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Roughly 40% of the proteome is intrinsically disordered; these proteins and domains behave differently from structured proteins, rarely adopting just one stable structure. Instead, it can be useful to think of them dynamically moving through a wide range of favorable conformations known as an ensemble.

About 40% of the proteome is thought to be disordered. Researchers are beginning to explore the evolutionary rules that govern these proteins.

Many structured proteins are under evolutionary pressure that constrains how much they can change over time. Too large a shift in amino acid sequence may compromise function, unless a secondary change happens to compensate. For intrinsically disordered proteins, however, it is more difficult to determine whether ensembles undergo the same selection pressure. Multiple sequence alignments often show significant changes to a disordered region’s primary amino acid sequence, sometimes including dramatic changes in length.

“If a protein is under selection, you expect (sequence) conservation, and we couldn’t see it,” said Luc铆a Chemes, a professor at Argentina’s Universidad Nacional de San Mart铆n. Still, studies of disordered proteins have made it clear over time that they have important conserved functions. Chemes said, “There’s evidence that there must be conservation of function — so how does this happen, if the sequence changes so much?”

To find out, Chemes’ team chose as a model system an adenovirus protein that disrupts a host transcription–repression complex. The viral protein must work rapidly early in infection, when its levels are low, to hijack the host cell cycle. Therefore, high-affinity binding is very important.

How much the viral protein can disrupt the complex depends on two short linear motifs that associate directly with the translation repressor, but the lab found that the strength of binding is also governed by an intrinsically disordered region that connects the two linear motifs. Using proteins constructed of the two motifs separated by different linkers, that the linkers have the optimal length to tether the linear motifs closely enough together to reach their binding sites and boost binding affinity at both sites. Any longer, and the linear motifs could flop through too many conformations, missing their binding target, while a shorter linker prevented both linear motifs from binding at once.

When comparing homologous proteins from viruses targeting different mammalian species, whose linear sequences varied in length from 40 to 75 amino acids, the lab was surprised to find that all-atom structural modeling predicted a consistent end-to-end distance. Closer packing and lower density of charge per residues enabled longer linear sequences to pack into the same length as shorter ones. That confirmed that evolutionary constraints on the ensemble structure ensured its function, even when its sequence fluctuated.

“It’s clear that there’s more conserved than meets the eye when you see an alignment,” Chemes said. But the conserved feature defining an ensemble may not always be end-to-end distance. The next challenge for the field will be to identify the features that need to be conserved in each intrinsically disordered protein.