Solution structure of the Pdp1 PWWP domain reveals its unique binding sites for methylated H4K20 and DNA

Abstract

Methylation of H4K20 (Lys(20) of histone H4) plays an important role in the regulation of diverse cellular processes. In fission yeast, all three states of H4K20 methylation are catalysed by Set9. Pdp1 is a PWWP (proline-tryptophan-tryptophan-proline) domain-containing protein, which associates with Set9 to regulate its chromatin localization and methyltransferase activity towards H4K20. The structure of the Pdp1 PWWP domain, which is the first PWWP domain identified which binds to methyl-lysine at the H4K20 site, was determined in the present study by solution NMR. The Pdp1 PWWP domain adopts a classical PWWP fold, with a five-strand antiparallel β-barrel followed by three α-helices. However, it differs significantly from other PWWP domains in some structural aspects that account, in part, for its molecular recognition. Moreover, we revealed a unique binding pattern of the PWWP domain, in that the PWWP domain of Pdp1 bound not only to H4K20me3 (trimethylated Lys(20) of histone H4), but also to dsDNA (double-stranded DNA) via an aromatic cage and a positively charged area respectively. EMSAs (electrophoretic mobility-shift assays) illustrated the ability of the Pdp1 PWWP domain to bind to the nucleosome core particle, and further mutagenesis experiments indicated the crucial role of this binding activity in histone H4K20 di- and tri-methylation in yeast cells. The present study may shed light on a novel mechanism of histone methylation regulation by the PWWP domain.

PWWP domains and their modes of sensing DNA and histone methylated lysines

“…Moreover, the PWWP domain o f D N M T 3 a e x h i b i t s n o c l e a r p r e f e r e n c e f o r hemimethylated, double-methylated, or CpG-containing double-stranded DNA sequences (Purdy et al 2010). Finally, consistent with previous results, the PWWP domains of Schizosaccharomyces pombe Pdp1, as well as the human proteins LEDGF/p75 and ZMNYD11 were shown to bind DNA nonspecifically (Qiu et al 2012;van Nuland et al 2013;Eidahl et al 2013;Wang et al 2014). In contrast to these data, Yang and Everett (2007) have shown that the PWWP domain of HGDF is able to specifically recognize a 37-bp DNA element common to the promoter of SMYD1, arguing about the specificity of interaction between PWWP domains and DNA.…”

“…The PWWP domains of DNMT3a/b contain a bundle of five α-helices (Qiu et al 2002;Wu et al 2011). On the other hand, those of BRPF1, HDGF, HDGF2, and Pdp1 are composed of two α-helices connected by a loop (Lukasik et al 2006;Vezzoli et al 2010;Wu et al 2011;Qiu et al 2012). Recently, the structure of the PWWP domain from LEDGF/ p75 revealed a C-terminal region with less helical content than its counterparts.…”

“…In addition, mutations on the PWWP domains of DNMT3a and DNMT3b abolish their interaction with heterochromatin and inhibit their ability to methylate DNA Ge et al 2004). From that time until now, numerous PWWP domains have been shown to exhibit DNA-binding activity, including those of LEDGF/p75 (Singh et al 2006;Eidahl et al 2013;van Nuland et al 2013), HDGF (Lukasik et al 2006;Yang and Everett 2007), MSH6 (Laguri et al 2008), DNMT3a (Purdy et al 2010), Pdp1 (Qiu et al 2012), and the Bromo-ZnF-PWWP domain of human ZMNYD11 . For most proteins, DNA binding occurs in a nonspecific manner, without sequence selectivity.…”

“…The stability of the PWWP domain arises from intramolecular hydrogen bonds and polar interactions. These interactions occur not only between residues located in the same substructure but also between residues on different substructures, such as those present in the β1 −β2 arch and β3−β4 loop and the ones in the helical region (Qiu et al 2002(Qiu et al , 2012Slater et al 2003;Nameki et al 2005;Vezzoli et al 2010;Wu et al 2011). …”

“…2) (Qiu et al 2002. The β2−β3 loop is the less conserved part of the β−barrel, both in terms of amino acid composition and length, allowing for the insertion of different secondary structural elements (Qiu et al 2002(Qiu et al , 2012Slater et al 2003;Nameki et al 2005;Vezzoli et al 2010;Wu et al 2011).…”