminus of different TM isoforms to protein fragments that correspond to the two TM-binding sites of different Tmod isoforms were analyzed. Several residues were determined to be responsible for the isoform-dependent differences in affinity. We suggest that changing a set of residues rather than a single residue is needed to alter the binding affinity of one isoform to mimic the affinity of another isoform. The general intrinsic disorder predictor, PONDR® VLXT, was shown to be a useful tool for analyzing regions involved in isoform-specific binding and for predicting the residues important for isoform differences in binding. Knowing the residues responsible for isoform-specific affinity creates a tool suitable for studying the influence of Tmod/TM interactions on sarcomere assembly in muscle cells or actin dynamics in non-muscle cells.

Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes

“…Mutations Chosen for Site 2-The TM-binding dissociation constant of Tmod2s2 is 4.4 Ϯ 0.5 M; it is the highest among other isoforms (1.3 Ϯ 0.3 M, 2.1 Ϯ 0.4 M, and 3.6 Ϯ 1.2 M for Tmod1s2, Tmod3s2, and Tmod4s2, respectively) (13).…”

“…We successfully used this approach in our previous studies (13). The CD spectra of all Tmod1 peptides (WT and mutated) were typical for the mostly unfolded polypeptide; the CD spectrum of ␣TM1azip was typical for the ␣-helical polypeptides.…”

“…Mutations Chosen for Site 1-It was shown that Tmod binding to TM is isoform dependent, and dissociation constants (K d ) for all possible complexes between Tmod isoforms and TM isoforms were determined (13). For example, the affinity of ␣TM1azip for Tmod2s1 was the lowest, whereas the affinity of ␣TM1azip was the highest for Tmod1s1 among other isoforms (K d ϭ 1.1 Ϯ 0.4 M, 4.7 Ϯ 0.4 M, 1.9 Ϯ 0.3 M, and 2.4 Ϯ 0.6 M for Tmod1s1, Tmod2s1, Tmod3s1, and Tmod4s1, respectively).…”

“…Based on these observations, it has been proposed that such "signatures" in the PONDR® prediction plots, where short regions of predicted order are bounded by extended regions of predicted disorder, might correspond to potential binding sites, called molecular recognition features, which undergo disorder-to-order transitions upon complex formation (37)(38)(39). We have already utilized this approach to compare different Tmod isoforms from nine species (13). That earlier analysis revealed that the N-terminal region of Tmods possess a large variation in the number and appearance of "dips" and that for a given Tmod, the disorder distribution was mostly conserved even for proteins from distant species.…”

“…TM binding to Tmod is isoform-specific; for example, Tmod1 binds to ␣-TM more strongly than all other Tmod isoforms (13). We hypothesized that changes in TM-binding ability should affect the actin-capping properties of Tmod.…”