Icated by the dashed black line. The Zn coordinating residues and hugely conserved Trp residue (W118 in PCAF_N domain of mGCN5) are drawn within the stick model. W118 types hydrogen bonding with all the primary chain carbonyl oxygens of E140 and G102. The two hydrogen bonds are indicated by a dashed purple line.Molecules 2021, 26,12 ofThe 3D structural comparison applying the DALI server [95] indicated that the all round structure is distinctive amongst the protein structures deposited into PDB. PCAF_N may very well be divided into three regions based on their characteristic structures. The N-terminal area (a. a. 8359) coordinating two Zn ions was termed the Zn region. The second area was termed the connecting area that forms an anti-parallel coiled-coil structure and connects the former and also the latter regions. The third area (a. a. 21672), folding into an -helix-rich structure, was termed the MORF4-related gene domain male-specific lethal3like (MSL3-like) domain (MSL3; PDB ID: 2y0n) [96], because the 3D structure comparison analysis by the program DALI indicated that the C-terminal region is homologous of MSL3, while the sequence identity in between them is low (about 22 ). The most intriguing structural function of PCAF_N will be the Zn area. Amino acid sequence analysis could not predict that the PCAF_N domain can coordinate Zn ions, indicating that they do not bear a common Zn binding motif including a RING finger. X-ray crystallography can be a valuable technique for identifying a metal ion. The XAFS analysis and anomalous Fourier map could clearly determine Zn ions that corresponded towards the sturdy electron densities observed inside the N-terminal region of PCAF. Strikingly, the coordination manner is special. The Zn region has a binuclear Zn-coordination structure (Zn2 Cys5 His2 ) (Figure 4B,C). The two Zn ions coordinate with seven residues (Cys107, Cys113, Cys115, Cys142, Cys145, His147, and His151). The sulfur atom of Cys145 coordinates each Zn ions (Figure 4C). Each of the Zn-coordinating residues on the PCAF_N domain are highly conserved among the proteins harboring the PCAF_N domain (Figure 4C). E3 ligase RAG1 has three Zn binding sites, and one of them is usually a related Zn coordination (Zn2 Cys5 His2 binuclear cluster) [97]. It should be noted that the binuclear Zn coordination in the RAG1 is Fmoc-Gly-Gly-OH Technical Information outdoors of your E3 ligase region, indicating that there is no provided proof to get a connection among E3 ligase activity and this binuclear Zn coordination. The HMM logo shows that the residues coordinating Zn ions and Trp residue are very conserved in PCAF_N family proteins (Figure 4D). The Trp is W118 inside the PCAF_N domain of mGCN5. W118 forms hydrogen bonding with the main chain carbonyl oxygens of E140 and G102, indicating that this extremely conserved Trp residue stabilizes the structure in the Zn region (Figure 4E). This compact two-Zn-ion coordination resembles the RING domain, but the Zn coordination pattern and ternary structure are one of a kind when compared with the RING domain. The lack of a Zn region lost the ubiquitin E3 activity of mGCN5 [18], indicating that the Zn area is accountable for exerting E3 activity. Nonetheless, it has not been unveiled no matter if the Zn region only has ubiquitin E3 activity as using a RING E3. All E3 ligases harbor an E2 ubiquitin-binding domain. In E2-RING structures, RINGs exhibit a common mode of interaction with E2 (Figure 2B). Within the PCAF_N domain, two loops coordinating Zn forms a