Metal Binding Sites In Metalloproteins Research Articles

Benchmarking polarizable and non-polarizable force fields for Ca2+-peptides against a comprehensive QM dataset.

Explicit description of atomic polarizability is critical for the accurate treatment of inter-molecular interactions by force fields (FFs) in molecular dynamics (MD) simulations aiming to investigate complex electrostatic environments such as metal-binding sites of metalloproteins. Several models exist to describe key monovalent and divalent cations interacting with proteins. Many of these models have been developed from ion-amino-acid interactions and/or aqueous-phase data on cation solvation. The transferability of these models to cation-protein interactions remains uncertain. Herein, we assess the accuracy of existing FFs by their abilities to reproduce hierarchies of thousands of Ca2+-dipeptide interaction energies based on density-functional theory calculations. We find that the Drude polarizable FF, prior to any parameterization, better approximates the QM interaction energies than any of the non-polarizable FFs. Nevertheless, it required improvement in order to address polarization catastrophes where, at short Ca2+-carboxylate distances, the Drude particle of oxygen overlaps with the divalent cation. To ameliorate this, we identified those conformational properties that produced the poorest prediction of interaction energies to reduce the parameter space for optimization. We then optimized the selected cation-peptide parameters using Boltzmann-weighted fitting and evaluated the resulting parameters in MD simulations of the N-lobe of calmodulin. We also parameterized and evaluated the CTPOL FF, which incorporates charge-transfer and polarization effects in additive FFs. This work shows how QM-driven parameter development, followed by testing in condensed-phase simulations, may yield FFs that can accurately capture the structure and dynamics of ion-protein interactions.

A Binuclear CuA Center Designed in an All α-Helical Protein Scaffold.

The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein coregulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here, we present a fully functional CuA center designed in a structurally nonhomologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopy that CuACcP is valence delocalized. Continuous wave and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from extended X-ray absorption fine structure spectroscopy and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu(II) and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties in a metal redox center.

Predicting disease-associated mutation of metal-binding sites in proteins using a deep learning approach

Metalloproteins play important roles in many biological processes. Mutations at the metal-binding sites may functionally disrupt metalloproteins, initiating severe diseases; however, there seemed to be no effective approach to predict such mutations until now. Here we develop a deep learning approach to successfully predict disease-associated mutations that occur at the metal-binding sites of metalloproteins. We generate energy-based affinity grid maps and physiochemical features of the metal-binding pockets (obtained from different databases as spatial and sequential features) and subsequently implement these features into a multichannel convolutional neural network. After training the model, the multichannel convolutional neural network can successfully predict disease-associated mutations that occur at the first and second coordination spheres of zinc-binding sites with an area under the curve of 0.90 and an accuracy of 0.82. Our approach stands for the first deep learning approach for the prediction of disease-associated metal-relevant site mutations in metalloproteins, providing a new platform to tackle human diseases. Metals can bind to proteins to fulfil important biological functions. Predicting the features of mutated binding sites can thus help us understand the connection between specific mutations and their role in diseases.

X-Ray Absorption Spectroscopy of Metalloproteins.

X-ray absorption spectroscopy (XAS) has been widely used as a powerful local structural tool for studying the metal binding sites in metalloproteins, owing to its element specificity, chemical sensitivity, and minimum requirement for sample preparation. The principle subject of this chapter is to provide a general introduction of this technique to molecular biologists and biochemists, with the intention to fill the knowledge gaps in interdisciplinary communication and collaboration. An update of the XAS-based technique applied recently to metalloproteins is also briefly introduced at the end of the chapter.

Aberrant coordination geometries discovered in the most abundant metalloproteins.

ABSTRACTMetalloproteins bind and utilize metal ions for a variety of biological purposes. Due to the ubiquity of metalloprotein involvement throughout 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. Toward these ends, we have improved our methodology for structurally and functionally characterizing metal binding sites in metalloproteins. Our new ligand detection method is statistically much more robust, producing estimated false positive and false negative rates of ∼0.11% and ∼1.2%, respectively. Additional improvements expand both the range of metal ions and their coordination number that can be effectively analyzed. Also, the inclusion of additional quality control filters has significantly improved structure‐function Spearman correlations as demonstrated by rho values greater than 0.90 for several metal coordination analyses and even one rho value above 0.95. Also, improvements in bond‐length distributions have revealed bond‐length modes specific to chemical functional groups involved in multidentation. Using these improved methods, we analyzed all single metal ion binding sites with Zn, Mg, Ca, Fe, and Na ions in the wwPDB, producing statistically rigorous results supporting the existence of both a significant number of unexpected compressed angles and subsequent aberrant metal ion coordination geometries (CGs) within structurally known metalloproteins. By recognizing these aberrant CGs in our clustering analyses, high correlations are achieved between structural and functional descriptions of metal ion coordination. Moreover, distinct biochemical functions are associated with aberrant CGs versus nonaberrant CGs. Proteins 2017; 85:885–907. © 2016 Wiley Periodicals, Inc.

HENRYK — An endless source of metal coordination surprises

The basic knowledge about biological inorganic chemistry, thermodynamics and metal binding sites of metalloproteins is crucial for the understanding of their metal binding–structure–function relationship. Metal–peptide complexes are useful and commonly used models of metal–enzyme active sites, among which copper and zinc models are one of the most extensively studied. HENRYK is a peptide sequence present in numerous proteins, and serves as a potentially tempting binding site for Cu2+ and Zn2+. Maybe more importantly, HENRYK also happens to be the first name of our group leader.The results of this work, which, at the first glance, might seem to be a ‘chemical scrabble’, went far beyond our expectations and surprised us with a novel, uncommon behavior of a Cu2+ complex with a peptide with a histidine in position one. At low pH, the binding is a typical histamine-like coordination, but with the increase of pH, the imidazole nitrogen is moved to the axial position and replaced with an amide; at basic pH, the binding mode is a {NH2, 3N−} one in the equatorial plane. It is important to note, that no dimeric species are formed in between. Such binding is thermodynamically much more stable than a simple complex with histamine, and quite comparable to complexes with several possible imidazole anchoring sites.

Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.

Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca(2+) binding proteins, and metallothioneins.In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, (113)Cd NMR, in conjunction with (13)C and (31)P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment.In the case of metallothionein, we will reflect upon how (113)Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear (1)H-(113)Cd multiple-quantum coherence methods (HMQC) was essential for the initial establishment of the 3D structure of metallothioneins, a protein family deficient in the regular secondary structural elements of α-helix and β-sheet and the first native protein identified with bound Cd. The (113)Cd NMR studies also enabled the characterization of the affinity of the individual sites for (113)Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.

Metalloproteomics, metalloproteomes, and the annotation of metalloproteins

Metalloproteomics includes approaches that address the expression of metalloproteins and their changes in biological time and space. Metalloproteomes are investigated by a combination of approaches. Experimental approaches include structural genomics, which provides insights into the architecture of metal-binding sites in metalloproteins and establishes ligand signatures from the types and spacings of the metal ligands in the protein sequence. Theoretical approaches employ these ligand signatures as templates for homology searches in sequence databases. In this way, the number of metalloproteins in the iron, copper, and zinc metalloproteomes in various phyla of life has been estimated. Yet, manganese metalloproteomes remain poorly defined. Metals have catalytic and structural functions in proteins. However, additional functions have evolved. Proteins that control metal homeostasis and proteins that are metal-regulated bind metal ions transiently and are generally not accounted for in estimates from bioinformatics. Thus, metalloproteomes are dynamic and likely to be larger than present estimates suggest. This account discusses the assignment of transition metals in metalloproteins and the ensuing issues facing analytical chemists and structural and computational biologists. Biological and chemical selectivities render metal selection by metalloproteins either more stringent or less stringent depending on the metal homeostatic system of the organism, the subcellular location of the protein, and environmental factors. Failure to recognize the principles of metal utilization has led to assigning the wrong metal in metalloproteins and has missed some of the regulatory functions of transition metal ions.

Metals in proteins: correlation between the metal-ion type, coordination number and the amino-acid residues involved in the coordination

Metal ions are constituents of many metalloproteins, in which they have either catalytic (metalloenzymes) or structural functions. In this work, the characteristics of various metals were studied (Cu, Zn, Mg, Mn, Fe, Co, Ni, Cd and Ca in proteins with known crystal structure) as well as the specificity of their environments. The analysis was performed on two data sets: the set of protein structures in the Protein Data Bank (PDB) determined with resolution <1.5 A and the set of nonredundant protein structures from the PDB. The former was used to determine the distances between each metal ion and its electron donors and the latter was used to assess the preferred coordination numbers and common combinations of amino-acid residues in the neighbourhood of each metal. Although the metal ions considered predominantly had a valence of two, their preferred coordination number and the type of amino-acid residues that participate in the coordination differed significantly from one metal ion to the next. This study concentrates on finding the specificities of a metal-ion environment, namely the distribution of coordination numbers and the amino-acid residue types that frequently take part in coordination. Furthermore, the correlation between the coordination number and the occurrence of certain amino-acid residues (quartets and triplets) in a metal-ion coordination sphere was analysed. The results obtained are of particular value for the identification and modelling of metal-binding sites in protein structures derived by homology modelling. Knowledge of the geometry and characteristics of the metal-binding sites in metalloproteins of known function can help to more closely determine the biological activity of proteins of unknown function and to aid in design of proteins with specific affinity for certain metals.

Dissecting the General Physicochemical Properties of Noncovalent Interactions Involving Tyrosine Side Chain as a Second‐Shell Ligand in Biomolecular Metal‐Binding Site Mimetics: An Experimental Study Combining Fluorescence, 13C NMR Spectroscopy and ESI Mass Spectrometry

Detailed physicochemical features inherent in the dynamic cation-pi interactions of aromatic amino acid side chains in the secondary coordination spheres around metal ions were extracted and mapped by intrinsic tyrosine fluorescence titration experiments with two homologous, artificially engineered metal-binding scaffolds which mimic metal-binding sites in metalloproteins. A newly formulated method for the treatment of fluorescence titration data allows straightforward assessment of both the magnitudes and properties of metal-chelation-assisted cation-aromatic interactions (K2) underlying a proposed two-step metallosupramolecular association process. The unprecedented linear platform-motif correlations between the two contrasting scaffolds in their changes in tyrosine fluorescence on binding of 3d metal cations help to elucidate the properties of general cation-arene recognition corresponding to the metal-responsive characteristics of the second-shell Tyr residue surrounding the metal-binding sites in the supramolecular context, and thereby define a new noncovalent design principle for metal-ion recognition in aqueous solution. As supported by NMR spectroscopic and ESI-MS analyses and molecular mechanics force field calculations, the systematic study exemplifies the concept of using steady-state tyrosine fluorescence as a powerful tool for comprehensive descriptions of cation-pi interactions in the extended environment of a metal-binding site. We established that the physicochemical properties pertaining to indirect metal-arene interactions are highly dependent on the electronic properties of the metal ions. This work suggests that second-shell cation-pi interactions may play more diverse roles, including modulation of structure, reactivity, and function of metal-binding sites, than the previously well-established direct cation-pi interactions involving hard cations (e.g., alkali metal ions). Moreover, such a study will continue to complement theoretical predications and/or the early experimental investigations in organic solvents.

Scaffolded amino acids as a close structural mimic of type-3 copper binding sites

We report the use of triazacyclophane (TAC)-scaffolded amino acids as a structural mimic for 3-histidine metal-binding sites in metalloproteins, especially for the mimicry of type-3 copper binding sites as are present in hemocyanin, tyrosinase and catechol oxidase.

XAFS Spectral Analysis of the Cadmium Coordination Geometry in Cadmium Thiolate Clusters in Metallothionein

We report the combination of measurement and prediction of X-ray absorption fine structure (XAFS) data, where the term XAFS refers to the overall spectrum that encompasses both the X-ray Absorption Near Edge Structure (XANES) region as well as the Extended X-ray Absorption Fine Structure (EXAFS) region, to evaluate the cadmium thiolate cluster structures in the metalloprotein metallothionein. XAFS spectra were simulated using coordinates from molecular models of the protein calculated by molecular mechanics/molecular dynamics (MM3/MD), from NMR analyses, and from analysis of X-ray diffraction data. XAFS spectra were also simulated using the coordinates from X-ray crystallographic data for [Cd(SPh)4]2-, CdS, [Cd2(mu-SPh)2(SPh)4]2-, and [Cd4(mu-SPh)6(SPh)4]2-. The simulated XAFS data that were calculated using the FEFF8 program closely resemble the experimental data reported for [Cd(SPh)4]2-, CdS, [Cd2(mu-SPh)2(SPh)4]2-, [Cd4(mu-SPh)6(SPh)4]2-, rabbit liver metallothionein cadmium alpha-domain (Cd4-alpha MT), and cadmium rabbit liver betaalpha metallothionein (Cd7-betaalpha MT). MM3 force field parameters were modified to include cadmium-sulfur bonding and were initially set to values derived from published X-ray diffraction and EXAFS experimental data. The force field was further calibrated and adjusted through comparison between experimental spectra taken from the literature and simulated XAFS spectra calculated using the FEFF8 program in combination with atomic coordinates from MM3/MD energy minimization. MM3/MD techniques were used with the calibrated force field to predict the high-resolution structure of the metal clusters in rabbit liver Cd7-MT. Structures for Cd3S9 (beta) MT and Cd4S11 (alpha) MT domains from MM3/MD calculations and those previously reported for Cd7-MT on the basis of 1H and 113Cd NMR data were compared. Structural differences between the different models for these cadmium thiolate clusters were evident. Combining the measurement and simulation of XAFS data provides an excellent method of assessing, modeling, and predicting metal-binding sites in metalloproteins when X-ray absorption spectroscopy (XAS) data are available.

De Novo Design of a Redox-Active Minimal Rubredoxin Mimic

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.

Coordination geometries of selected transition metal ions (Co 2+, Ni 2+, Cu 2+, Zn 2+, Cd 2+, and Hg 2+) in metalloproteins

In order to determine preferred coordination geometries of six divalent cations (Co 2+, Ni 2+, Cu 2+, Zn 2+, Cd 2+, and Hg 2+), two sources of experimental data were exploited: Protein Data Bank and Cambridge Structural Database. Metal-binding sites of approximately 100 metalloproteins and 3000 smaller transition metal complexes were analyzed and classified. The correlation between the geometries of small-molecule crystal structures and the metal-binding sites in metalloproteins was investigated. The abundance of amino acid residues participating in coordination metal-protein bonds of metalloproteins was evaluated. From the performed analysis it follows that the octahedral arrangement is preferred by Co 2+ and Ni 2+, tetrahedral by Zn 2+, square planar by Cu 2+, and linear by Hg 2+. Cadmium (II) cation tends to bind in both tetrahedral and octahedral arrangements and single coordination geometry cannot be unambiguously ascribed to it.

Use of near-infrared Yb 3+ vibronic side band spectroscopy to probe protein metal-binding sites

Near infrared Yb 3+ vibronic sideband spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This novel technique, using near-infrared excitation and detection (970–1200 nm) provides a selective well-resolved “infrared-like” vibrational spectrum of those ligands which are chelated to the Yb 3+ ion. Weak vibronic sidebands (VSB) whose energy shifts, with respect to the main 2 F 5 2 → 2 F 7 2 Yb 3+ zero-phonon electronic transition, represent the vibrational frequencies of the Yb 3+ ligands. As a model complex, we have studied the VSB spectrum of Yb 3+ complexed with Eriochrome Cyanine R (Yb:ECR), a ligand which possesses COO −, CO, and CO functional groups. VSB arising from the vibrational modes of these chemical groups are readily observed and well resolved. VSB fluorescence spectra of Yb 3+ reconstituted into the Fe 3+ binding sites of the iron-transporting protein human serum transferrin indicates that these two sites are not spectroscopically identical. The chemical nature of the ligands of the Yb 3+ in this protein, as deduced by the observed VSB frequencies, are entirely in agreement with the known crystal structure. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.

Direct vibrational structure of protein metal-binding sites from near-infrared Yb3+ vibronic side band spectroscopy.

Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This technique provides a selective "IR-like" vibrational spectrum of those ligands chelated to the Yb3+ ion. VSB spectra of various model complexes of Yb3+ representing different ligand types were studied to provide references for the VSB spectra of Yb(3+)-reconstituted metalloproteins. Ca2+ in the calcium-binding protein parvalbumin and Fe3+ in the iron-transporting protein transferrin were replaced with Yb3+. The fluorescence of Yb3+ reconstituted into these two proteins exhibits weak VSBs whose energy shifts, with respect to the main 2F5/2-->2F7/2 Yb3+ electronic transition, represent the vibrational frequencies of the Yb3+ ligands. The chemical nature of the ligands of the Yb3+ in these proteins, as deduced by the observed VSB frequencies, is entirely in agreement with their known crystal structures. For transferrin, replacement of the 12CO3(2-) metal counterion with 13CO3(2-) yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.

113Cd NMR spectroscopy of coordination compounds and proteins

Despite the proven utility of 113Cd NMR chemical shifts for qualitatively or, in some cases, semiquantitatively assessing the nature of ligands at metal binding sites in metalloproteins and for studying enzyme function and mechanisms, it is likely that the full potential of 113Cd spectroscopy as a metallobioprobe has yet to be realized. The development of a more quantitative relationship between structural parameters and 113Cd chemical shift has been hampered in part by the lack of suitable model compounds and chemical dynamics [6]. As described throughout this review, these challenges are currently being met through applications of solid-state and low-temperature solution NMR methods and the design and study of less labile protein model compounds. However, even for cases where dynamics and solvent or anion effects can be ruled out, it appears that ligand atom type and number, bond lengths, coordination geometry, and neighboring atoms (including other metals) all influence 113Cd shifts. Studies of coordination compounds in solution have thus far provided useful information on the influence of donor-atom type and number on isotropic Cd chemical shifts. The influence on shift of coordination geometry and CdL bond lengths will probably only be adequately evaluated using the single crystal approach pioneered by Ellis and co-workers. Studies of the Cd ion bound exclusively by O-donor ligands will likely also be only adequately addressed through solid-state NMR methods due to lability problems with these ligands. In this area, model compounds with O-donor ligands which exhibit the unusual high-field shifts found for ICaBP and HRP are lacking. 113Cd NMR chemical shift information alone is at present inadequate for unambiguous determination of structural features or structural changes at the metal binding sites of metalloproteins. In this regard, HMQC NMR spectroscopy promises to be a particularly useful tool for identifying the ligands which are coordinated to Cd. For cases where ligand exchange or nuclear relaxation rates are high (relative to 1/ J), however, this method will not work and determination of structural features may rely exclusively on chemical shift analyses. It is therefore important that both techniques be developed and utilized.