Nesfatin-3 possesses divalent metal ion binding properties, which remain hidden in the nucleobindin-2 precursor protein.

BACKGROUND: Nucleobindin-2 (Nucb2) is a multidomain protein that, due to its structure, participates in many physiological processes. It was originally identified in several regions of the hypothalamus. However, more recent studies have redefined and extended the function of Nucb2 far beyond its initially observed role as a negative modulator of food intake. RESULTS: Previously, we described Nucb2 as structurally divided into two parts: the Zn2+-sensitive N-terminal half and the Ca2+-sensitive C-terminal half. Here, we investigated the structural and biochemical properties of its C-terminal half, which, after posttranslational processing, yields the formation of a fully uncharacterized peptide product known as nesfatin-3. Nesfatin-3 likely contains all the key respective structural regions of Nucb2. Hence, we expected that its molecular properties and affinity toward divalent metal ions might resemble those of Nucb2. Surprisingly, the obtained results showed that the molecular properties of nesftain-3 were completely different from those of its precursor protein. Moreover, we designed our work as a comparative analysis of two nesfatin-3 homologs. We noticed that in their apo forms, both proteins had similar shapes and existed in solution as extended molecules. They both interacted with divalent metal ions, and this interaction manifested itself in a compaction of the protein molecules. Despite their similarities, the differences between the homologous nesfatin-3s were even more informative. Each of them favored interaction with a different metal cation and displayed unique binding affinities compared either to each other or to Nucb2. CONCLUSIONS: The observed alterations suggested different from Nucb2 physiological roles of nesfatin-3 and different impacts on the functioning of the tissues and on metabolism and its control. Our results clearly demonstrated that nesfatin-3 possessed divalent metal ion binding properties, which remained hidden in the nucleobindin-2 precursor protein.

Relations between Structure and Zn(II) Binding Affinity Shed Light on the Mechanisms of Rad50 Hook Domain Functioning and Its Phosphorylation.

The metal binding at protein-protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence-structure-stability analysis based on human Rad50 presented here separates the individual structural components that increase the stability of the complex, pointing to amino acid residues far away from the Zn(II) binding site as being largely responsible for the complex stabilization. The influence of the individual components is very well reflected by the previously published crystal structure of the human Rad50 zinc hook (PDB: 5GOX). In addition, we hereby report the effect of phosphorylation of the zinc hook domain, which exerts a destabilizing effect on the domain. This study identifies factors governing the stability of metal-mediated protein-protein interactions and illuminates their molecular basis.

InterMetalDB: A Database and Browser of Intermolecular Metal Binding Sites in Macromolecules with Structural Information.

InterMetalDB is a free-of-charge database and browser of intermolecular metal binding sites that are present on the interfaces of macromolecules forming larger assemblies based on structural information deposited in Protein Data Bank (PDB). It can be found and freely used at https://intermetaldb.biotech.uni.wroc.pl/. InterMetalDB collects the interfacial binding sites with involvement of metal ions and clusters them on the basis of 50% sequence similarity and the nearest metal environment (5 Å radius). The data are available through the web interface where they can be queried, viewed, and downloaded. Complexity of the query depends on the user, because the questions in the query are connected with each other by a logical AND. InterMetalDB offers several useful options for filtering records including searching for structures by particular parameters such as structure resolution, structure description, and date of deposition. Records can be filtered by coordinated metal ion, number of bound amino acid residues, coordination sphere, and other features. InterMetalDB is regularly updated and will continue to be regularly updated with new content in the future. InterMetalDB is a useful tool for all researchers interested in metalloproteins, protein engineering, and metal-driven oligomerization.

Galvanization of Protein-Protein Interactions in a Dynamic Zinc Interactome.

The presence of Zn2+ at protein-protein interfaces modulates complex function, stability, and introduces structural flexibility/complexity, chemical selectivity, and reversibility driven in a Zn2+-dependent manner. Recent studies have demonstrated that dynamically changing Zn2+ affects numerous cellular processes, including protein-protein communication and protein complex assembly. How Zn2+-involved protein-protein interactions (ZPPIs) are formed and dissociate and how their stability and reactivity are driven in a zinc interactome remain poorly understood, mostly due to experimental obstacles. Here, we review recent research advances on the role of Zn2+ in the formation of interprotein sites, their architecture, function, and stability. Moreover, we underline the importance of zinc networks in intersystemic communication and highlight bioinformatic and experimental challenges required for the identification and investigation of ZPPIs.

Structural zinc binding sites shaped for greater works: Structure-function relations in classical zinc finger, hook and clasp domains.

Metal ions are essential elements present in biological systems able to facilitate many cellular processes including proliferation, signaling, DNA synthesis and repair. Zinc ion (Zn(II)) is an important cofactor for numerous biochemical reactions. Commonly, structural zinc sites demonstrate high Zn(II) affinity and compact architecture required for sequence-specific macromolecule binding. However, how Zn(II)-dependent proteins fold, how their dissociation occurs, and which factors modulate zinc protein affinity as well as stability remains not fully understood. The molecular rules governing precise regulation of zinc proteins function are hidden in the relationship between sequence and structure, and hence require deep understanding of their folding mechanism under metal load, reactivity and metal-to-protein affinity. Even though, this sequence-structure relationship has an impact on zinc proteins function, it has been shown that other biological factors including cellular localization and Zn(II) availability influence overall protein behavior. Taking into account all of the mentioned factors, in this review, we aim to describe the relationship between structure-function-stability of zinc structural sites, found in a zinc finger, zinc hook and zinc clasps, and reach far beyond a structural point of view in order to appreciate the balance between chemistry and biology that govern the protein world.