Structural Biochemistry/PHD Finger

PHD Zinc Fingers
Plant homeodomain (PHD) zinc fingers are conserved modules found in proteins that modify chromatin and mediate the molecular interactions in gene transcription. PHD zinc fingers were originally discovered to play a role in gene transcription and recognition of lysine-methylated histone H3. Recently, studies have shown that PHD fingers also have a sophisticated histone sequence reading ability that is set by the interplay between various histone modifications. These studies emphasize the functional aspects of PHD fingers as genome readers that can control gene expression through molecular recruitment of multiprotein complexes of chromatin regulators and other transcription factors.

Ligand Recognition
The plant homeodomain consists of approximately 50 - 80 amino acid residues of various sequences containing a certain zinc-binding motif that shows up in many chromatin-associated proteins. The PHD folding pattern consists of two anti parallel β-sheets and a C-terminal α-helix, which is stabilized by two zinc atoms. The PHD fingers read the N-terminal tail of histone H3 (methlyation of H3K4 and to a smaller extent the methylation state of H3R2 and the acetylation of H3K14). It has been reported that all PHD finger structures bind histone H3 through interactions with the first six N-terminal residues of H3, with the exception of two residues.

Conclusion
The reason that PHD fingers are so fascinating to scientists is that these domains are the smallest in size, which allows them to be versatile epigenome readers. More importantly, the structure-function relationships of the PHD fingers revealed from these recent studies illustrate how functional diversity of a protein module can be achieved by evolutionary changes to the structural features or amino acid residues near ligand binding sites. Also, because of PHD's low sequence conservation and adaptable structural plasticity, it will not be surprsing to see other modifications occur in the PHD recognition domains in the future.

Reading of H3K4me3
The BPTF-PHD structures reveal the main characteristics of PHD fingers that can read H3K4me3. The binding occurs through an aromatic cage where a trimethyl ammonium group is stabilized by van der Waals and cation-–π interaction, which is similar to the ones observed in chromodomain, MBT, PWWP, and Tudor domains. This aromatic cage is composed of one Trp and three Tyr residues; and it has three faces and a 'lid' that is beyond the tip of H3K4me3. Subsequently determined structures of other fingers in complex with the H3K4me3 peptides show that the cage varies and can contain a combination of two to four aromatic and hydrophobic residues. These residues that participate in aromatic cages tend to appear at similar positions within the sequence. At the most conserved position is the invariable Trp residue that is at the beginning of the β1 strand which is then followed by the aromatic or hydrophobic residue that starts at the β2 position. Generally, the residues that are used to form the aromatic cage exists in parts of the structure that are rigid, such as β-strand or close to the Zn-coordinating Cys residue. At minimum, two aromatic residues that include the invariable Trp at position 1, appears to be necessary for the H3K4me3 binding.

The simplest observed so far aromatic cage is that of jumonji, which is the AT-rich interactive domain 1A (JARID1A), and is composed of only two Trp residues that are at both positions 1 and 2. Together with JARID1A, the aromatic cage of recombination active gene 2 (RAG2) and myeloid/lymphoid or mixed-lineage leukemeia-1 (MLL1) lack the 'lid' residues that are present at position 3 in all other PHD finger aromatic cages. Additionally at Y1581, which is position V, in MLL1 undergoes a conformation change when binding that is not observed within the other PHD fingers. There is a slight variation in H3K4 binding region which is observed in the PHD fingers of yeast homolog of mammalian ING1 (Yng1), transcription initiation factor TFIID subunit 3 (TAF3), pygopus homolog 1(PYGO), inhibitor of group protein (ING4), and other PHD fingers of the ING family that have charged (Asp) or hydrophilic (Ser) residues that are close to the H3K4 residue at position 4 or 5. Of those, only the charged residues at position 5 in PYGO plays a role in the methylated lysine binding by slightly shifting the affinity in favor of the dimethlyated form of K4 (H3K4me2), making the affinities of the free PHD for both H3K4me and H3K4me2 virtually identical, and it shows a slight preference for H3K4me2 over both H3K4me1 and H3k4me3 in the PHD-HD1 complex. Even though the Ser and ASP residues in the other PHDs contribute to H3 binding, they do so through interactions with residues other then the K4.

Mutational studies in the BPTF suggest that the presence of a negatively charged residue in aromatic boxes can alter the binding selectivity of the other PHD fingers. Affinities in the wild-BPTF, which shows a preference for the H3K4me3 over the H3K4me2, change when mutated thus resulting a preference in H3K4me2 over the H3K4me3. Thus the relative affinity of H3K4me3 versus H3K4me2 can be modulated by subtle changes within the sequence of the PHD fingers. The biological impact of these small differences in affinity however, are not clear.

Reference
Sanchez, Roberto, and Zhou Ming-Ming. "The PHD Finger: A Versatile Epigenome Reader." Trends Biochem Sci. 2011 Jul;36(7):364-72. Epub 2011 Apr 21. < http://www.sciencedirect.com/science/article/pii/S0968000411000491>