Hopeful (Protein InDel) Monsters?

In this issue of Structure, Arpino and colleagues describe in atomic detail how a protein stomachs a deletion within a helix, an event that rarely occurs in nature or in the lab. Can insertions and deletions (InDels) trigger dramatic structural transitions

Malleable Machines in Transcription Regulation: The Mediator Complex

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein–protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function

Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations

<div><p>Systematic mappings of the effects of protein mutations are becoming increasingly popular. Unexpectedly, these experiments often find that proteins are tolerant to most amino acid substitutions, including substitutions in positions that are highly conserved in nature. To obtain a more realistic distribution of the effects of protein mutations, we applied a laboratory drift comprising 17 rounds of random mutagenesis and selection of M.HaeIII, a DNA methyltransferase. During this drift, multiple mutations gradually accumulated. Deep sequencing of the drifted gene ensembles allowed determination of the relative effects of all possible single nucleotide mutations. Despite being averaged across many different genetic backgrounds, about 67% of all nonsynonymous, missense mutations were evidently deleterious, and an additional 16% were likely to be deleterious. In the early generations, the frequency of most deleterious mutations remained high. However, by the 17th generation, their frequency was consistently reduced, and those remaining were accepted alongside compensatory mutations. The tolerance to mutations measured in this laboratory drift correlated with sequence exchanges seen in M.HaeIII’s natural orthologs. The biophysical constraints dictating purging in nature and in this laboratory drift also seemed to overlap. Our experiment therefore provides an improved method for measuring the effects of protein mutations that more closely replicates the natural evolutionary forces, and thereby a more realistic view of the mutational space of proteins.</p></div

Dynamics of the laboratory drift.

<p><b>a</b>. Cumulative mutational loads (average number of mutations per gene) along the 17 rounds of the laboratory neutral drift. <i>N</i>_<i>t</i> is the average number of total mutations per gene (shown as ‘diamonds), <i>N</i>_<i>a</i> is the average number of nonsynonymous mutations per gene (shown as ‘squares’). Mutational loads were derived from deep-sequencing of G0, G3, G7 and G17 repertoires (full points) as well as by Sanger sequencing—standard, full-length sequencing of randomly selected variants from each round (empty points). Error bars show the standard error for the calculated averages. The lines illustrate the observed trends (not a fit for a specific equation). <b>b.</b><i>N</i>_<i>a</i><i>/N</i>_<i>s</i> is ratios of nonsynonymous to synonymous mutations (shown as ‘triangles); and the average number of compensatory mutations per gene (<i>W</i>_<i>rel</i> >1.1, shown as ‘circles’). Compensatory mutations are listed in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004421#pcbi.1004421.s013" target="_blank">S2 Table</a></b> and were defined as enriched mutations, either by assigned beneficial fitness effect for individual mutations by (<i>W</i>_<i>rel</i> >1.1) or high positional fitness effect (the averaged <i>W</i>_<i>rel</i> per position as calculated in <b><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004421#pcbi.1004421.g003" target="_blank">Fig 3A</a></b>, <i>W</i>_{rel (Positional)} >1.1). The effect of compensatory mutations discussed in the section of “<i>Dynamics of the laboratory drift</i>”. <b>c.</b> The cumulative mutational load for mutations with different fitness effects: ‘deleterious’ (<i>W</i>_<i>rel</i> ≤0.6), ‘Nearly-neutral’ (<i>W</i>_<i>rel</i> 0.61–0.8), ‘Neutral’ (<i>W</i>_<i>rel</i> 0.81–1.1) and ‘Beneficial’ (<i>W</i>_<i>rel</i> >1.1).</p

The theoretically possible <i>vs</i>. observed mutational space of M.HaeIII.

<p>The theoretically possible <i>vs</i>. observed mutational space of M.HaeIII.</p

The distribution of fitness effects of mutations in M.HaeIII's drift.

<p><b>a</b>. The distributions of fitness effects of all mutations observed in the sequenced rounds of the drift (G3, G7 and G17) by their relative fitness values, <i>W</i>_<i>rel</i>. Mutations were binned by unit interval values of <i>W</i>_<i>rel</i> = 0.1, ranging from <i>W</i>_<i>rel</i> = 0 to > 1.4 (missense: n = 1,957; nonsense: n = 125; synonymous: n = 321). <b>b</b>. Distribution of the frequencies of mutations within each given range of <i>W</i>_<i>rel</i> values. The frequencies of all mutations within a given <i>W</i>_<i>rel</i> range were summed up and divided by the sum of frequencies for all mutations within the same round. <b>c</b>. Log-values of the fold changes in the frequencies per each <i>W</i>_<i>rel</i> range in G17 relative to G3.</p