NMR studies of metalloproteins

Metalloproteins represent a large share of the proteomes, with the intrinsic metal ions providing catalytic, regulatory, and structural roles critical to protein functions. Structural characterization of metalloproteins and identification of metal coordination features including numbers and types of ligands and metal-ligand geometry, and mapping the structural and dynamic changes upon metal binding are significant for understanding biological functions of metalloproteins. NMR spectroscopy has long been used as an invaluable tool for structure and dynamic studies of macromolecules. Here we focus on the application of NMR spectroscopy in characterization of metalloproteins, including structural studies and identification of metal coordination spheres by hetero-/homo-nuclear metal NMR spectroscopy. Paramagnetic NMR as well as (13)C directly detected protonless NMR spectroscopy will also be addressed for application to paramagnetic metalloproteins. Moreover, these techniques offer great potential for studies of other non-metal binding macromolecules.postprin

Structure-function analysis and characterization of metalloproteins.

Metalloproteins are proteins that can bind at least one metal ion as a cofactor. They utilize metal ions for a variety of biological purposes, and are essential for all domains of life. Due to the ubiquity of metalloprotein’s involvement across these processes across all domains of life, how proteins coordinate metal ions for different biochemical functions is of great relevance to understanding the implementation of these biological processes. One of the most important aspects of metal binding is its coordination geometry (CG), which often implies functional activities. Most of the current studies are based on the assumption of previously reported CG models founded mainly in a non-biological chemical context. While this general procedure provides us with great measures on the closest CG model a metal site adopts, it also biases and limits the binding ligand selection and coordination results to the canonical CG models examined. Thus, if a CG model exists that has never be reported previously or is not accounted for in a study, instances from the CG would either be misclassified into an expected model and cause a high in-class variation or considered as outliers. To solve this problem, we have developed our analysis, where the less-biased low-variation measure, bond-length, was used determine the binding ligands and the higher-variation measure, angle, was used to cluster the metal shells into canonical or novel CGs with functional associations. This methodology is model-free, and allows us to derive the CG models from the data itself. Thus, we can handle unknown CGs that may cause problems to the classification methods. This new methodology has enabled the discovery of several previously uncharacterized CGs for zinc and other top abundant metalloproteins. By recognizing these novel/aberrant CGs in our clustering analyses, high correlations were achieved between structural and functional descriptions of metal ion coordination

Structure and Dynamics of Metalloproteins in Live Cells

X-ray absorption spectroscopy (XAS) has emerged as one of the premier tools for investigating the structure and dynamic properties of metals in cells and in metal containing biomolecules. Utilizing the high flux and broad energy range of X-rays supplied by synchrotron light sources, one can selectively excite core electronic transitions in each metal. Spectroscopic signals from these electronic transitions can be used to dissect the chemical architecture of metals in cells, in cellular components and in biomolecules at varying degrees of structural resolution. With the development of ever-brighter X-ray sources, X-ray methods have grown into applications that can be utilized to provide both a cellular image of relative distribution of metals throughout the cell as well as a high-resolution picture of the structure of the metal. As these techniques continue to grow in their capabilities and ease of use, so to does the demand for their application by chemists and biochemists interested in studying the structure and dynamics of metals in cells, in cellular organelles and in metalloproteins

EPR of Co(II) as a Structural and Mechanistic Probe of Metalloprotein Active Sites: A Review of Studies on Aminopeptidase

Co(II) can often be substituted for Zn(II) in zinc-dependent metalloenzymes to provide spectroscopically accessible forms of the enzymes. Co(II) is an excellent spectroscopic probe as it is both optically active and EPR active. Further, its fast relaxation properties make it a useful paramagnetic shift reagent in NMR. In EPR, the dependence of the spectra of high-spin Co(II) on E/D and the sensitivity of the resolvability of the 59Co hyperfine structure to strain terms allow structural information to be inferred from the EPR spectra. In addition to its useful spectroscopic properties, Co(II) is often an extremely good functional mimic of Zn(II), and Co(II)-substituted zinc-dependent enzymes often display catalytic activities analogous to the native Zn(II)-containing enzyme forms. It is therefore somewhat surprising that there are few examples of EPR studies of Co(II)-substituted enzymes. The most detailed studies carried out to date are those on the aminopeptidase from Aeromonas proteolytica. Therefore, the methodology of extracting structural information from EPR of Co(II)-containing proteins is described using studies on A. proteolytica aminopeptidase as an example

The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia

Background: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb2+, Al+3, SeO2, Cu2+, AsO4, and Zn2+. With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. Results: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. Conclusions: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils

Metallomics in environmental and health related research: Current status and perspectives

Metals and metalloids play distinct roles in human health, either beneficial or toxic, depending on their concentrations and species. There is an increasing interest in metals uptake, trafficking, function, and exertion in microorganisms to maintain and advance human health. Metallomics, an emerging research area, focuses on elucidation of metals/metalloids location, distribution, speciation, and behavior in living organisms. This paper briefly summarized the recent progress on the methodology development of metallomics including various techniques, i. e. multiple dimensional liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICPMS), gel electrophoresis-laser ablation-inductively coupled plasma mass spectrometry (GE-LA-ICPMS), synchrotron X-ray fluorescent spectroscopy (XFS), and the applications of metallomics in environmental and health care. © 2012 The Author(s).published_or_final_versio

A Less-Biased Analysis of Metalloproteins Reveals Novel Zinc Coordination Geometries

Zinc metalloproteins are involved in many biological processes and play crucial biochemical roles across all domains of life. Local structure around the zinc ion, especially the coordination geometry (CG), is dictated by the protein sequence and is often directly related to the function of the protein. Current methodologies in characterizing zinc metalloproteins\u27 CG consider only previously reported CG models based mainly on nonbiological chemical context. Exceptions to these canonical CG models are either misclassified or discarded as outliers. Thus, we developed a less-biased method that directly handles potential exceptions without pre-assuming any CG model. Our study shows that numerous exceptions could actually be further classified and that new CG models are needed to characterize them. Also, these new CG models are cross-validated by strong correlation between independent structural and functional annotation distance metrics, which is partially lost if these new CGs models are ignored. Furthermore, these new CG models exhibit functional propensities distinct from the canonical CG models

De novo design of multi-domain metalloenzymes

The course of evolution required the recombination of protein domains to perform ever-growing complex functions. The presence of an additional domain in a multi-domain protein expands, alters, or modulates the functionality with respect to the isolated one-domain protein (1). In particular, small molecule binding domains have shown a strong propensity to form multi-domain proteins and regulate enzymatic, transport, and signal-transducing domains (2). This modulation is referred to as allostery (from Greek, other solid body), as the properties of a functional site are affected by a small molecule bound to a distinctive protein site (3). Taking inspiration from Nature, artificial proteins have been engineered combining different domains to develop bioinspired molecular machines, able to respond to external stimuli (4). This Ph.D. project, born from the collaboration of the Artificial Metallo-Enzyme Group and the DeGradoLab, was devoted to the development of a multi-domain protein. This represents the first example of an artificial multi-domain protein, in which allostery was designed completely from scratch (5,6). DF (Due Ferri), a diiron phenol oxidase domain, and PS (Porphyrin-binding Sequence), a zinc porphyrin binding domain, were selected as starting proteins to be combined and give DFP (Due Ferri Porphyrin).7 The multiple junctions were exploited to link the two domains, and obtain a more extensive structural coupling between them. While the two metalloproteins present the same kind of domain, the two four-helix bundles are characterized by different geometrical parameters. Therefore, a structural-based methodology was firstly developed in order to identify the best colocalization and helical junctions to accommodate the changes in interhelical separation and registry between the bundles. The x-ray structure of the first analogue, DFP1, was determined, bound to its metal cofactors. The superposition of the 120 residues comprising binding sites gave an excellent fit to the design model, with an overall backbone RMSD of less than 1.4 Å. However, DFP1 was designed to maximize structural stability with a tight and uniform packing, which hindered the access to organic substrates at the DF domain and, thus, its functional characterization. The channel-lining residues of the dimetal-binding site in DF domain were mutated in Gly residues to create a pocket for a substrate. The introduction of helix-breaking residues, that gave oligomerization promiscuity, required also the mutation of DF loop, leading to the final candidate DFP3. An extensive spectroscopic characterization was performed to investigate the functional properties of the multi-domain proteins. DFP3 was demonstrated to bind the designed zinc porphyrin ZnP (Zn-meso-(trifluoromethyl)porphin) at the PS domain with nanomolar affinity. The strong negative Cotton Effect in the ZnP Soret region confirmed the tight and single-mode binding in the rigid asymmetric protein core. On the other side of the multi-domain metalloprotein, cobalt binding experiments confirmed the preservation of the DF penta-coordinating environment. Indeed, the dizinc form was able to stabilize the semiquinone form of 3,5-ditertbutylcatechol/quinone couple, and DFP3 showed ferroxidase and phenoloxidase activities. Although these reactivities were still present upon ZnP binding, a modulation effect was observed. The catalytic characterization of 4-aminophenol oxidation demonstrated a Michaelis-Menten mechanism in the phenoloxidase activity, and high-lightened a 4-fold tighter Km and a 7-fold decrease in kcat upon binding of ZnP. Molecular Dynamics simulations suggested that the presence of ZnP restrains the conformational freedom of a second-shell Tyr, that have been previously shown to largely affect the reactivity of the diiron center. Subsequently, the binding fitness of the zinc porphyrin was changed to investigate the bidirectionality of the allosteric regulation. In the presence of the different zinc porphyrin ZnDP (ZnDP, Zn-Deuteroporphyrin IX), DFP3 resulted to be more flexible, as demonstrated by thermal and chemical denaturations. Nevertheless, the dizinc center continued to stabilize the seminiquinone, and the ferroxidase and phenol oxidase activities were still modulated by the presence of ZnDP. DFP3 showed an excellent affinity for ZnDP, only one order lower in magnitude compared to the designed ZnP. More importantly, the ZnDP affinity was modulated by the presence of zinc bound to DFP3, showing a 3-fold decrease in KD, and demonstrating the presence of a back-regulation. In final instance, the photosensitizing properties of zinc porphyrin-DFP3 complexes were tested in the oxidation of the biological redox cofactor NADH. The photocatalytic characterization highlighted the paramount role of the protein scaffold not only in increasing the reaction rate, but also in protecting the zinc porphyrins from highly reactive species. The lower binding fitness DFP3 towards ZnDP hindered this protection, enabling a major permeability of these species and leading to the zinc porphyrin photobleaching. Although only a preliminary characterization of photocatalysis has been performed, the high reactivity and versatility of such systems are a promising starting point for the de novo design of artificial photosystems for the storage of light energy in chemical fuels.   References (1) Bashton, M. & Chothia, C. The Generation of New Protein Functions by the Combination of Domains. Structure 15, 85–99 (2007). (2) Anantharaman, V., Koonin, E. V. & Aravind, L. Regulatory potential, phyletic distribution and evolution of ancient, intracellular small-molecule-binding domains11Edited by F. Cohen. J. Mol. Biol. 307, 1271–1292 (2001). (3) Monod, J., Wyman, J. & Changeux, J.-P. On the nature of allosteric transitions: A plausible model. J. Mol. Biol. 12, 88–118 (1965). (4) Ostermeier, M. Engineering allosteric protein switches by domain insertion. Protein Eng. Des. Sel. 18, 359–364 (2005). (5) Researchers design allosteric protein from scratch. Chemical & Engineering News https://cen.acs.org/biological-chemistry/Researchers-design-allosteric-protein-scratch/98/i48. 6. Pirro, F. et al. Allosteric cooperation in a de novo-designed two-domain protein. Proc. Natl. Acad. Sci. 117, 33246–33253 (2020). (7) Lombardi, A., Pirro, F., Maglio, O., Chino, M. & DeGrado, W. F. De Novo Design of Four-Helix Bundle Metalloproteins: One Scaffold, Diverse Reactivities. Acc. Chem. Res. 52, 1148–1159 (2019)