Crystal Structure and Solution NMR Studies of Lys48-linked Tetraubiquitin at Neutral pH

Abstract

Ubiquitin modification of proteins is used as a signal in many cellular processes. Lysine side-chains can be modified by a single ubiquitin or by a polyubiquitin chain, which is defined by an isopeptide bond between the C terminus of one ubiquitin and a specific lysine in a neighboring ubiquitin. Polyubiquitin conformations that result from different lysine linkages presumably differentiate their roles and ability to bind specific targets and enzymes. However, conflicting results have been obtained regarding the precise conformation of Lys48-linked tetraubiquitin. We report the crystal structure of Lys48-linked tetraubiquitin at near-neutral pH. The two tetraubiquitin complexes in the asymmetric unit show the complete connectivity of the chain and the molecular details of the interactions. This tetraubiquitin conformation is consistent with our NMR data as well as with previous studies of diubiquitin and tetraubiquitin in solution at neutral pH. The structure provides a basis for understanding Lys48-linked polyubiquitin recognition under physiological conditions.

Introduction

The covalent attachment of ubiquitin to substrate proteins helps to regulate numerous cellular pathways.1., 2., 3. Specific lysine residues on the substrate can be modified with a single ubiquitin (monoubiquitination) or with a polyubiquitin chain. Ubiquitin conjugation involves a series of enzymatic steps that end with the formation of an isopeptide bond between the C-terminal carboxylate of ubiquitin and the ε-amine of the substrate lysine. The 76 residue ubiquitin protein itself contains seven surface lysine residues that can each be modified by ubiquitin conjugation via Lys–Gly76 isopeptide bonds, leading to the formation of linear polyubiquitin chains.³ There are different types of polyubiquitin chains, each distinguished by the particular Lys residue to which each successive ubiquitin is conjugated. Although all seven surface lysine residues of ubiquitin can be used in the formation of polyubiquitin chains, Lys63 and Lys48-linked chains are more frequently observed and are the best characterized. Lys63-linked polyubiquitin chains play a role in DNA damage tolerance and in the inflammatory response through non-degradative signaling pathways.⁴ Lys48-linked polyubiquitin chains target their substrates to the 26 S proteasome for degradation,⁵ and the minimal chain needed for efficient proteasomal recognition and substrate degradation has been shown to consist of four ubiquitin molecules.⁶ The different types of polyubiquitin chains must have distinct structural characteristics that target their substrates to specific cellular pathways.

A variety of protein domains called ubiquitin-binding domains recognize and bind ubiquitin, including UIMs (ubiquitin-interacting motifs), UBAs (ubiquitin-associated domains), and others.⁷ Some of these ubiquitin-binding domains bind monoubiquitin, while others recognize polyubiquitin chains.⁸ Ubiquitin-binding domains that recognize monoubiquitin all bind to a hydrophobic patch in ubiquitin, burying it at the complex interface.9., 10. This hydrophobic patch is centered around three residues on the ubiquitin surface: Leu8, Ile44, and Val70.⁷ In the case of polyubiquitin binding, however, the way in which ubiquitin-binding domains distinguish among different chain linkages is not understood. In particular, there are UBA domains that are able to distinguish among the different types of polyubiquitin chains and bind preferentially to chains with a specific linkage.⁸ The specific topology of the polyubiquitin chain, or the surfaces of the polyubiquitin chain that are exposed upon formation of a particular lysine linkage, are possible structural elements that could be recognized.9., 10.

There are three crystal structures of Lys48-linked polyubiquitin that have been reported. The first crystal structure reported was of diubiquitin (1AAR),¹¹ which adopts a conformation that buries the ubiquitins' hydrophobic patches at the interface. This arrangement, referred to as the closed conformation, obstructs the binding surface of ubiquitin that interacts with ubiquitin-binding domains.⁷ In the other conformations of polyubiquitin, referred to as open conformations, the hydrophobic patches of the ubiquitins are exposed and available for binding. The other two previously reported Lys48-linked polyubiquitin crystal structures are of tetraubiquitin (1TBE,¹² 1F9J¹³), and differ in overall organization and topology. In one case, the chain is in an open conformation, with the hydrophobic patch of each ubiquitin exposed instead of buried at an interface,¹² while the other tetraubiquitin structure has a configuration that buries the hydrophobic patch of each ubiquitin.¹³ In addition, these tetraubiquitin structures differ from the diubiquitin crystal structure in the way in which the proximal ubiquitin (the first ubiquitin in the polyubiquitin chain that is conjugated to a substrate lysine residue, and which we denote as 1) and the next ubiquitin (which we denote as 2) are oriented. Different conformations are observed in each structure for the covalent linkages, which consist of the C-terminal ubiquitin residue, Gly76 (on 2), and the covalently linked Lys48 side-chain (on 1). A similar difference in the orientation and the linkage is observed for the other two ubiquitins, 3 and 4. Another complication affecting the interpretation of the previous tetraubiquitin crystal structures is that both were determined from crystals containing only half the tetraubiquitin in the asymmetric unit, making the linkage between ubiquitins 2 and 3 ambiguous. In one case (1F9J), this led to two possible conformations for tetraubiquitin.¹³ Distinguishing among the different possible conformations is important to understanding how Lys48-linked polyubiquitin chains are recognized in the cell.

NMR studies of Lys48-linked diubiquitin suggest that its conformation is dynamic and pH sensitive.¹⁴ At neutral pH, diubiquitin adopts a predominantly closed conformation similar to that seen in the diubiquitin crystal structure,14., 15. while at more acidic pH the diubiquitin takes on a more open conformation that exposes the hydrophobic patches.14., 16. A similar dependence on pH was observed for tetraubiquitin.¹⁴ Solution studies of Lys48-linked tetraubiquitin at neutral pH have yielded useful insights into tetraubiquitin topology, but ambiguities regarding pair-wise contacts between ubiquitins remain, and the overall chain topology is still not understood.14., 17. In addition, the tetraubiquitin crystal structures that were crystallized at acidic pH do not account for the solution study data. This leaves the overall topology and inter-ubiquitin contacts for Lys48-linked tetraubiquitin at physiological pH still in question.

We report here a new crystal structure of Lys48-linked tetraubiquitin that differs in several respects from previously reported structures. The crystals were grown at near-neutral pH (6.7) and contain two complete tetraubiquitin chains in the asymmetric unit. The tetraubiquitin is in a conformation that differs from that in previously reported structures and clearly shows all the linkages between the ubiquitins, which were not seen in the previous structures. The overall conformation is a dimer of diubiquitins, consistent with results from solution studies at neutral pH. We present additional, site-directed spin-labeling studies that further support the closed compact structure seen in the crystal. This new structure provides molecular details on the relative orientation, and specific contacts of the ubiquitin monomers in the chain, lending insight into the overall shape and surface of Lys48-linked tetraubiquitin that may be used in its roles within the cell.