Insight into the Coordination and the Binding Sites of Cu²⁺by the Histidyl-6-Tag using Experimental and Computational Tools

Abstract

His-tags are specific sequences containing six to nine subsequent histydyl residues, and they are used for purification of recombinant proteins by use of IMAC chromatography. Such polyhistydyl tags, often used in molecular biology, can be also found in nature. Proteins containing histidine-rich domains play a critical role in many life functions in both prokaryote and eukaryote organisms. Binding mode and the thermodynamic properties of the system depend on the specific metal ion and the histidine sequence. Despite the wide application of the His-tag for purification of proteins, little is known about the properties of metal-binding to such tag domains. This inspired us to undertake detailed studies on the coordination of Cu(2+) ion to hexa-His-tag. Experiments were performed using the potentiometric, UV-visible, CD, and EPR techniques. In addition, molecular dynamics (MD) simulations and density functional theory (DFT) calculations were applied. The experimental studies have shown that the Cu(2+) ion binds most likely to two imidazoles and one, two, or three amide nitrogens, depending on the pH. The structures and stabilities of the complexes for the Cu(2+)-Ac-(His)6-NH2 system using experimental and computational tools were established. Polymorphic binding states are suggested, with a possibility of the formation of α-helix structure induced by metal ion coordination. Metal ion is bound to various pairs of imidazole moieties derived from the tag with different efficiencies. The coordination sphere around the metal ion is completed by molecules of water. Finally, the Cu(2+) binding by Ac-(His)6-NH2 is much more efficient compared to other multihistidine protein domains.

The unusual metal ion binding ability of histidyl tags and their mutated derivatives

“…Our previous studies on copper complexes with the His 6 -tag have shown similar results: the presence of polymorphic binding states (with the participation of maximum two His residues in metal ion binding) and the formation of helical structures for some binding modes. 12,13 Since our recent studies on the specificity of the metal ion interactions with His-tags containing 6 or 9 His residues have shown very unusual behavior and incredible efficiency of binding metal ions, 11,12,14 this work extends our studies to new analogues in which the His residues mutated to Ala residues. Therefore the aim of this study is to examine the properties and the structural characterization of these new poly-(His-Ala) analogues of the pHG peptide.…”

Conformational Transitions of the Amyloid‐β Peptide Upon Copper(II) Binding and pH Changes

“…Various approaches have been developed to describe the interactions between metal ions and coordinated residues in classical MD simulations, including nonbonded, semibonded, and bonded models. Cu 2+ bonded models already exist for the CHARMM force field,,, and a semibonded Cu 2+ model that includes the Jahn−Teller effect is available for the OPLS‐AA/L force field ,. More than twenty years ago, Hancock already used bonded models to study systems including copper and nickel ,.…”

Ruthenium coordination preferences in imidazole‐containing systems revealed by electrospray ionization mass spectrometry and molecular modeling: Possible cues for the surprising stability of the Ru (III)/tris (hydroxymethyl)‐aminomethane/imidazole complexes

“…The (1,2) connectivity is the only other type that allows all three metal atoms to be accommodated using identical connectivity patterns for all of them: (1,2/3,4/5,6). Since metal binding is known to induce alpha‐helical conformation within poly‐histidine sequences, we chose the alpha‐helix as the initial conformation of HHHHHH prior to introducing three ruthenium atoms (each coordinated to two tris (hydroxymethyl)‐aminomethane molecules in the O‐ cis /N‐ trans conformation) to the peptide. Both (1,4/2,5/3,6) and (1,2/3,4/5,6) configurations were constructed and energy‐minimized, followed by molecular dynamics (MD) simulations.…”

“…(1,2/3,4/5,6). Since metal binding is known to induce alpha-helical conformation within poly-histidine sequences, 35 Figure 8). The more compact, helix-like conformation of (1,4/2,5/3,6) is also more energetically favorable, with its energy calculated to be 127 kcal/mol lower compared with that of (1,2/3,4/5,6).…”