Ubiquitin chains

Ubiquitin chains (or polyubiquitin chains) are protein post-translational modifications that regulate proteasome dependent protein degradation, the cellular response to DNA damage, the inflammatory response and other cellular functions ^[1][2]. Chains begin as a single ubiquitin attached to the modified protein via an isopeptide linkage between a lysine side chain within the substrate protein and the C-terminal glycine of ubiquitin. The chain is built and extended by ubiquitination of ubiquitin on one of the lysines of ubiquitin or the N-terminus. There are a total of seven lysines in ubiquitin (K6, K11, K27, K29, K33, K48, and K63) and chains using all seven lysines have been identified ^[3]. Chains are frequently referred to in the literature by the lysine position in ubiquitin that connects one ubiquitin to the next. For example, chains built on lysine 48 of ubiquitin are called K48-linked chains. N-terminal to C-terminal connection of ubiquitin molecules is called a linear ubiquitin chain and chains which contain linkages through several lysine positions are called mixed chains ^[4]. Ubiquitin chains with different linkages can have different cellular functions some of which are summarized below. The basis for the functional differences between polyubiquitin chains of different linkages were not apparent until the X-ray and NMR structures of several chains were solved.

K48-linked ubiquitin chains

(2o6v).

K48-linked ubiquitin chains are the primary signal for proteasome dependent degradation of proteins. The attachment of a chain of four or more ubiquitin molecules to a protein is required for efficient degradation. The X-ray crystal structure of K-48 linked diubiquitin^[5] showed inter-ubiquitin interactions between the (residues L8, I44, V70) of the two molecules. A later X-ray crystal structure of K48-linked tetraubiquitin^[6] showed that the ubiquitin chain adopted a globular tertiary structure which has been described as a . The interactions between the hydrophobic patches of ubiquitin molecules in 1 and 2 or 3 and 4 were similar to those observed in the diubiquitin structure ^[5].

Since the initial structure, several K48-linked tetraubiquitin crystal structures ^{[7] [8] [9]} have shown there are slight rearrangements of the tertiary structure of the chain dependent upon the pH of the crystallization solution. At pH 6.7, the chain adopts what is known as the closed conformation, because the chain remains in a largely compact form. At pH values less than 4.5, the interaction between diubiquitin molecules becomes weaker and the chain is less compact and there are fewer inter-ubiquitin contacts ^[8]. The different tertiary conformations of the polyubiquitin chain are thought to be indicative of the dynamics of the K48-linked ubiquitin chain in the cell. These changes would allow ubiquitin binding proteins to interact with the hydrophobic patches of the ubiquitin molecules^[10]. The structure of cyclic K48-linked tetraubiquitin adopts the same dimer of ubiquitin dimer structure seen in the linear chains ^[11]. The authors of this structure suggest this structure demonstrates the inherent flexibility of the ubiquitin chain.

K63-linked ubiquitin chains

chains bound to proteins are associated with the DNA damage response and NF-κB signaling ^[12]. In contrast to K48-linked tetraubiquitin, the structure of K63-linked ubiquitin is linear, with no inter-ubiquitin contacts apparent in the crystal ^[13][14]. Small angle X-ray scattering of K63-linked tetraubiquitin confirmed the observations from crystal structures but suggested that a small percentage of chains adopted a partially compacted structure ^[13]. The specific inter-ubiquitin contacts were not apparent from this experiment. The K63-linked ubiquitin chain binding domains in the signaling proteins NEMO ^[15] and Rap80 ^[16] bridge the hydrophobic patches of consecutive ubiquitins in the chain through a single alpha helix. The work of Sims and coworkers ^[17] showed that decreasing the distance between the ubiquitin interacting motifs in Rap80 decreased the affinity of the Rap80 binding domain for the ubiquitin chain.

K11-linked ubiquitin chains

K11-linked ubiquitin chains are linked to proteasomal degradation and possibly endoplasmic reticulum associated degradation ^[3]. The X-ray crystal structure of K11-linked chains shows a compact structure like K48-linked ubiquitin chains ^[18]. However, the hydrophobic patches which mediate the inter-ubiquitin interactions in K48-linked chains are surface exposed in K11-linked chains. The interaction surface between the two ubiquitins consists of a number of charged and hydrogen bond forming residues. NMR or crystal structures of longer K11-linked chains have not been published to confirm whether this conformation persists with additional ubiquitins.

Linear ubiquitin chains

Linear ubiquitin chains are linked by a peptide bond between the N-terminal alpha-amino group of one ubiquitin and the C-terminus of another ubiquitin. These chains are associated with NF-κB signaling ^[19] and some cellular ubiquitin is expressed as linear chains of ubiquitin before processing to monoubiquitin by ubiquitin proteases ^[20]. Structurally, linear ubiquitin chains are similar to the extended conformation observed for K63-linked chains ^[21].

Ubiquitin chains of other linkages

As of June 2011, there are not structures available for ubiquitin chains with K6, K27, K29, or K33 linkages. Molecular modeling by Fushman and Walker ^[22] suggest K6 and K27 linked chains will adopt a compact structure similar to that of K48-linked chains, while K29 and K33-linked chains will adopt a more open conformation. The model of K11-linked chains in this study suggests inter-ubiquitin contact between the hydrophobic patches ^[22], while the crystal structure suggests these patches are not interacting ^[18]. This discrepancy may be because burial of the hydrophobic patch was a restraint in the modeling. Further structural and biochemical work is necessary to determine the correct conformation.

Chains of Ubiquitin-like proteins (Ubls)

(1z2m).

SUMO (Small Ubiquitin-like Modifying Object) does form chains^[23], however no structural information on these chains is available. Chains of other Ubls may exist, but there is not significant data on their structure and function. The tertiary structure of ISG15 is comprised of two beta grasp folds and is similar in appearance to diubiquitin ^[24]. Whether this influences the function of ISG15 is not clear at this time.