Metalloproteins and metalloproteomics in health and disease.

Metalloproteins represents more than one third of human proteome, with huge variation in physiological functions and pathological implications, depending on the metal/metals involved and tissue context. Their functions range from catalysis, bioenergetics, redox, to DNA repair, cell proliferation, signaling, transport of vital elements, and immunity. The human metalloproteomic studies revealed that many families of metalloproteins along with individual metalloproteins are dysregulated under several clinical conditions. Also, several sorts of interaction between redox- active or redox- inert metalloproteins are observed in health and disease. Metalloproteins profiling shows distinct alterations in neurodegenerative diseases, cancer, inflammation, infection, diabetes mellitus, among other diseases. This makes metalloproteins -either individually or as families- a promising target for several therapeutic approaches. Inhibitors and activators of metalloenzymes, metal chelators, along with artificial metalloproteins could be versatile in diagnosis and treatment of several diseases, in addition to other biomedical and industrial applications.

The mechanistic insights into different aspects of promiscuity in metalloenzymes.

Enzymes are nature's ultimate machinery to catalyze complex reactions. Though enzymes are evolved to catalyze specific reactions, they also show significant promiscuity in reactions and substrate selection. Metalloenzymes contain a metal ion or metal cofactor in their active site, which is crucial in their catalytic activity. Depending on the metal and its coordination environment, the metal ion or cofactor may function as a Lewis acid or base and a redox center and thus can catalyze a plethora of natural reactions. In fact, the versatility in the oxidation state of the metal ions provides metalloenzymes with a high level of catalytic adaptability and promiscuity. In this chapter, we discuss different aspects of promiscuity in metalloenzymes by using several recent experimental and theoretical works as case studies. We start our discussion by introducing the concept of promiscuity and then we delve into the mechanistic insight into promiscuity at the molecular level.

Single-cell transcriptomic analysis to identify endomembrane regulation of metalloproteins and motor proteins in autoimmunity.

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue.

Gut microbial metalloproteins and its role in xenobiotics degradation and ROS scavenging.

The gut microbial metalloenzymes play an important role in maintaining the balance between gut microbial ecosystem, human physiologically processes and immune system. The metals coordinated into active site contribute in various detoxification and defense strategies to avoid unfavourable environment and ensure bacterial survival in human gut. Metallo-ß-lactamase is a potent degrader of antibiotics present in periplasmic space of both commensals and pathogenic bacteria. The resistance to anti-microbial agents developed in this enzyme is one of the global threats for human health. The organophosphorus eliminator, organophosphorus hydrolases have evolved over a course of time to hydrolyze toxic organophosphorus compounds and decrease its effect on human health. Further, the redox stress responders namely superoxide dismutase and catalase are key metalloenzymes in reducing both endogenous and exogenous oxidative stress. They hold a great importance for pathogens as they contribute in pathogenesis in human gut along with reduction of oxidative stress. The in-silico study on these enzymes reveals the importance of point mutation for the evolution of these enzymes in order to enhance their enzyme activity and stability. Various mutation studies were conducted to investigate the catalytic activity of these enzymes. By using the "directed evolution" method, the enzymes involved in detoxification and defense system can be engineered to produce new variants with enhance catalytic features, which may be used to predict the severity due to multi-drug resistance and degradation pattern of organophosphorus compounds in human gut.

Exploring metalloproteins found in the secretion of venomous species: Biological role and therapeutical applications.

Several species during evolution suffered random mutations in response to various environmental factors, which resulted in the formation of venom in phylogenetically distant species. The composition of the venom of most species is poorly known. Snake venom is well characterized while most species have poorly known composition. In contrast, snake venoms are well characterized which proteins and peptides are the main active and most abundant constituents. 42 protein families have been identified, including metalloproteins known as metalloproteinases. These macromolecules are enzymes with zinc in their active site derived from the disintegrin A and metalloproteinase (ADAM) cellular family and are categorized into three classes (PI, PII and PIII) according to their domain organization. The snake venom metalloproteinases (SVMP) are cytotoxic, neurotoxic, myotoxic and/or hematotoxic with a crucial role in the defense and restraint of prey. In this scenario envenoming represents a danger to human health and has been considered a neglected disease worldwide, particularly in tropical and subtropical countries. Nevertheless, recently advances in "omics" technologies have demonstrated interesting biological activities of SVMPs such as antimicrobial, anticancer, against cardiovascular diseases and nervous system disorders. Metalloproteins have the therapeutic potential to be converted into drugs as other components of the venom have undergone this process (e.g., captopril, tirefiban and eptifibatide). So, this chapter is focused on the metalloproteins found in the secretions of venomous species, highlight some aspects such as structure, biological activity, pharmacological therapeutic potential and on.

Metalloproteins structural and functional insights into immunological patterns.

Metalloproteins play a crucial role in regulating different aspects of the immune system in humans. They have various functions in immunity, including recognizing and presenting antigens, aiding in the movement and effectiveness of immune cells, and facilitating interactions between the host and pathogens. Understanding how these proteins work can help us develop new methods to control the immune response in different diseases. Metalloproteins contain metal ions in their structure, which allows them to perform these diverse functions. They encompass a wide range of enzymes, signaling molecules, and structural proteins that utilize metal ions as cofactors for their activities. Examples of metalloproteins include superoxide dismutase, catalase, and metalloproteases, which regulate oxidative stress, inflammation, and tissue remodelling processes associated with immune activation. By studying their functions and the effects of their dysfunction, researchers can develop strategies to improve immune function and combat various diseases. This review explores the diverse functions of metalloproteins in immune processes, highlighting their significance in both health and disease.

C-H functionalization reactions catalyzed by artificial metalloenzymes.

CH functionalization, a promising frontier in modern organic chemistry, facilitates the direct conversion of inert CH bonds into many valuable functional groups. Despite its merits, traditional homogeneous catalysis, often faces challenges in efficiency, selectivity, and sustainability towards this transformation. In this context, artificial metalloenzymes (ArMs), resulting from the incorporation of a catalytically-competent metal cofactor within an evolvable protein scaffold, bridges the gap between the efficiency of enzymatic transformations and the versatility of transition metal catalysis. Accordingly, ArMs have emerged as attractive tools for various challenging catalytic transformations. Additionally, the coming of age of directed evolution has unlocked unprecedented avenues for optimizing enzymatic catalysis. Taking advantage of their genetically-encoded protein scaffold, ArMs have been evolved to catalyze various CH functionalization reactions. This review delves into the recent developments of ArM-catalyzed CH functionalization reactions, highlighting the benefits of engineering the second coordination sphere around a metal cofactor within a host protein.

Perspective: multi-configurational methods in bio-inorganic chemistry.

Transition metal ions play crucial roles in the structure and function of numerous proteins, contributing to essential biological processes such as catalysis, electron transfer, and oxygen binding. However, accurately modeling the electronic structure and properties of metalloproteins poses significant challenges due to the complex nature of their electronic configurations and strong correlation effects. Multiconfigurational quantum chemistry methods are, in principle, the most appropriate tools for addressing these challenges, offering the capability to capture the inherent multi-reference character and strong electron correlation present in bio-inorganic systems. Yet their computational cost has long hindered wider adoption, making methods such as density functional theory (DFT) the method of choice. However, advancements over the past decade have substantially alleviated this limitation, rendering multiconfigurational quantum chemistry methods more accessible and applicable to a wider range of bio-inorganic systems. In this perspective, we discuss some of these developments and how they have already been used to answer some of the most important questions in bio-inorganic chemistry. We also comment on ongoing developments in the field and how the future of the field may evolve.

Rationalization of a Streptavidin Based Enantioselective Artificial Suzukiase: An Integrative Computational Approach.

An Artificial Metalloenzyme (ArM) built employing the streptavidin-biotin technology has been used for the enantioselective synthesis of binaphthyls by means of asymmetric Suzuki-Miyaura cross-coupling reactions. Despite its success, it remains a challenge to understand how the length of the biotin cofactors or the introduction of mutations to streptavidin leads the preferential synthesis of one atropisomer over the other. In this study, we apply an integrated computational modeling approach, including DFT calculations, protein-ligand dockings and molecular dynamics to rationalize the impact of mutations and length of the biotion cofactor on the enantioselectivities of the biaryl product. The results unravel that the enantiomeric differences found experimentally can be rationalized by the disposition of the first intermediate, coming from the oxidative addition step, and the entrance of the second substrate. The work also showcases the difficulties facing to control the enantioselection when engineering ArM to catalyze enantioselective Suzuki-Miyaura couplings and how the combination of DFT calculations, molecular dockings and MD simulations can be used to rationalize artificial metalloenzymes.

AcrIIA28 is a metalloprotein that specifically inhibits targeted-DNA loading to SpyCas9 by binding to the REC3 domain.

CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal viruses have developed countermeasures, employing anti-CRISPR (Acr) proteins to counteract CRISPR-Cas systems. Despite the revolutionary impact of CRISPR-Cas systems on genome editing, concerns persist regarding potential off-target effects. Therefore, understanding the structural and molecular intricacies of diverse Acrs is crucial for elucidating the fundamental mechanisms governing CRISPR-Cas regulation. In this study, we present the structure of AcrIIA28 from Streptococcus phage Javan 128 and analyze its structural and functional features to comprehend the mechanisms involved in its inhibition of Cas9. Our current study reveals that AcrIIA28 is a metalloprotein that contains Zn2+ and abolishes the cleavage activity of Cas9 only from Streptococcus pyrogen (SpyCas9) by directly interacting with the REC3 domain of SpyCas9. Furthermore, we demonstrate that the AcrIIA28 interaction prevents the target DNA from being loaded onto Cas9. These findings indicate the molecular mechanisms underlying AcrIIA28-mediated Cas9 inhibition and provide valuable insights into the ongoing evolutionary battle between bacteria and phages.

Computational modelling of supramolecular metallopeptide assemblies.

Among the important questions in supramolecular peptide self-assemblies are their interactions with metallic compounds and ions. In the last decade, intensive efforts have been devoted to understanding the structural properties of these interactions including their dynamical and catalytic impact in natural and de novo systems. Since structural insights from experimental approaches could be particularly challenging, computational chemistry methods are interesting complementary tools. Here, we present the general multiscale strategies we developed and applied for the study of metallopeptide assemblies. These strategies include prediction of metal binding site, docking of metallic moieties, classical and accelerated molecular dynamics and finally QM/MM calculations. The systems of choice for this chapter are, on one side, peptides involved in neurodegenerative diseases and, on the other, de novo fibrillar systems with catalytic properties. Both successes and remaining challenges are highlighted so that the protocol could be apply to other system of this kind.

Machine Learning-Enhanced Quantum Chemistry-Assisted Refinement of the Active Site Structure of Metalloproteins.

Understanding the fine structural details of inhibitor binding at the active site of metalloenzymes can have a profound impact on the rational drug design targeted to this broad class of biomolecules. Structural techniques such as NMR, cryo-EM, and X-ray crystallography can provide bond lengths and angles, but the uncertainties in these measurements can be as large as the range of values that have been observed for these quantities in all the published structures. This uncertainty is far too large to allow for reliable calculations at the quantum chemical (QC) levels for developing precise structure-activity relationships or for improving the energetic considerations in protein-inhibitor studies. Therefore, the need arises to rely upon computational methods to refine the active site structures well beyond the resolution obtained with routine application of structural methods. In a recent paper, we have shown that it is possible to refine the active site of cobalt(II)-substituted MMP12, a metalloprotein that is a relevant drug target, by matching to the experimental pseudocontact shifts (PCS) those calculated using multireference ab initio QC methods. The computational cost of this methodology becomes a significant bottleneck when the starting structure is not sufficiently close to the final one, which is often the case with biomolecular structures. To tackle this problem, we have developed an approach based on a neural network (NN) and a support vector regression (SVR) and applied it to the refinement of the active site structure of oxalate-inhibited human carbonic anhydrase 2 (hCAII), another prototypical metalloprotein target. The refined structure gives a remarkably good agreement between the QC-calculated and the experimental PCS. This study not only contributes to the knowledge of CAII but also demonstrates the utility of combining machine learning (ML) algorithms with QC calculations, offering a promising avenue for investigating other drug targets and complex biological systems in general.

Functional metalloenzymes based on myoglobin and neuroglobin that exploit covalent interactions.

Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.

Redox-reversible siderophore-based catalyst anchoring within cross-linked artificial metalloenzyme aggregates enables enantioselectivity switching.

The immobilisation of artificial metalloenzymes (ArMs) holds promise for the implementation of new biocatalytic reactions. We present the synthesis of cross-linked artificial metalloenzyme aggregates (CLArMAs) with excellent recyclability, as an alternative to carrier-based immobilisation strategies. Furthermore, iron-siderophore supramolecular anchoring facilitates redox-triggered cofactor release, enabling CLArMAs to be recharged with alternative cofactors for diverse selectivity.

Shared functions of Fe-S cluster assembly and Moco biosynthesis.

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein.

Lanmodulin's EF 2-3 Domain: Insights from Infrared Spectroscopy and Simulations.

Lanmodulins are small, ∼110-residue proteins with four EF-hand motifs that demonstrate a picomolar affinity for lanthanide ions, making them efficient in the recovery and separation of these technologically important metals. In this study, we examine the thermodynamic and structural complexities of lanthanide ion binding to a 41-residue domain, EF 2-3, that constitutes the two highest-affinity metal-binding sites in the lanmodulin protein from Methylorubrum extorquens. Using a combination of circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we characterize the metal binding capabilities of EF 2-3. ITC demonstrates that binding occurs between peptide and lanthanides with conditional dissociation constants (Kd) in the range 20-30 µM, with no significant differences in the Kd values for La3+, Eu3+, and Tb3+ at pH 7.4. In addition, CD spectroscopy suggests that only one binding site of EF 2-3 undergoes a significant conformational change in the presence of lanthanides. 2D IR spectroscopy demonstrates the presence of both mono- and bidentate binding configurations in EF 2-3 with all three lanthanides. MD simulations, supported by Eu3+ luminescence measurements, explore these results, suggesting a competition between water-lanthanide and carboxylate-lanthanide interactions in the EF 2-3 domain. These results underscore the role of the core helical bundle of the protein architecture in influencing binding affinities and communication between the metal-binding sites in the full-length protein.

Consensus guidelines for the diagnosis and management of isolated sulfite oxidase deficiency and molybdenum cofactor deficiencies.

Sulfite intoxication is the hallmark of four ultrarare disorders that are caused by impaired sulfite oxidase activity due to genetic defects in the synthesis of the molybdenum cofactor or of the apoenzyme sulfite oxidase. Delays on the diagnosis of these disorders are common and have been caused by their unspecific presentation of acute neonatal encephalopathy with high early mortality, followed by the evolution of dystonic cerebral palsy and also by the lack of easily available and reliable diagnostic tests. There is significant variation in survival and in the quality of symptomatic management of affected children. One of the four disorders, molybdenum cofactor deficiency type A (MoCD-A) has recently become amenable to causal treatment with synthetic cPMP (fosdenopterin). The evidence base for the rational use of cPMP is very limited. This prompted the formulation of these clinical guidelines to facilitate diagnosis and support the management of patients. The guidelines were developed by experts in diagnosis and treatment of sulfite intoxication disorders. It reflects expert consensus opinion and evidence from a systematic literature search.

A database overview of metal-coordination distances in metalloproteins.

Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.

Integration of metalloproteome and immunoproteome reveals a tight link of iron-related proteins with COVID-19 pathogenesis and immunity.

Increasing clinical data show that the imbalance of host metallome is closely associated with different kinds of disease, however, the intrinsic mechanisms of action of metals in immunity and pathogenesis of disease remain largely undefined. There is lack of multiplexed profiling system to integrate the metalloproteome-immunoproteome information at systemic level for exploring the roles of metals in immunity and disease pathogenesis. In this study, we build up a metal-coding assisted multiplexed proteome assay platform for serum metalloproteomic and immunoproteomic profiling. By taking COVID-19 as a showcase, we unbiasedly uncovered the most evident modulation of iron-related proteins, i.e., Ft and Tf, in serum of severe COVID-19 patients, and the value of Ft/Tf could work as a robust biomarker for COVID-19 severity stratification, which overtakes the well-established clinical risk factors (cytokines). We further uncovered a tight association of transferrin with inflammation mediator IL-10 in COVID-19 patients, which was proved to be mainly governed by the monocyte/macrophage of liver, shedding light on new pathophysiological and immune regulatory mechanisms of COVID-19 disease. We finally validated the beneficial effects of iron chelators as anti-viral agents in SARS-CoV-2-infected K18-hACE2 mice through modulation of iron dyshomeostasis and alleviating inflammation response. Our findings highlight the critical role of liver-mediated iron dysregulation in COVID-19 disease severity, providing solid evidence on the involvement of iron-related proteins in COVID-19 pathophysiology and immunity.

Flexibility of Binding Site is Essential to the Ca2+ Selectivity in EF-Hand Calcium-Binding Proteins.

High binding affinity and selectivity of metal ions are essential to the function of metalloproteins. Thus, understanding the factors that determine these binding characteristics is of major interest for both fundamental mechanistic investigations and guiding of the design of novel metalloproteins. In this work, we perform QM cluster model calculations and quantum mechanics/molecular mechanics (QM/MM) free energy simulations to understand the binding selectivity of Ca2+ and Mg2+ in the wild-type carp parvalbumin and its mutant. While a nonpolarizable MM model (CHARMM36) does not lead to the correct experimental trend, treatment of the metal binding site with the DFTB3 model in a QM/MM framework leads to relative binding free energies (ΔΔGbind) comparable with experimental data. For the wild-type (WT) protein, the calculated ΔΔGbind is ∼6.6 kcal/mol in comparison with the experimental value of 5.6 kcal/mol. The good agreement highlights the value of a QM description of the metal binding site and supports the role of electronic polarization and charge transfer to metal binding selectivity. For the D51A/E101D/F102W mutant, different binding site models lead to considerable variations in computed binding affinities. With a coordination number of seven for Ca2+, which is shown by QM/MM metadynamics simulations to be the dominant coordination number for the mutant, the calculated relative binding affinity is ∼4.8 kcal/mol, in fair agreement with the experimental value of 1.6 kcal/mol. The WT protein is observed to feature a flexible binding site that accommodates a range of coordination numbers for Ca2+, which is essential to the high binding selectivity for Ca2+ over Mg2+. In the mutant, the E101D mutation reduces the flexibility of the binding site and limits the dominant coordination number of Ca2+ to be seven, thereby leading to reduced binding selectivity against Mg2+. Our results highlight that the binding selectivity of metal ions depends on both the structural and dynamical properties of the protein binding site.