PARP_1

Poly(ADP-ribose) polymerase-1 (PARP-1) (PDB ID: 5DS3) from Homo sapiens belongs to the PARP family that modifies ADP-ribose after translation using NAD+ to control biological processes, especially those involving cell’s future. PARP-1 serves a major role biologically in responding to cellular stress by synthesizing poly(ADP-ribose) (PAR) at DNA damaged sites, thereby activating DNA repair factors (1, 2). For instance, PARP-1 could bind to DNA to discover DNA impaired regions, thus avoiding cell death. On the other hand, when interleukin 1β-converting enzyme (ICE) homologues cleave PARP-1, cell death would hasten. In the experiment, PARP-1-inactivated mutant demonstrated approximately 30-fold more chromosome breakage compared to that of the native structure because the sister chromatid exchange (SCE) rate was increased, thus precluding the repair of DNA (3).

In addition to DNA repair, PARP-1 also participates in apoptosis and in chromatin structure management. While sudden increase in PAR concentration from PARP-1 activation would speed up DNA repair, when DNA damage endures for a long period, PARP-1 is hyperactivated, depleting NAD+ (1). Moreover, excessive activity of PARP-1 precludes hexokinase function, which in turn causes mitochondria to malfunction; ultimately, failed glycolysis leads to bioenergetics collapse, causing parthanatos, which is a process for apoptosis (4).

The protein structure is further analyzed by crystallization, though not all protein portions were crystallized. Vapor diffusing sitting drop was the method for forming the crystals. The structural data were from ADSC QUANTUM 315r diffraction type using CCD detector, with X-ray diffraction for improving the shell resolution. The crystallized domains are PARP-type 1-zinc finger region, PARP-type-2-zinc finger region, nuclear localization signal-short sequence motif and automodification domain-region of interest (5).

PARP-1’s secondary structures include alpha helices, beta sheets and random coils. Initially, the HD domain composed of alpha-F and ADP-ribosyltransferase (ART) domain create a pocket in which the entrance of NAD+ is prohibited. Upon PARP-1 activation through deterioration of DNA or protein interactions, the secondary structure within the HD domain unfolds to open the pocket and accept NAD+ in order to manufacture poly(ADP-ribose) (1).

Lys-587 and Trp-589 are two of important residues close to the 5’ end that precludes the effects of hydrogen exchange (HX), thus preventing the DNA damage. In addition, Asp-770, Asp-776, and Glu-763 are the three basic residues located on alpha-F and directly face the binding site for NAD+. Asp-770 disrupts binding of adenine base, while Asp-776 and Glu-763 disrupt the phosphate groups of NAD+ using their charged side chains. Also, as the three residues are near the region with high HX concentrations with DNA presence, they mediate autoinhibition (1).

PARP-1 has three ligands: olaparib, PEG-400, and sulfate ion. Out of three, only olaparib performs a biological function, while the other ligands were employed for crystallization process. Olaparib is an inhibitor complexed at the site of PARP-1 that causes conformational change when bound. At PARP-1’s active site for catalysis, olaparib has its pyrimidine ring within the binding pocket for stability. As for PEG-400, along with water, it generates solvent that enhances the solubility of polar reactants (5-6). Moreover, to bind the substrate poly(ADP-ribose), the sulfate ion is attached to the pyrophosphate binding site (7).

PARP-1 consists of one unit forming six domains. DNA binding occurs through F1 and F2, while F3 is also significant for catalytic activity. An automodification domain (AD) is involved in automofication of PARP-1, and WGR both binds DNA and connects domains to activated DNA repair factors. Also, the catalytic domain (CAT) includes a helical domain and ADP-ribosyltransferase (ART) that control catalysis (1). PARP-1’s secondary structures include alpha helices, beta sheets and random coils. Initially, the HD domain composed of alpha-F and ADP-ribosyltransferase (ART) domain create a pocket in which the entrance of NAD+ is prohibited. Upon PARP-1 activation through deterioration of DNA or protein interactions, the secondary structure within the HD domain unfolds to open the pocket and accept NAD+ in order to manufacture poly(ADP-ribose). Of those six, four domains were critical for DNA damage-dependent catalysis (F1, F3, WGR, CAT) by binding to damaged DNA sites concertedly to collapse and change DNA’s conformation (1). One unit provides the DNA-binding site while the other is the BRCT domain involved in cell-cycle regulation. The other two subunits are PARP alpha-helical and PARP catalytic site working in conjunction to bind NAD+ (8). As HD contains a hydrophobic core, deleting two leucine residues causes PARP-1 to be hyperactive such that NAD+ binds to the active site, initiating PAR synthesis (1).

In order to disclose information about structurally similar proteins, the bioinformatics servers Position-Specific-Iterated Basic Local Alignment Search Tool (PSI-BLAST) and Dali server were used. Moreover, ExPASy provided that PARP-1 has the molecular weight of 29955.21 Da and the isoelectric point of 9.09 (9). PSI-BLAST sought to discover the primary structure closely matching that of the protein of interest. It uses a protein query to discover subjects, which are proteins with resembling primary structures. After analyzing sequence homology and identifying gaps, which are the differences in sequence between comparison amino acids, the E value is assigned. Whereas gaps result in higher E value, sequence homology lowers it. The E value less than 0.05 is significant. Also, Max Score specifies the sequence, and the program also factors in mutation, which lowers sequence similarity, thus raising the E value (10). On the other hand, the Dali server compares the tertiary structures. The structural comparison server works by having a huge database of PDB entries along with information about protein folding. It updates new structures weekly. Instead of comparing three-dimensional structures and calculating discrepancy between them with a least-squares approach, a sum-of-pairs method is used. It takes intermolecular distances into account, producing Z-scores that reflect closeness of folds (11).

With PSI-BLAST and the Dali Server, a comparison structure called the catalytic fragment of PARP (Poly[ADP-ribose] polymerase) complexed with carba-NAD (1A26), which is a same protein in a different organism, was found. Unlike PARP-1 that originated from Homo sapiens, it came from Gallus gallus (chicken) (5). However, both serve the same function as a catalytic enzyme that binds NAD+ or as a protein that binds DNA. The E value from PSI-BLAST was 1x10-122, which is less than 0.05 implying that the sequence homology is significant (10-11).

Moreover, the Z-score from the Dali server was 35.6; as the Z-score exceeding two indicates significant similarity, the two proteins have similar tertiary structures (11). Also, compared to PARP-1, PARP complexed with carba-NAD has a longer chain (361 vs. 271 residues). In terms of secondary structures, whereas PARP-1 contains 3/10 helix at residue number 958-960, PARP has a beta strand (2).

Additional to PARP complexed with carba-NAD found in Gallus gallus, another comparison structure is a different protein in the same organism, Homo sapiens. Poly[ADP-ribose] polymerase 2 (PARP-2) (PDB ID: 5DSY) has the Z-score of 35.6 and the E value of 4x10^-127 (5-8). The values indicated that the primary structures are significantly similar as well as the tertiary structures. The associated ligand for PARP-2 is 2-[4-[(2S,3S,4R,5R)-5-(6-aminopurin-9-yl)- 3,4-bis(oxidanyl)oxolan-2-yl]carbonylpiperazin- 1-yl]-N-(1-oxidanylidene-2,3-dihydroisoindol- 4-yl)ethanamide, or nicotinamide binding pocket (Nic) (5). While the ligands for PARP-1 and PARP-2 are different, the function as an inhibitor is identical; like olaparib of PARP-1, EB-47 in Nic of PARP-2 is an inhibitor that is structurally similar to NAD+’s adenosine group and is also located in the catalytic domain, posing steric hindrance (1, 12).

In essence, PARP-1 responds to damage in DNA to control the process of cell death. Its ligand olaparib serve as an inhibitor for NAD+ binding, and its six-domain structure work together to perform enzymatic activity. Its comparison structure PARP-2 has a ligand with the same function, and the structure that is similar to that of PARP-1 also responds to the DNA damage.