E3ubiquitin_proteinligaseCHIP

E3 ubiquitin-protein ligase carboxy terminus heat shock 70 kDa (HSC70)-Interacting Protein or CHIP (PDB ID: 6EFK) is a ubiquitin ligase found in Homo sapiens that can target chaperone substrates for degradation (1). As a general class, E3 ubiquitin ligase proteins help provide specificity to the ubiquitin proteasome by linking the process of ubiquitination and substrate recognition (2). Ubiquitination is a type of post-translation modification of proteins that is necessary for a variety of different cellular processes, such as cell-cycle progression, protein degradation, DNA repair, signal transduction, and many others. Ubiquitination occurs when a ubiquitin protein is covalently bonded to the terminus of a lysine's ε-amino group. Ubiquitin occasionally can bond to the N terminus of a protein in order for ubiquitination to occur, but this happens less frequently than the C-terminus binding (3). Ubiquitination requires a series of enzymes to act for the entire process to be completed. E1 enzymes activate ubiquitin, E2 enzymes then conjugate the protein to a catalytic cysteine, and E3 enzymes end the process by binding the ubiquitin to the substrate (3). CHIP is one of the E3 ligases that targets misfolded or damaged proteins for degradation by tagging them with ubiquitin and facilitating the movement of the labelled protein to the proteasome (4).

The exact mechanism that CHIP uses to target proteins is not fully understood, but is being heavily researched because of the impact these processes have in diseases, such as Alzheimer's disease, Huntington's disease, and Parkinson's disease. The inadequate degradation of different substrates such as tau or huntingtin are known to be directly correlated to the diseases mentioned above (4). Research has already shown that CHIP-knockout mice show an increase in soluble phosphorylated tau accumulation in the brain. Therefore, an understanding of the mechanism by which CHIP marks substrates could help scientists and healthcare researchers determine novel ways to slow down the progression of diseases associated with defective CHIP coding genes (5).

E3 ubiquitin-protein ligase CHIP from Homo sapiens was expressed in Escherichia coli (E. coli) BL21 (DE3) by implementing bacterial transformation techniques (6). The protein was crystallized via hanging drop method at room temperature by mixing 100 nL of a CHIP TPR solution with 100 µl of the crystallization condition of heat shock protein 70 (Hsp70) at 0.4 M CaCl₂, 0.1 M HEPES (pH 7.5), 25% PEG 4,000 and CHIPOpt at 0.05 M CaCl₂, 0.1 M HEPES (pH 7.0), 28% PEG 4,000, 0.01 M CoCl₂ (6). The calcium ligands coming from CaCl₂ were only used to enhance crystallization and are not generally associated with the protein. X-ray diffraction was used to obtain the structure of CHIP (6). The entire crystal could not be crystallized. It appears that both the amino-terminus and carboxy-terminus are missing as well as the U-box domain and part of the CHIP_TPR_N domain. The domain and residues roughly between 25 and 150 are resolved.

The Expasy database determined the molecular weight of the protein to be 31,774.46 Da, and the isoelectric point is at pH 8.53 (7). CHIP is a heterotetramer consisting of seven α-helices made up of 102 residues or 77% α-helix structure, with the remainder consisting of random coils. The heterotetramer is two pairs of identical subunits (8). CHIP is an E3 protein containing a U-box and a tetratricopeptide repeat (TPR) domain (6). The primary structure of CHIP is 276 residues long with a composition of acidic, basic, hydrophobic, and uncharged residues. The U-box name refers to a section of the primary structure of the protein and specifically, to a 70 amino acid stretch on the carboxyl terminus of the protein, while the TPR domain is a protein-protein interaction module selective for a certain motif of Hsp70. This primary sequence is found in a wide range of E3 proteins and is generally related to recruitment and colocalization of E2 and substrate molecules (9).

While not fully understood, the TPR domain is recognized to play a vital role in the binding of CHIP to Hsp70 and other chaperones and to its tertiary and quaternary structure. The TPR domain contains a hydrophobic pocket caused by an extra turn in helix 6, an extended linker between helix 5 and 6, and hydrophobic collapse. These two bent motifs in CHIP orient critical residues Phe-98, Phe-131, and Ile-135 in such a way to form the hydrophobic pocket which is an area of increased binding affinity for many hydrophobic chaperones (6).

CHIP has another binding domain called the carboxy clamp that is also a part of the TPR region and is considered the main active site for substrate binding. The carboxylate clamp, much like the hydrophobic pocket, is a result of the kinked backbone mentioned above. This clamp is formed by critical residues Lys-30 and Lys-95 and is a place of increased hydrogen bonding in the protein (10). When functioning, this clamp commonly interacts with co-chaperones, proteins that assist in the folding of chaperone protein. Mutagenesis studies at the carboxylate clamp have shown that mutations resulting in incorrect formation of the carboxylate clamp affect the binding affinity of co-chaperones which then leads to improper folding of chaperone proteins (11). If there is an abundance of ineffective chaperone proteins, then this could also affect how much certain proteins are successfully degraded. The tertiary and quaternary structure, as represented by the hydrophobic pocket and carboxylate clamp, of CHIP has an extreme importance on its binding affinity, and even small changes in its structure can have a lasting impact. The hydrophobic pocket is an example of quaternary structure because it is the interaction of two tertiary structure units.

While not a major component of functioning, CHIP can associate with sodium ions (Na+) which allows for the substrate to locate and bind to the carboxylate clamp more easily (12). Sodium ions are not required for the protein to function, but they are frequently associated. CHIP does not have an alternate conformation, but it has been complexed with acetylated pentamer peptides to better understand the molecular determinants of CHIP specificity (13). The complex is not functionally different.

In order to find proteins structurally similar to E3 ubiquitin-protein ligase CHIP, the basic local alignment search tool (BLAST) and the 3D conservation mapping Dali server were used. Positron-Specific Iterative BLAST, or PSI-BLAST, uses an algorithm to compare primary structures of the input sequence to a library of documented sequences to identify other proteins with similar primary structures. Comparison of these sequences results in a given E-value which indicates similarity between proteins and is calculated by determining gaps in total sequence homology (14). The smaller the E-value, the more closely related the primary structure is, with an E-value less than 0.05 indicating a significant match for our purposes. PSI-BLAST results for the primary structure of CHIP gave many results with two comparison proteins being selected: RNA polymerase II-associated protein 3 (PDB ID: 6FD7) with an E-value of 2*10^-8 and Stress-induced-phosphoprotein 1 (PDB ID: 2LNI) with an E-value of 5*10^-10 (14).

The Dali server compares proteins based off of their tertiary structure and calculates the differences in intramolecular distances using the sum-of-pairs method. Similarity of structures using the Dali servers gives a comparison based on Z-score, with any Z-score greater than two indicating a significant match for our purposes. The same two comparison proteins were indicated to be similar in the Dali server results. RNA polymerase II-associated protein 3 had a Z-score of 19.6 and Stress-induced-phosphoprotein 1 had a Z-score of 15.7 (15).

RNA polymerase II-associated protein 3 (RPAP3) and Stress-induced-phosphoprotein 1 were chosen as comparison proteins for CHIP. RPAP3 is a protein involved in the assembly process of many molecular machines by forming an interface between RNA polymerase II and other chaperone proteins (16). This protein is similar to CHIP due to the presence of a TPR domain that allows for binding to chaperone proteins. In the same manner as CHIP, RPAP3 can recruit Hsp90 as a scaffolding protein to stabilize the protein complex around RNA polymerase II. RPAP3 is also made up of seven α-helices, each 100 residues, which is strikingly similar to CHIP's seven 102 residue α-helices. However, these proteins differ in that RPAP3 is only a single chain while CHIP is four (17). The hydrophobic pocket of RPAP3 is made of critical residues Phe-293, Lys-294, Typ-305, and Met-324, indicating this tertiary structure is located in a different portion of the protein than it is in CHIP (17). This is an example of how small changes in protein structure can affect the function of a protein and its job within our body, while also showing that main domains are conserved across differing protein types.

Stress-induced-phosphoprotein 1 is a co-chaperone for heat shcok protein 90 (Hsp90), helping it fold properly so that it can associate with other protein complexes. While CHIP itself is not generally involved in the folding process, it is frequently associating with co-chaperones involved in protein folding. As in CHIP and RPAP3, a TPR domain is responsible for its ability to locate and bind to the appropriate molecules. Stress-induced-phosphoprotein 1, much like RPAP3, is a single chain protein, but it consists of eight α-helices, each of which is 93 residues in length (18). Clearly, these two proteins were going to be structurally similar to CHIP due to the PSI-BLAST and Dali server results, but comparison of the protein function indicates how structure is related to function. Stress-induced-phosphoprotein 1 had values indicating its structure was not as similar to CHIP as the structure of RPAP3 was. An investigation into the function of stress-induced-phosphoprotein 1 showed that the function of this protein exhibits less overlap with the function of CHIP than RPAP3, suggesting once more that structure is related to function.

E3 ubiquitin-protein ligase CHIP serves to ubiquitinate chaperones that are misfolded for degradation. The inability to mark chaperones for degradation can result in chaperones building up in a body system and ultimately causing serious harm. CHIP is the subject of heavy research at the moment because it is a known participant in Alzheimer's disease as well as other neurodegenerative diseases. Researchers are working towards understanding CHIP's interaction with the specific chaperones causing these diseases in the hope of finding a cure or a means to slow disease progression.