Conformational Selection of Ubiquitin Quaternary Structures Driven by Zinc Ions

Abstract

Zinc ions bridging two ubiquitin molecules (with His68 at the interface) contribute to select a subset of conformers from the noncovalent dimer ensemble, thus restricting quaternary structure dynamics, which hampers apo-protein crystallization. The type of selected conformer is shown to determine the crystal packing, which varies from orthorhombic to cubic symmetry.

Aggregation Pathways of Native‐Like Ubiquitin Promoted by Single‐Point Mutation, Metal Ion Concentration, and Dielectric Constant of the Medium

“…The E16V mutant crystallized under conditions analogous to those used for wild-type hUb, [15,17] andd isplays as imilarc rystallization behavior.U sing az inca cetate concentrationr anging from 35 to below 100 mm,o nly orthorhombic crystals were obtained (Table S1, see the Supporting Information). At 100 mm salt concentration both cubic and orthorhombic crystals were obtained.…”

“…At 100 mm salt concentration both cubic and orthorhombic crystals were obtained. [17] These metal ions in Zn-hUb crystals are coordinated to Met1 (terminal amino group) and Glu16 of one protein molecule( Figure S2 a, see the Supporting Information) and to Glu51 of a protein molecule from an underlying triangulars ubset (A'B*'C'; Figure S2 b, see the Supporting Information). The orthorhombic crystals, isomorphous with the hUb crystals, contain three protein chains (A, B, and C) in the asymmetric unit (Figure3).…”

“…[10][11][12][13] Indeed, it has been shown that Cu II binding,c oupledw ith ar educed dielectric constant of the medium obtained by adding2 ,2,2-trifluoroethanol( TFE) to water,d rives hUb through well-defined aggregation pathways. [15][16][17] In these sites, the metali on (Hg 2 + ,C d 2 + ,P t 2 + ,o rZ n 2 + )b ridges the residues of two or more hUb molecules forming different macromolecular clusters.I nt he particular case of zinc ions, changes in Zn 2 + concentration alters the solution concentration of synthons, leadingt od ifferent crystal forms (orthorhombic and cubic for Zn 2 + /hUb molar ratios lower and highert han 8, respectively). [15][16][17] In these sites, the metali on (Hg 2 + ,C d 2 + ,P t 2 + ,o rZ n 2 + )b ridges the residues of two or more hUb molecules forming different macromolecular clusters.I nt he particular case of zinc ions, changes in Zn 2 + concentration alters the solution concentration of synthons, leadingt od ifferent crystal forms (orthorhombic and cubic for Zn 2 + /hUb molar ratios lower and highert han 8, respectively).…”

“…[14] Using X-ray crystallography,s everalm etal-binding sites have been identified on the hUb surface. [17] Interestingly,t he relevance of Zn 2 + -hUb interactions is supported by the detection of high quantities of zinc ions in neurons [18,19] and these quantities are even greateri nt he case of pathologies such as Parkinson's or Alzheimer's diseases. [17] Interestingly,t he relevance of Zn 2 + -hUb interactions is supported by the detection of high quantities of zinc ions in neurons [18,19] and these quantities are even greateri nt he case of pathologies such as Parkinson's or Alzheimer's diseases.…”

“…To this purpose, dockingc alculations were used to search for potential hUb prefibrillar noncovalentd imers dissimilar to physiological and crystallographic dimers, [9,17] potentially promoting aggregation. To this purpose, dockingc alculations were used to search for potential hUb prefibrillar noncovalentd imers dissimilar to physiological and crystallographic dimers, [9,17] potentially promoting aggregation.…”