Protein Phosphatase-1
Created by: Hannah Dawson
Protein phosphatase-1 with tautomycin (PDB ID: 3E7B) is protein phosphatase-1 bound to an inhibitor toxin tautomycin. The protein phosphatase-1 (PP1) in its normal functioning state is one of seven major enzymes in total that dephosphorylate serine/threonine residues in eukaryotic cells (1). This means PP1 is a serine-threonine phosphatase, which is a hydrolase enzyme with the function of cleaving phosphates from serine and threonine residues by hydrolyzing phosphoric acid monoesters into a phosphate ion and a serine or threonine with a free hydroxyl group (2). PP1 has over 180 known targeting proteins and also binds proteins that localize PP1 to distinct regions of the cell and inhibitor proteins that bind and block the PP1 active site (2). PP1 is found throughout the human body and its function is the regulation of many cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling (2). The ubiquitous distribution of PP1 in all human tissues suggests it plays an important role in bodily function and thus is worth studying. PP1 is a member of the PPP (phosphoprotein phosphatases) family, which includes PP1, PP2A, PP4, PP6, PP2B/calcineurin, PP5, and PP7, which all specifically dephosphorylate serine and threonine residues (3).
The general function of protein phosphatase-1 is to remove phosphates, dephosphorylate, phosphothreonine or phosphoserine resides in proteins. This occurs when two metal ions bind to the metal binding sites of PP1 and activate water, which initiates a nucleophilic attack on the phosphorus atom, thus removing it from the substrate that in this case is a serine or threonine residue (4). Since PP1 is involved in numerous pathways, only one pathway is considered here: PP1 involvement in the regulation of the mitotic spindle checkpoint (Image 1). The kinase Aurora B is a serine/threonine kinase that is activated by MPF (mitosis promoting factor) phosphorylation (5). Once Aurora B is activated it phosphorylates serine and threonine residues on proteins that are involved in maintaining the mitotic spindle checkpoint, such as those that make up the MCC (mitotic checkpoint complex) (5). This MCC keeps the anaphase-promoting complex (APC) inactive until the correct mitotic spindle formation is achieved (5). Once the chromosomes are all attached correctly in the mitotic spindle the Aurora B kinase shuts down and its activity is reversed by protein phosphatase-1 (PP1) (5). PP1 dephosphorylates those proteins Aurora B phosphorylated, including Mad 1 and Mad 2, spindle checkpoint proteins (5). Once the inhibition of the MCC is removed and the APC is activated through PP1 dephosphorylation of cdc20, the mitotic spindle can proceed into anaphase and thus the cell can continue through the cell cycle (5). This is represented in the panel labeled spindle checkpoint silencing in Image 1.
PP1 is a serine/threonine phosphatase that is composed of two identical chains A and B both 299 amino acids in length and the total length of the protein is 598 amino acids (2). The secondary structure of each chain is 34% helical and 18% beta sheet. Of the 11 helices in each chain 10 are alpha helices and 1 is a 3/10 helix (2). Of the 16 beta strands 14 are beta strands and 2 are beta bridges (2). The regions that connect these helices and beta strands and comprise the rest of the protein are either turns, bends, or empty with no secondary structure (2). Each catalytic subunit of PP1, chains A and B, then adopts a compact alpha-beta fold with a beta sandwich wedged between two alpha-helical domains (4). These features of secondary structure can be readily seen in the crystal structure of Protein phosphatase-1 with tautomycin 1.
In the PDB structure (PDB ID: 3E7B), protein phosphatase-1 is bound to tautomycin. Tautomycin is a PP1 toxin inhibitor found in soil bacteria Streptomyces sp. (2). Tautomycin functions as an inhibitor by directly inhibiting the catalytic activity of PP1. This happens when the diacid functionality of tautomycin coordinates with the active site of PP1, blocking its ability to interact with substrates (2). An additional image of tautomycin bound to PP1 is provided in parts c and d of Image 2.
A BLAST search was performed on the entire length of chain A and B of PP1, which are identical. The results show that most of the protein length is conserved, especially the region between amino acids 50-250 (6). This suggests that the entire length of each chain is needed for correct protein function and thus much of the sequence is conserved. Also the high level of conservation in the 50-250 region suggests that this portion of the protein is particularly important to protein function and is thus conserved. This region of the protein may contain the active site or metal binding site, both key to the protein function. Conserved domains within protein phosphatase-1 include those of the MPP (metallophosphatase) superfamily (6). This means that PP1 shares homology in domains commonly conserved in proteins within the MPP superfamily. The MPP superfamily is a group of enzymes with a conserved domain containing an active site consisting of two metal ions coordinated with octahedral geometry by a cage of histidine, aspartate, and asparagine resides (6). This suggests that PP1 has a di-metal active site that requires metal ions for functioning. BLAST results also show that PP1 is homologous with PP2Ac, which is the catalytic subunit of PP2A, a closely related protein (6). This suggests the functioning of the PP1 catalytic subunit is the same or very similar to that of PP2A. Additionally, the BLAST search shows a conserved domain of PP1 is similar to that of calcineurin-like phosphoesterase, which includes protein phosphoserine phosphatases, nucleotidases, sphingomyelin phosphodiesterases and 2'-3' cAMP phosphodiesterases (6). All of these proteins are hydrolases involved in removing phosphate groups from molecules, which confirms PP1 has a similar role (6). Homology is also seen between a conserved PP1 domain and the domain of polynucleotide kinase-phosphatase, an enzyme active in bacterial RNA repair (6). This suggests PP1 may function in pathways involving RNA.
Each polypeptide chain in PP1 comprises a serine/threonine phosphatase PP1-alpha catalytic subunit and there are at least 100 regulatory subunits that can bind to a catalytic subunit and regulate PP1 activity (4). Within each catalytic subunit there is an active site, which is where dephosphorylation of substrates occurs. This active site lies at the bifurcation point of a Y-shaped surface feature formed by three shallow surface grooves that roughly follow the domain boundaries of the beta sandwich and two helical domains (4). As seen in Image 2, these three surface grooves are the hydrophobic groove, acidic groove, and C-terminal groove, which are formed by the C-terminal subdomain sitting on the surface of the N-terminal subdomain (7). Two metal ions, Fe2+ and Mn2+ are located at the active site and needed for protein function (4). Also seen in Image 2 is the ß12/ß13 loop which has an important role in inhibition of PP1 by toxins, specifically the Tyr-272 in the loop is involved in binding to toxins (7).
Mutations to certain amino acid residues in PP1 can reduce or eliminate PP1 function. These include Asn-124 and His-248 that are involved in metal binding, Arg-96 and Arg-221 that are involved in phosphosubstrate binding, and Asp-95 and Asp-208 that are both located in the active site (7). Mutations at these residues resulted in large loss of enzyme activity and thus are likely to play an important role in the functioning of the PP1 protein (7).
The connection between PP1 structure and function can largely be seen in the catalytic mechanism of the protein, which displays how the active site structure and amino acids contribute to the function of PP1 as a serine/threonine phosphatase. Catalysis in PP1 involves nucleophilic attack on a phosphosubstrate by a metal-activated water molecule (7). Asn-124, His-248, Asp-92, His-66, and Asp-64 act in binding the metal ions used for the activation of water (7). His-125 acts as a general acid that protonates the leaving group serine or threonine (7). Asp-95 in the active site has a carboxyl group that hydrogen bonds with a nitrogen in the His-125 and thus confines and stabilizes its protonated state (7). Arg-96, Asn-124 and Arg-221 bind to phosphosubstrate oxygens and help in substrate binding and stabilization of a pentacoordinate transition state and Asp-208 helps maintain the Arg-221 conformation (7). Thus the specific amino acids and their arrangement in the active site are key to the phosphatase function of PP1. Additionally, the acidic groove residues of the PP1 catalytic subunit may influence substrate specificity of the protein (7).