An Extremely Stable Interprotein Tetrahedral Hg(Cys) ₄ Core Forms in the Zinc Hook Domain of Rad50 Protein at Physiological pH

In nature, thiolate-based systems are the primary targets of divalent mercury (HgII ) toxicity. The formation of Hg(Cys)x cores in catalytic and structural protein centers mediates mercury's toxic effects and ultimately leads to cellular damage. Multiple studies have revealed distinct HgII -thiolate coordination preferences, among which linear HgII complexes are the most commonly observed in solution at physiological pH. Trigonal or tetrahedral geometries are formed at basic pH or in tight intraprotein Cys-rich metal sites. So far, no interprotein tetrahedral HgII complex formed at neutral pH has been reported. Rad50 protein is a part of the multiprotein MRN complex, a major player in DNA damage-repair processes. Its central region consists of a conserved CXXC motif that enables dimerization of two Rad50 molecules by coordinating ZnII . Dimerized motifs form a unique interprotein zinc hook domain (Hk) that is critical for the biological activity of the MRN. Using a series of length-differentiated peptide models of the Pyrococcus furiosus zinc hook domain, we investigated its interaction with HgII . Using UV-Vis, CD, PAC, and 199 Hg NMR spectroscopies as well as anisotropy decay, we discovered that all Rad50 fragments preferentially form homodimeric Hg(Hk)2 species with a distorted tetrahedral HgS4 coordination environment at physiological pH; this is the first example of an interprotein mercury site displaying tetrahedral geometry in solution. At higher HgII content, monomeric HgHk complexes with linear geometry are formed. The Hg(Cys)4 core of Rad50 is extremely stable and does not compete with cyanides, NAC, or DTT. Applying ITC, we found that the stability constant of the Rad50 Hg(Hk)2 complex is approximately three orders of magnitude higher than those of the strongest HgII complexes known to date

Deciphering the Enigmatic Function of Pseudomonas Metallothioneins

Metallothioneins (MTs) are low molecular weight, Cys-rich proteins that sequester both essential and non-essential metal ions. Despite being highly conserved in the Pseudomonas genus of Gram-negative bacteria, knowledge of their physiological function in this species is scarce. Using the strain P. fluorescens Q2-87 as a model organism, we investigated the role of a conserved MT in zinc homeostasis, cadmium detoxification as well as its implications in stress response. We show that MT expression is only induced in the stationary phase and provides a fitness benefit for long-term starvation survival, while it is not required for metal resistance and acquisition, oxidative or nitrosative stress response, biofilm formation or motility

Impact of naturally occurring serine/cysteine variations on the structure and function of Pseudomonas metallothioneins

Metallothioneins (MTs), small cysteine-rich metal-binding proteins, support the viability of organisms under normal physiological conditions and help them to respond to different environmental stressors. Upon metal coordination (e.g. ZnII, CdII, CuI) they form characteristic polynuclear metal–thiolate clusters that are known for their high thermodynamic stability and kinetic lability. However, despite numerous studies, it is still not understood how MTs modulate their metal-binding properties. Pseudomonas MTs are an emerging subclass of bacterial MTs, distinct for their high number of His residues and for several unique features such as an intrinsically disordered long C-terminal tail and multiple variations in the number and nature of coordinating amino acids. These variations might provide the bacteria with a functional advantage derived from evolutionary adaptation to heterogeneous environments. Nearly 90% of the known Pseudomonas MT sequences feature a central YC[C with combining low line]xxC motif, that is altered to YC[S with combining low line]xxC in the rest. We demonstrate that the additional Cys residue serves as a coordinating ligand without influencing the metal-binding capacity, the overall metal-binding stability or the structure. However, the additional ligand changes intra-cluster dynamics and, as a consequence, modulates metal transfer reactions that could be functionally advantageous in vivo

A histidine-rich Pseudomonas metallothionein with a disordered tail displays higher binding capacity for cadmium than zinc

Metallothioneins (MTs) are crucial players in metal-related physiological processes. They are characterized by a high cysteine content and unique metal binding properties resulting in specific metal–thiolate clusters formation. Here we present the first NMR solution structure of a Pseudomonas MT, PflQ2 MT, using the strain P. fluorescens Q2-87. It consists of a metal binding domain and an intrinsically disordered C-terminal tail, that was not observed in other MTs so far. While not influencing the structure or function of the metal binding domain, the tail contains a potential binding motif that might be important in so far undiscovered biological interactions. Unusual is the different metal binding capacity for three ZnIIversus four CdII ions that results in two novel metal-cluster topologies. Nevertheless, the affinity for the fourth CdII ion is reduced due to transient coordination. PflQ2 MT contains an unusually large number of four histidine residues, of which only one is involved in metal ion binding. The three non-coordinating histidine residues influence neither the protein fold nor the stability in vitro. We demonstrate that reinstatement of a second coordinating histidine residue, observed for cyanobacterial MTs, in place of a non-coordinating residue in Pseudomonas MTs, decreases the kinetic lability of the cluster, while preserving the overall metal ion binding stability and the protein fold. Since high thermodynamic stability combined with high kinetic lability of metal binding are mechanistic features critical for the function of MTs, the observed replacement might be advantageous for Pseudomonas MTs with respect to metal ion handling in vivo

Impact of naturally occurring serine/cysteine variations on the structure and function of Pseudomonas metallothioneins

Metallothioneins (MTs), small cysteine-rich metal-binding proteins, support the viability of organisms under normal physiological conditions and help them to respond to different environmental stressors. Upon metal coordination (e.g. ZnII, CdII, CuI) they form characteristic polynuclear metal–thiolate clusters that are known for their high thermodynamic stability and kinetic lability. However, despite numerous studies, it is still not understood how MTs modulate their metal-binding properties. Pseudomonas MTs are an emerging subclass of bacterial MTs, distinct for their high number of His residues and for several unique features such as an intrinsically disordered long C-terminal tail and multiple variations in the number and nature of coordinating amino acids. These variations might provide the bacteria with a functional advantage derived from evolutionary adaptation to heterogeneous environments. Nearly 90% of the known Pseudomonas MT sequences feature a central YC[C with combining low line]xxC motif, that is altered to YC[S with combining low line]xxC in the rest. We demonstrate that the additional Cys residue serves as a coordinating ligand without influencing the metal-binding capacity, the overall metal-binding stability or the structure. However, the additional ligand changes intra-cluster dynamics and, as a consequence, modulates metal transfer reactions that could be functionally advantageous in vivo.ISSN:1756-5901ISSN:1756-591

Conformational triggers associated with influenza matrix protein 1 polymerization

A central role for the influenza matrix protein 1 (M1) is to form a polymeric coat on the inner leaflet of the host membrane that ultimately provides shape and stability to the virion. M1 polymerizes upon binding membranes, but triggers for conversion of M1 from a water-soluble component of the nucleus and cytosol into an oligomer at the membrane surface are unknown. While full-length M1 is required for virus viability, the N-terminal domain (M1NT) retains membrane binding and pH-dependent oligomerization. We studied the structural plasticity and oligomerization of M1NT in solution using NMR spectroscopy. We show that the isolated domain can be induced by sterol-containing compounds to undergo a conformational change and self-associate in a pH-dependent manner consistent with the stacked dimer oligomeric interface. Surface-exposed residues at one of the stacked dimer interfaces are most sensitive to sterols. Several perturbed residues are at the interface between the N-terminal subdomains and are also perturbed by changes in pH. The effects of sterols appear to be indirect and most likely mediated by reduction in water activity. The local changes are centered on strictly conserved residues and consistent with a priming of the N-terminal domain for polymerization. We hypothesize that M1NT is sensitive to changes in the aqueous environment and that this sensitivity is part of a mechanism for restricting polymerization to the membrane surface. Structural models combined with information from chemical shift perturbations indicate mechanisms by which conformational changes can be transmitted from one polymerization interface to the other