Domain structure of CDY family proteins

All CDY family members consist of a chromodomain, a connecting hinge region, an enoyl-CoA-hydratase domain and a short C-terminal part (compare with Figure 1-7). The chromodomain consists of 55 amino acids and the enoyl-CoA-hydratase domain has about 173 amino acids.

Figure 1-7 Domain structure of CDY family members

Chromodomain

Chromodomains are evolutionarily highly conserved regions of about 40 amino acids, which are present in various proteins involved in chromatin organization and gene regulation [108].

The domain folds into a globular conformation of about 30 Å in diameter. It consists of an antiparallel three-stranded -sheet packed against an -helix in the carboxy-terminal segment of the domain (Figure 1-8). Overall the -sheets have a negative net charge and form a hydrophobic groove on one side, which is composed of conserved residues.

Connected to these observations the first defined molecular function could be assigned to the HP1 chromodomain. Primarily it was suggested that chromodomains are RNA-binding modules [109]. But then several groups showed that the main function of the HP1 chromodomain is the binding of the histone H3 tail methylated on lysine 9 [98, 110].

Accordingly, it was demonstrated that canonical chromodomains in general recognize methylated H3.

The binding of the HP1 chromodomain to methylated lysine 9 of histone H3 is maintained by its hydrophobic pocket by providing an appropriate environment for docking onto the histone H3 tail. The H3 tail adopts a -sheet conformation and builds together with two antiparallel -sheets of the chromodomain a three -sheet bundle.

Figure 1-8 HP1 chromodomain binding to trimethylated lysine 3 of histone H3.

HP1 chromodomain (orange) binds to the H3 peptide carrying a trimethylation on lysine 9 (red). The three blue residues Y24, W45 and Y48 build a tri-aromatic cage binding the methylation. The glutamate 23 (green) makes a hydrogen bond to the peptide backbone, whereas glutamate 56 (green) contacts serine 10 of the histone H3 tails. Figure was adapted from PDB: 1kne and [110].

Additionally, three aromatic residues (Figure 1-8) cage the methylammonium group of histone H3 methylated on lysine 9. Glutamate 23 of the chromodomain makes a hydrogen bond to the H3 peptide backbone and glutamate 56 interacts with the serine 10 adjacent to the methylated lysine 9 (Figure 1-8).

Methylations of lysines occur also on other sites within histones (compare with Table 1-2) but interestingly chromodomains studied to this point can only recognize lysines embedded in ARKS motifs (e.g. H3K9me, H3K27me) or ARTKQ motifs (e.g. H3K4me). The chromodomain of the Polycomb protein binds specifically to methylated lysine 27 of histone H3 [111]. The overall folds of the HP1 and the Polycomb chromodomain are similar, but their peptide-binding grooves show distinct features that provide the discrimination between the two marks [111].

Figure 1-9 Alignment of chromodomains.

Alignment of chromodomains from the human CDY family CDY, CDYL1a, CDYL1b and CDYL2 with human HP1, HP1 and HP1 as well as with CBX2, CBX4, CBX7 and CBX8 the Polycomb homologs of Homo sapiens. Red stars indicate the three residues forming the tri-aromatic cage for histone methylation binding.

The Polycomb chromodomain interacts with more amino acids of the histone tail surrounding the methylation mark and the HP1 chromodomain recognizes the residues in close vicinity of the PTM in a more precise manner. An alignment of the CDY family of chromodomains with the chromodomains of the three human isoforms of HP1 (, , ) and with the chromodomain of the human Polycomb proteins CBX2, CBX4, CBX7 and CBX8 is presented in Figure 1-9.

CDY family chromodomains show a high homology to the HP1 and Polycomb chromodomains. The three aromatic residues described as important for the function are present in the CDY family chromodomains, exept for in the CDYL1a splicing variant (see also 1.2.4). A superposition of the CDYL2 chromodomain with the HP1 chromodomain bound to the H3 peptide carrying a methylation on lysine 9 shows that CDYL2 and by homology also CDY and CDYL1 may have a tertiary structure similar to HP1 (Figure 1-10).

Therefore it is possible that the chromodomain of CDY family proteins may interact with histones methylated on certain lysine residues.

Figure 1-10 Alignment of HP1 and the human CDYL2 chromodomain.

HP1 (PDB: 1kne) chromodomain (pink) is bound to a histone H3 peptide carrying a trimethylation on lysine 9 (black). The overlayed CDYL2 (PDB: 2dnt) chromodomain is shown in green. Figure was adapted from [95].

Enoyl-CoA-hydratase domain

Enoyl-CoA-hydratases (also termed as crotonases) can act as isomerases, dehydrogenases and hydratases [112].

Figure 1-11 Alignment of the Enoyl-CoA-hydratase domains of the CDY family.

Dark orange indicates identical residues, whereas light orange labels homologous residues. Red stars label the three important enzymatic residues in enoyl-CoA-hydratases. ECHP: human peroxisomal ECH, ECHM: human mitochondrial ECH. Blue box: Adenine binding site, Red box: active loop.

The substrates of crotonases are mainly double bonds of unsaturated fatty acids during -oxidation in mitochondria (short fatty acids) as well as in peroxisomes (very long fatty acids).

An alignment of the CDY family enoyl-CoA-hydratase with the mitochondrial and peroxisomal enzymatic domains reveal only a moderate overall homology (Figure 1-11). The adenine binding pocket interacting with Acetyl-CoA in enoyl-CoA-hydratases shows a slightly higher degree of homology. In agreement, it was demonstrated that CDYL1 ECH domain interacts with coenzyme A [101].

Despite a good consistence of amino acids within the active loop, the three important residues (one glycine and two glutamates see Figure 1-11) maintaining the enzymatic reaction of enoyl-CoA-hydratases are exchanged in the ECH domain of the CDY family.

Figure 1-12 CDY family enoyl-CoA hydratase domains build trimeric structures

A, trimeric fold of the CDYL1 ECH domain (PDB: 1gtr). Each monomer has a different color. B, hexameric fold of CDY ECH domain (PDB: 2fw2). C, rat peroxisomal enoyl-CoA hydratase (PDB:

1dub). D, superposition of A and C.

Nevertheless, the ECH domains of the CDY family fold into the typical arrangement of enoyl-CoA-hydratase enzymes (Figure 1-12). Figure 1-12A shows the compact trimeric

structure formed by three CDY family proteins. Two of the homotrimers are able to form loose dimers (Figure 1-12B).

Figure 1-12C and D represent a superposition of the CDY-family ECH domain with the rat enoyl-CoA-hydratase enzyme and reveal that at least the crotonase-like fold of the ECH domain is conserved.