During protein evolution, some amino acid substitutions modulate protein function (tuneability). In most proteins, the accessible tuneable range is wide and can be sampled by a set of protein variants that each contain multiple amino acid substitutions. In other proteins, the full range can be accessed by a set of variants that each contains a single substitution. Indeed, site-saturating substitutions at individual rheostat positions can sample the full range for some folded globular proteins. In proteins with intrinsically disordered regions (IDRs), many functional studies, which would also detect tuneability, used multiple substitutions or deleted small regions. These results have led to proposed mechanisms such as the acidic exposure model for transcriptional activation domains (AD). Only a few IDRs have been assessed with single substitutions. Results have been mixed: (i) The disordered ADs of two full-length transcription factors did not show tuneability, yet (ii) a fragment of another AD was tuneable by single substitutions. Here, we tested tuneability in the AD of a full-length, non-DNA-binding transcription factor, human class II transactivator (CIITA). Sequence analyses and experiments showed that AD of CIITA is an IDR. Functional assays of singly-substituted variants showed that this IDR was highly tuneable, and some outcomes were not predicted by the acidic exposure model. Instead, four tested positions showed rheostat behaviour, with a wide range of effects on transcriptional activation. Thus, tuneability of different IDRs can vary widely; future studies are needed to illuminate the biophysical features that govern whether an IDR can be tuned by single substitutions.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new rule?

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