New crystal form of human ubiquitin in the presence of magnesium

Abstract

Ubiquitin is a small globular protein that has a considerable number of lysine residues on its surface. This results in a high surface entropy that precludes the formation of crystal-packing interactions. To date, only a few structures of the native form of ubiquitin have been solved, and most of the crystals that led to these structures were obtained in the presence of different divalent metal cations. In this work, a new crystallographic structure of human ubiquitin solved from crystals grown in the presence of magnesium is presented. The crystals belonged to a triclinic space group, with unit-cell parameters a = 29.96, b = 30.18, c = 41.41 Å, α = 88.52, β = 79.12, γ = 67.37°. The crystal lattice is composed of stacked layers of human ubiquitin molecules with a large hydrophobic interface and a smaller polar interface in which the magnesium ion lies at the junction between adjacent layers in the crystal. The metal ion appears in a hexa-aquo coordination, which is key to facilitating the crystallization of the protein.

Uranyl Binding to Proteins and Structural-Functional Impacts

“…Ubiquitin (Ub) is a small protein (76 amino acids) in different organisms, which plays crucial roles in degradation of misfolded proteins via the ubiquitin proteasome system (UPS), and maintains the protein homeostasis in living cells [50]. Although Ub is not a metalloprotein, crystallographic studies showed that various divalent metal ions potentially bind to the protein, such as Cu 2+ , Zn 2+ , Cd 2+ and Hg 2+ [51], as well as Mg 2+ [52]. Using molecular modeling and dynamics simulations, Lin and co-workers showed that two surface residues of Ub, Glu18 and Asp21, may coordinate to UO 2 2+ , as well as water molecules ( Figure 2d) [53].…”

The Carcinogen Cadmium Activates Lysine 63 (K63)-Linked Ubiquitin-Dependent Signaling and Inhibits Selective Autophagy

“…Of interest, both Cd and Zn are group IIb metals that share a common affinity for sulfhydryl groups and likely the same molecular targets. By displacing Zn, one might assume from the so-called ionic mimicry concept that Cd would inhibit the Zn-dependent enzymes, disturb folding of Zn-domain, and interfere with Zn-regulated physiological processes [28,[56][57][58][59].…”

“…As Cd and Zn are in the same group of the chemical periodic table, it can be inferred from the so-called ionic mimicry concept that Cd may share with Zn the same targets [28,56]. In this regard, Cd is able to replace Zn and binds to ubiquitin [57,58], Zn finger domains, and the SH group within the active sites of ubiquitin ligases and deubiquitinases [59], a property that may help explain the dramatic upregulation of ubiquitination. However, the results of in vitro assays using free tetraubiquitin suggested that the addition of Cd (5 µM) had little, if any, direct effect on the activity of the ubiquitin ligases and deubiquitinases (Figure 3E,F).…”

The FUSION protein crystallization screen

“…The counterions from set 1 of additive mixes such as sodium, potassium and others (Table 1) can alter the physico-chemical properties of proteins in solution (Bostro ¨m et al, 2011;Medda et al, 2012;Salis & Ninham, 2014;McPherson, 2001). Additionally, they can facilitate crystallization through the media-tion of contact sites, especially with divalent metal cations (Camara-Artigas et al, 2016;Hegde et al, 2017). The additives from set 2 (Table 2) are more likely to alter protein-water interactions (Timasheff, 2002) than be part of the crystal structure.…”