Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism
The author:Go Top Peptide Biotech    Released in:2021年04月12日
摘要:Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II.

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the “CTD code” that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4–5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism

How do Ess1 and Pin1 bind to multivalent CTD substrates?

Both the WW and PPIase domains of Ess1 and Pin1 bind pSer/pThr-Pro motifs. This dual-binding capacity, and the fact that most substrates contain multiple binding motifs complicates affinity measurements. Deciphering the mechanism of action of these proteins has been challenging and controversial. Early models suggested the WW domain tethers Ess1/Pin1 to protein substrates, increasing the local concentration of the PPIase domain, which then isomerizes nearby pSer-Pro motifs. This is based on the >10-fold higher binding affinity of the WW domain for single-site peptides in vitro17,23. However, it is not known whether the WW and PPIase domains bind multisite (multivalent) substrates simultaneously and/or cooperatively, or whether the domains compete with each other for occupancy. Nor has the stoichiometry and arrangement of Ess1/Pin1 proteins on long, physiologically relevant substrates been determined. To address these questions and gain a mechanistic understanding of how Ess1 interacts with its major in vivo target, the Rpb1 CTD, we determined the affinity and stoichiometry of Ess1–CTD interaction using multiple orthogonal approaches.

Ess1 binds better to longer CTD peptides

Prior studies of Ess1/Pin1–CTD interaction were limited to peptides bearing only a single heptad repeat (Y1S2P3T4S5P6S7). To provide a more realistic model of CTD interaction, we generated a series of CTD peptides of increasing length ranging from 1 to 5 heptad repeats (1R–5R) (Table 2). To simplify the analysis, phosphorylation (incorporated during synthesis) was restricted to only the outermost repeats, and positioned exclusively on the Ser5-Pro6 motif. The pSer5-Pro6 position was chosen because it has a higher binding affinity and turnover rate than does pSer2-Pro318,23, and because mutations of Ser5 show a stronger genetic interaction with Ess1 in vivo12.

Ess1 binds as a monomer, favoring 5R-CTD peptides

Ess1–CTD interactions were also analyzed using sedimentation velocity analytical ultracentrifugation (SV-AUC), a method that maintains the equilibrium between free and bound species as the complex sediments in a gravitational field43. As such, it is possible to measure binding affinities, stoichiometry, cooperativity and potential conformational changes upon binding43. Ess1 is a stable globular protein that sediments as a monodisperse monomer that did not change over a 4-fold concentration range (Fig. 4a). The sedimentation coefficient of Ess1 (s ~ 1.45; f/f0 ~ 1.35) indicates there is added hydrodynamic drag consistent with an elongated shape in solution, vs. a spherical protein of this size, which would have a higher s value (s = 1.65; SEDNTERP)44, consistent with the SAXS and NMR results.

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism

WW and PPIase domain contacts are enhanced with longer CTD peptides

To identify individual residues associated with the binding interface on Ess1, we titrated unlabeled CTD peptides and used NMR to monitor the backbone amide chemical shifts of residues in Ess1. From these spectra, we calculated chemical shift perturbations (CSPs) and mapped these onto the structure of Ess1

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism

Structural and functional differences between Ess1 and Pin1

The structure of the S. cerevisiae Ess1 reveals conserved folds for the WW domains and PPIase domains, consistent with the fact that orthologs ranging from C. albicans Ess1 to human Pin1 complement ess1 deletion mutants in S. cerevisiae. However, the elongated structure of Ess1 and distinct linker region raises a number of important questions. How does the more rigid structure of the fungal enzymes and distinct juxtaposition of the two protein domains influence (or restrict) substrate interactions? Put another way, why does the mammalian Pin1 enzyme lack a highly structured linker found in the fungal Ess1 enzymes, and what possible evolutionary advantage might that confer? Finally, what is the role of the prominent linker α-helix found in the fungal enzymes?


We suggest that the interdomain flexibility of the mammalian orthologs of Ess1 increases the diversity of substrates that can be recognized using a concerted simultaneous binding mechanism. Indeed, human Pin1 is thought to recognize hundreds of potential targets, while multiple genetic studies in yeast have only revealed a limited number of targets8,48,49. For more rigid proteins like Ess1 and CaEss1, the flexibility required for simultaneous binding may instead reside in the substrates themselves, for example, in long polymeric targets like the CTD, whose pSer-Pro-binding motifs are less spatially constrained than in globular proteins. The potential differences in substrate preferences makes the explicit prediction that, unlike the ability of Pin1 to complement in yeast, the fungal enzymes would not be capable of fully substituting for Pin1 in mammals. Finally, the prominent solvent-exposed α-helix found in the Ess1 and CaEss1 enzymes could mediate fungal-specific protein–protein interactions.


Original source:

https://www.nature.com/articles/s42003-021-01906-8


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