S. Cerevisiae SGF29 Tudor Domain

Babiker Higazi '23 and Daniel Frank '23

Contents:

I. Introduction

The Sgf29 protein is a novel element of the SAGA (Spt–Ada–Gcn5 acetyltransferase) complex, an evolutionarily conserved multiprotein complex that has been studied extensively due to its important role in gene regulation. SAGA was first identified as a histone acetyltransferase complex in yeast and later on observed in humans as well. Of the 20 protein subunits that compose the SAGA complex, Sgf29 is the only one with a tandem-Tudor domain that is conserved across species (from yeast to humans). Using mass spectrometry technology to screen for trimethyl mark interactors in H3 and H4 histones in human cells, Sgf29 was determined to bind to both H3K4me3 and H3K4me2 sites, with a partiality for the H3K4me3 site. Chromatin immunoprecipitation (ChIP)-sequencing studies have also shown Sgf29 present at gene promoters that mostly overlap with H3K4me3, the ligand responsible for epigenetic modification of DNA packaging protein H3. This H3K4me2/3-specific binding by the Sgf29 Tudor domain has been established as the structural basis behind the linkage between the human SAGA complex and H3K4me3 site since these structures come unbound when Sgf29 is knocked down. Sgf29 and the larger group of methyl mark recognizing proteins are essential for conducting the proper acetylation of their HAT complexes. The Sgf29 reader domain is thus crucial to targeting the misregulation and nonfunctioning of human acetyltransferase function, which can potentially result in impairment of chromatin dynamics.  

II. General Structure

TThe X-ray diffraction image in question is that of the tandem Tudor domains present in the Sgf29 proteins of Saccharomyces cerevisiae and humans. The protein consists of two parts, and , which are both found at the protein’s C-terminus. Unlike tandem Tudor domains observed elsewhere, the domains in Sgf29 pack tightly in a way that they face one another. Both parts of the domain consist of a barrel-like-fold created by the five twisted antiparallel b strands that compose both Tudor1 and Tudor2. Along with this domain, there is also a coiled-coil domain at the N-terminus of the protein. Both of these domains preferentially bind to methylated peptides, as they both selectively recognize H3K4me2/3 markings on the histone H3. The first four residues of the H3K4me3 peptide are lodged between the tandem Tudor domains.    

III. C-Terminus

The tandem-Tudor domain of Sgf29 is located at its in both yeast and humans. Both of these two domains contain an alanine residue which interacts with an adjacent K4 residue of a methylated H3 histone. This interaction is specific to methylated H3 histones, and so grants this subunit the level of substrate specificity appropriate for its role as reader complex. If not for these unique interactions at each domain’s binding “pockets” ( respectively), it would be impossible for the SAGA complex to bind and acetylate these methylated components. This association is highly secure since, due to its negative charge and inflexibility, the acetylation of the H3A1 residue would disassociate binding between H3K4me3 and hsSGF29. The foundational amine group of the H3A1 binding pocket is responsible for forming a key interaction that is the formed between an aspartic acid (D163) in the Tudor domain and the carboxylic group of an alanine on the backbone of the histone. Without this specific interaction, the bond would be about seventy times weaker. This means that this singular salt bridge is nearly a prerequisite for a successful interaction overall. The other residue, K4m3, is also bound to a negatively-charged, small pocket that is located on the Tudor 2 domains in hsSGF29 and scSgf29 and bordered by two aromatic residues. The foundational amine group of K4m3 forms a hydrogen bond with the oxygen atom of Y245 and Y212 in hsSGF29 and scSgf29 respectively. Like H3A1, K4m3 also interacts with a salt bridge that lies between its methyllysine and the negatively charged D266 residue on the slip side of the binding pocket.

IV. N-Terminus

At its , Sgf29 contains a coiled-coil domain that binds to a SAGA component important in modulating Gcn5 activity called Ada3. In determining hsSgf29 structure, longer versions of the protein were used during the crystallization process experiments for structural quality purposes. In these versions of hsSgf29, the N-terminus contains an extra alpha-helix located between the two Tudor domains. Thus, this alpha-helix is located outside of the histone binding cleft and has no effect on histone binding.

V. References

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