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The genetic information of eukaryotic organisms is packaged into a compacted chromatin structure containing nucleosome core particles with DNA wrapped around two copies each of the four histone proteins H2A, H2B, H3 and H4 (ref. 1). Proteins that regulate chromatin are involved in DNA processes including transcription, replication, recombination and repair. Chromatin regulatory proteins can be grouped into four broad classes: histone chaperone proteins that assemble and disassemble histones and replace variant histones in chromatin2; ATP-dependent chromatin-remodeling enzymes that physically move the histone proteins about the DNA3; post-translational modification enzymes that covalently modify histones, predominantly on the N-terminal histone tail regions4; and chromatin-targeting proteins or modules that recruit proteins to DNA and modified or unmodified histones5.

In the last category, many protein modules that recognize sequence- and type-specific histone modifications have been identified5. The first such domain to be characterized was the bromodomain, which remains the only known domain to specifically recognize acetylated lysines within histones6. Recognition of methyllysine modifications is more complex than for acetyllysine modifications because lysine residues can be mono-, di- or trimethylated. Given this increased complexity, it is perhaps not surprising that several folded domains have been shown to recognize methylated lysines including chromodomains, tudor domains, WD40 domains and PHD fingers5. On page 245 of this issue of Nature Structural & Molecular Biology, Collins et al.7 now report yet another methyllysine-recognition module, and it comes from an unlikely source—ankyrin repeats.

Ankyrin repeats are segments of 32–33 amino acids typically found in clusters of four

Michael M. Brent and Ronen Marmorstein are at the Wistar Institute and the Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. e-mail: marmor@wistar.org

or more contiguous repeats. Each repeat unit comprises a helix-turn-helix-β-turn element, with the helices stacked in a linear fashion to form a side-by-side helical bundle from which β-hairpins project orthogonally (Fig. 1a). Ankyrin repeats are found predominately in eukaryotes and occur in functionally diverse proteins including cytoskeletal proteins, enzymes, toxins and transcription factors8,9. Ankyrin repeats mediate protein-protein interactions10, but until now they were not reported to selectively bind covalently modified histones. Collins et al.7 use fluorescence polarization studies to show that the ankyrin repeat domains of G9a and G9a-like protein (GLP) preferentially bind to mono- and

dimethylated H3 lysine 9 (H3K9me1 and H3K9me2, respectively) with dissociation constants in the 10 µM range, and determine the crystal structure of the GLP ankyrin repeat domain bound to an H3K9me2-containing peptide. The structure reveals that the methylated lysine is sandwiched between the β-hairpins and helices of the fourth and fifth ankyrin repeats of the six-repeat GLP protein. A unique aspect of this finding is that these methyllysine-specific targeting domains are found within the same polypeptide chain that also produces the methyllysine marks. The only other histone-modifying enzyme that contains a recognition module for the same type of modification that it produces is the Gcn5

Ankyrin for methylated lysinesMichael M Brent & Ronen Marmorstein

The ankyrin repeats of the G9a and GLP histone methyltransferases have now been shown to be binding modules for mono- and dimethyllysine histone H3 lysine 9 (H3K9), revealing a new function for an ankyrin repeat domain and showing that a polypeptide chain can both create and recognize the same histone mark.

Figure 1 Structures of methyllysine binding domains in complex with their methyllysine-containing target peptides, showing the overall domain structure (above), a surface representation of the methyllysine binding pocket (middle) and a schematic representation of the aromatic cage (below). (a) The GLP ankyrin repeat (PDB 3B7B)7. Only ankyrin repeats 3, 4 and 5 of the six in total are shown. (b) The heterochromatin protein 1 (HP1) chromodomain (PDB 1KNA)19. (c) The bromodomain PHD finger transcription factor (BPTF; PDB 2F6J)20. The bromodomain is not shown. (d) The 53BP1 tandem tudor domain (PDB 2IG0)21. Some WD40 domains also seem to have a preference for methyllysine but are not represented here as they do not show extensive methyllysine interactions comparable to the other domains14–16,22,23.

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222 VOLUME 15 NUMBER 3 MARCH 2008 NATURE STRUCTURAL & MOLECULAR BIOLOGY

(or PCAF) family of histone acetyltransferase proteins, which contain an acetyllysine-targeting bromodomain. However, although Gcn5 and PCAF proteins acetylate H3K14 (ref. 11), their bromodomains recognize acetylated H4K16 (ref. 12).

In the structure of the GLP ankyrin repeat domain7, the dimethylammonium group of H3K9me2 is bound in a cage formed by three tryptophan residues and a glutamate residue that forms a salt bridge with the methylammonium proton (Fig. 1a). The structure suggests how mono- but not trimethyllysine can be accommodated in the binding pocket. Biochemical studies using G9a reveal that mutation of any of the tryptophan residues or the glutamate residue abrogates peptide binding. A comparison of the GLP–H3K9me2 complex with the other methyllysine-targeting modules bound to their respective substrates reveals several similarities and differences (Fig. 1). Specifically, all methyllysine binding domains reported to date form a remarkably similar pocket, with two to four aromatic residues and a glutamate or aspartate residue oriented in optimal positions to bind methyllysine by a combination of van der Waals, charge-π and charge-charge interactions (Fig. 1). The acidic residue in the pocket seems to have an important role in determining selectivity for lower over higher methylation states. Domains that bind mono- and dimethyllysine have a more closed pocket in which the acidic side chain forms a direct salt bridge with a methylammonium proton of a mono- and dimethyllysine, but poses a steric obstacle for trimethyllysine binding (Fig. 1a,d). Domains that bind with a preference for trimethyllysine have a more distant acidic residue to widen the pocket for

this modification (Fig. 1b,c). Taken together, it seems that at least four divergent protein folds use similar molecular strategies for the recognition of methylated lysine residues of histones5, although it has been proposed that some of these domains may have evolved from a common ancestor13.

An important question that arises from this study regards the biological advantage of having both the methyltransferase domain that produces the H3K9me1 and H3K9me2 marks and the ankyrin repeats that recognize them within the same polypeptide chain. One possible reason is to facilitate propagation of the H3K9me1 and H3K9me2 modifications. That is, once the substrate is converted to product, the enzyme rebinds to the product via the recognition module so that the enzymatic domain can convert the next substrate within an adjacent nucleosome to product and so on. Another possibility is that the modified substrate can be ‘protected’ from demethylation until an appropriate time. The answer to this question may have something to do with why G9a and GLP form heterodimers. Does the same polypeptide chain actually make and recognize the modification, or does one subunit of the complex create the modification whereas the other subunit binds the modification? Consistent with the latter possibility, the G9a ankyrin repeats have a slight preference for H3K9me2, whereas GLP ankyrin repeats have a slight preference for H3K9me1 (ref. 7).

The findings by Collins et al. have broader implications for other protein modules that bind histone modifications. It seems likely that still other methyllysine binding modules will be identified. In addition, the ankyrin repeat fold could be suitable for targeting unmodified or other histone modifications

such as acetylation or phosphorylation. Indeed, the WDR5 member of the WD40 module and the BHC80 member of the PHD finger have been shown to target unmodified histones14–17. Some bromodomains and chromodomains also have poor conservation within their respective ligand binding regions, suggesting that they may recognize other protein modifications as well12,18. With the arrival of the ankyrin repeat as a new methyllysine-targeting module, it is likely that other histone-targeting modules, also from unlikely sources, will follow.

1. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. & Richmond, T.J. Nature 389, 251–260 (1997).

2. De Koning, L., Corpet, A., Haber, J.E. & Almouzni, G. Nat. Struct. Mol. Biol. 14, 997–1007 (2007).

3. Wang, G.G., Allis, C.D. & Chi, P. Trends Mol. Med. 13, 373–380 (2007).

4. Bhaumik, S.R., Smith, E. & Shilatifard, A. Nat. Struct. Mol. Biol. 14, 1008–1016 (2007).

5. Taverna, S.D., Li, H., Ruthenburg, A.J., Allis, C.D. & Patel, D.J. Nat. Struct. Mol. Biol. 14, 1025–1040 (2007).

6. Dhalluin, C. et al. Nature 399, 491–496 (1999).7. Collins, R.E. et al. Nat. Struct. Mol. Biol. 15, 245–250

(2008).8. Sedgwick, S.G. & Smerdon, S.J. Trends Biochem. Sci.

24, 311–316 (1999).9. Schultz, J., Milpetz, F., Bork, P. & Ponting, C.P.

Proc. Natl. Acad. Sci. USA 95, 5857–5864 (1998).10. Mosavi, L.K., Cammett, T.J., Desrosiers, D.C. &

Peng, Z.Y. Protein Sci. 13, 1435–1448 (2004).11. Kuo, M.H. et al. Nature 383, 269–272 (1996).12. Owen, D.J. et al. EMBO J. 19, 6141–6149 (2000).13. Maurer-Stroh, S. et al. Trends Biochem. Sci. 28, 69–74

(2003).14. Couture, J.F., Collazo, E. & Trievel, R.C. Nat. Struct.

Mol. Biol. 13, 698–703 (2006).15. Ruthenburg, A.J. et al. Nat. Struct. Mol. Biol. 13,

704–712 (2006).16. Schuetz, A. et al. EMBO J. 25, 4245–4252 (2006).17. Lan, F. et al. Nature 448, 718–722 (2007).18. Nielsen, P.R. et al. Nature 416, 103–107 (2002).19. Jacobs, S.A. & Khorasanizadeh, S. Science 295,

2080–2083 (2002).20. Li, H. et al. Nature 442, 1058–1061 (2006).21. Botuyan, M.V. et al. Cell 127, 1361–1373 (2006).22. Han, Z. et al. Mol. Cell 22, 137–144 (2006).23. Wysocka, J. et al. Cell 121, 859–872 (2005).

With more than 100 protein factors and five small nuclear RNAs (snRNAs), the size and complexity of the spliceosome easily rival

those of most cellular machineries. The collection of associated proteins changes throughout spliceosome assembly, and even the catalytically active complex may change its composition between the two transesterification reactions of splicing. This, together with several conformational changes accompanying consecutive transitions in

Maria M. Konarska is at the Rockefeller University, New York, New York 10065, USA. e-mail: konarsk@mail.rockefeller.edu

the assembly, catalytic and disassembly phases of spliceosome action, creates a remarkable complexity that poses substantial experimental challenges.

Recent work by Bessonov et al.1 describes the purification and characterization of fully assembled, active spliceosome C complexes that contain first-step products—a lariat

A purified catalytically competent spliceosomeMaria M Konarska

The compositional complexity of the spliceosome creates a serious obstacle for its experimental analysis. Purification of a compositionally defined splicing complex C capable of completing the second step of splicing in the absence of additional proteins opens the door for future mechanistic and structural analyses.

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