Interpreting the structural mechanism of action for MT7 and human muscarinic acetylcholine receptor 1 complex by modeling protein–protein interaction

MT7 is a selective human muscarinic acetylcholine receptor 1 (hM1) allosteric binder with subnanomolar affinity. Understanding the binding mode of hM1–MT7 will give insights to discover small molecular ligand for hM1. MT7 is a peptide, and hM1 is a G-protein-coupled membrane receptor. Therefore, we have employed homology modeling, protein–protein docking, explicit membrane molecular dynamics (MD) simulations, and molecular mechanic/Poisson-Boltzmann surface area energy decomposition analysis approaches to reveal the hM1–MT7 binding mode. The binding mode is consistent with the experimental data. We have discovered that the binding mode consists of three interaction regions in five residue interaction clusters. By analyzing the cluster representative structures, the cluster residues form an interaction network, which shows a multiple-point-to-site binding mode. Hydrogen binding statistical analysis reveals that E170 (hM1) and R34 (MT7) are both locked in electrostatic cages with counter charges, respectively. This is confirmed by the dynamic distances calculation between these residues, and biological mutant experiments.     

Structural model of ligand-G protein-coupled receptor (GPCR) complex based on experimental double mutant cycle data: MT7 snake toxin bound to dimeric hM1 muscarinic receptor

The snake toxin MT7 is a potent and specific allosteric modulator of the human M1 muscarinic receptor (hM1). We previously characterized by mutagenesis experiments the functional determinants of the MT7-hM1 receptor interaction (Fruchart-Gaillard, C., Mourier, G., Marquer, C., Stura, E., Birdsall, N. J., and Servent, D. (2008) Mol. Pharmacol. 74, 1554–1563) and more recently collected evidence indicating that MT7 may bind to a dimeric form of hM1 (Marquer, C., Fruchart-Gaillard, C., Mourier, G., Grandjean, O., Girard, E., le Maire, M., Brown, S., and Servent, D. (2010) Biol. Cell 102, 409–420). To structurally characterize the MT7-hM1 complex, we adopted a strategy combining double mutant cycle experiments and molecular modeling calculations. First, thirty-three ligand-receptor proximities were identified from the analysis of sixty-one double mutant binding affinities. Several toxin residues that are more than 25 Å apart still contact the same residues on the receptor. As a consequence, attempts to satisfy all the restraints by docking the toxin onto a single receptor failed. The toxin was then positioned onto two receptors during five independent flexible docking simulations. The different possible ligand and receptor extracellular loop conformations were described by performing simulations in explicit solvent. All the docking calculations converged to the same conformation of the MT7-hM1 dimer complex, satisfying the experimental restraints and in which (i) the toxin interacts with the extracellular side of the receptor, (ii) the tips of MT7 loops II and III contact one hM1 protomer, whereas the tip of loop I binds to the other protomer, and (iii) the hM1 dimeric interface involves the transmembrane helices TM6 and TM7. These results structurally support the high affinity and selectivity of the MT7-hM1 interaction and highlight the atypical mode of interaction of this allosteric ligand on its G protein-coupled receptor target.     

Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+

The cadmium-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-alpha MT and Zn4-alpha MT, and calf liver apo-beta MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-alpha MT, and apo-beta MT), and is observed to a much lesser extent in the titration of Zn-alpha MT. Comparison between the band centers of the Cd-alpha MT and Cd-beta MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the alpha domain, with little direct contribution from the beta domain. Analysis of the spectra recorded during titrations of Zn7-MT 2 with Cd2+ suggests: (i) that Cd2+ replaces Zn2+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspecific, "distributed" manner across both domains; (iii) that cluster formation in the alpha domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from "synthetic" Cd4,Zn3-MT following a treatment cycle that allows the redistribution of cadmium into the alpha domain; warming the synthetic "native," Cd4,Zn3-MT, to 65 degrees C results in cadmium being preferentially bound in the alpha domain; and (v) Zn7-MT will bind Cd2+ quite normally at up to 65 degrees C but with greater specificity for the alpha domain compared with titrations carried out at 25 degrees C. These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn-MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.     

Muscarinic toxin 7 selectivity is dictated by extracellular receptor loops

Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe --> Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent Ki = 0.26 nM in a functional assay) compared with the M1 receptor (apparent Ki = 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.     

Cadmium binding and metal cluster formation in metallothionein: a differential modification study

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively [Otvos, J.D., & Armitage, I. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. The formation of these oligonuclear complexes designated as Cd4 and Cd3 clusters has now been monitored in MT reconstituted with varying amounts of Cd by using differential chemical modification of Cys with [14C]iodoacetamide. At ratios below 2-3 mol of Cd/mol of MT bound, no differential protection of Cys by the metal, and hence no preferred binding, is detectable. At Cd-to-protein ratios between 3 and 5 mol of Cd/mol of MT, the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain, indicating formation of the Cd4 cluster. At still higher ratios, formation of the Cd3 cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labeling of the different Cys in Cd7-MT indicates that the ligands of the Cd3 cluster are 3 times as accessible to iodoacetamide than those of the Cd4 cluster, suggesting a greater thermodynamic or kinetic stability of the latter.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Structural determinants for the interactions between muscarinic toxin 7 and muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors (mAChRs) have five subtypes and play crucial roles in various physiological functions and pathophysiological processes. Poor subtype specificity of mAChR modulators has been an obstacle to discover new therapeutic agents. Muscarinic toxin 7 (MT7) is a natural peptide toxin with high selectivity for the M1 receptor. With three to five residues substituted, M3, M4, and M5 receptor mutants could bind to MT7 at nanomolar concentration as the M1 receptor. However, the structural mechanisms explaining MT7–mAChRs binding are still largely unknown. In this study, we constructed 10 complex models of MT7 and each mAChR subtype or its mutant, performed molecular dynamics simulations, and calculated the binding energies to investigate the mechanisms. Our results suggested that the structural determinants for the interactions on mAChRs were composed of some critical residues located separately in the extracellular loops of mAChRs, such as Glu4.56, Leu4.60, Glu/Gln4.63, Tyr4.65, Glu/Asp6.67, and Trp7.35. The subtype specificity of MT7 was attributed to the non‐conserved residues at positions 4.56 and 6.67. These structural mechanisms could facilitate the discovery of novel mAChR modulators with high subtype specificity and enhance the understanding of the interactions between ligands and G‐protein‐coupled receptors. Copyright © 2015 John Wiley & Sons, Ltd.     

M?ssbauer studies on the metal-thiolate cluster formation in Fe(II)-metallothionein

The stepwise 57Fe(II)-thiolate cluster formation in rabbit liver metallothionein-2 (MT) has been followed at pH 8.5 using M?ssbauer spectroscopy. The zero-field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting delta EQ and isomer shift delta as found for reduced rubredoxin (Rdred) or the model compound [Fe(II)(SPh)4]2-, thus indicating an Fe(II)-tetrathiolate coordination. A similar conclusion was reached also in previous electronic absorption studies [M. Good and M. Vasák (1986) Biochemistry 25,8353--8356]. The M?ssbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin-Hamiltonian formalism resulting in M?ssbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2-. The identity of the M?ssbauer parameters of partially and fully metal-occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal-occupied 57Fe(II)4-MT form appear to be magnetically isolated, whereas in the fully metal-saturated 57Fe(II)7-MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existence of three isolated metal binding sites and a metal-thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187, 125-129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704-710], which revealed two metal-thiolate clusters containing three and four metal ions, respectively, and involving all 20 cysteine residues in metal binding, the appearance of M?ssbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7-MT is peculiar and deserves further studies. It is concluded, moreover, that the four-metal cluster is diamagnetic with the four Fe(II) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two-step process.     

Silver binding to rabbit liver metallothionein. Circular dichroism and emission study of silver-thiolate cluster formation with apometallothionein and the alpha and beta fragments

We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo alpha and -beta fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 degrees C and 55 degrees C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the alpha and beta fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The alpha and beta fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3- alpha MT 1 and Ag6- alpha MT 1 is suggested by the spectral data obtained at 20 and 55 degrees C. Formation of Ag3- beta MT 1 is suggested by the spectral data recorded at 20 degrees C for the beta fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. A band at 575 nm reaches a maximum for Ag16-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.     

MT1-MMP hemopexin domain exchange with MT4-MMP blocks enzyme maturation and trafficking to the plasma membrane in MCF7 cells

The hemopexin-like domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) enables MT1-MMP to form oligomers that facilitate the activation of pro-matrix metalloproteinase-2 (pro-MMP-2) at the cell surface. To investigate the role of the MT1-MMP hemopexin domain in the trafficking of MT1-MMP to the cell surface we have examined the activity of two MT1-MT4-MMP chimaeras in which the hemopexin domain of MT1-MMP has been replaced with that of human or mouse MT4-MMP. We show that MT1-MMP bearing the hemopexin domain of MT4-MMP was incapable of activating pro-MMP-2 or degrading gelatin in cell based assays. Furthermore, cell surface biotinylation and indirect immunofluorescence show that transiently expressed MT1-MT4-MMP chimaeras failed to reach the plasma membrane and were retained in the endoplasmic reticulum. Functional activity could be restored by replacing the MT4-MMP hemopexin domain with the wild-type MT1-MMP hemopexin domain. Subsequent analysis with an antibody specifically recognising the propeptide of MT1-MMP revealed that the propeptides of the MT1-MT4-MMP chimaeras failed to undergo proper processing. It has previously been suggested that the hemopexin domain of MT4-MMP could exert a regulatory mechanism that prevents MT4-MMP from activating pro-MMP-2. In this report, we demonstrate unambiguously that MT1-MT4-MMP chimaeras do not undergo normal trafficking and are not correctly processed to their fully active forms and, as a consequence, they are unable to activate pro-MMP-2 at the cell surface.     

Metallothionein can function as a chaperone for zinc uptake transport into prostate and liver mitochondria

In mammalian cells the cytosolic concentration of free Zn(2+) ions is extremely low (nM-fM range) and unlikely to provide an adequate pool for the uptake and accumulation of zinc in mitochondria. We previously identified a mitochondrial uptake transport process that effectively transports zinc directly from low molecular weight zinc ligands independent of and in the absence of available free Zn(2+) ions. Since metallothionein (MT) is an important ligand form of cellular zinc, we determined if Zn(7)-MT was an effective chaperone and donor for delivery and uptake of zinc by prostate and liver mitochondria. The results reveal that both intact mitochondria and mitoplasts effectively accumulated zinc from Zn(7)-MT. The study confirms and extends our previous report that the putative zinc transporter is associated with the inner mitochondrial membrane and involves a direct exchange of zinc from the ligand to the transporter. The ventral prostate cells contain no detectable MT; so that ligands (such as citrate, aspartate) other than MT are zinc donors for mitochondrial zinc accumulation. However, in liver and perhaps other cells, Zn(7)-MT is probably important in the cytosolic trafficking of zinc to the mitochondria for the uptake of zinc into the mitochondrial matrix by the putative zinc uptake transporter.     

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu, Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)alpha(Cu4Zn1)beta-MT, (Cu3Zn2)alpha(Cu4Zn1)beta-MT and (Cu4Zn1)alpha(Cu6)beta-MT. The cooperative formation of (Cu3Zn2)alpha(Cu4Zn1)beta-MT from (Zn4)alpha(Cu4Zn1)beta-MT indicates that the preference of Cu(I) for binding to the beta domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent alpha and beta fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)alpha(Cu4Zn1)beta-MT], while that of alpha MT has rendered a mixture of Cu4Zn1-alpha MT (40%), Cu5Zn1-alpha MT (20%) and Cu7-alpha MT (40%). In the case of beta MT, a mixture of Cu6-beta MT (25%) and Cu7-beta MT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-beta MT from native sources may not be adequate.     

锌_7与镉_7－金属硫蛋白清除羟自由基的比较

分离及纯化兔肝金属硫蛋白制备去金属金属硫蛋白、锌７与镉７金属硫蛋白．在不同ｐＨ条件下,比较后二者清除羟自由基能力;在ｐＨ６条件下,比较锌７－金属硫蛋白与有关蛋白和无机锌盐清除羟自由基效果．结论是在近生理ｐＨ条件下锌７－金属硫蛋白清除羟自由基能力远强于镉７－金属硫蛋白．金属硫蛋白清除羟自由基的能力主要来源于蛋白中处于还原态的流基．     

113Cd NMR studies on metal-thiolate cluster formation in rabbit Cd(II)-metallothionein: evidence for a pH dependence

The formation of two metal-thiolate clusters in rabbit liver metallothionein 2 (MT) has been examined by 113Cd NMR spectroscopy at pH 7.2 and 8.6. The chemical shifts of the 113Cd resonances developing in the course of apoMT titration with 113Cd(II) ions have been compared with those of fully metal occupied 113Cd7-MT. At pH 7.2 and at low metal occupancy (less than 4), a cooperative formation of the four-metal cluster (cluster A) occurs. Further addition of 113Cd(II) ions generates all the resonances of the three-metal cluster (cluster B) in succession, suggesting cooperative metal binding to this cluster also. In contrast, similar studies at pH 8.6, at low metal occupancy (less than 4), reveal a broad NMR signal centered at 688 ppm. This observation indicates that an entirely different protein structure exists. When exactly 4 equiv of 113Cd(II) are bound to apoMT, the 113Cd NMR spectrum changes to the characteristic spectrum of cluster A. Further addition of 113Cd(II) ions again leads to the cooperative formation of cluster B. These results stress the determining role of the cluster A domain on the overall protein fold. The observed pH dependence of the cluster formation in MT can be rationalized by the different degree of deprotonation of the cysteine residues (pKa approximately 8.9), i.e., by the difference in the Gibbs free energy required to bind Cd(II) ions to the thiolate ligands at both pH values.     

Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.     

Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe^II₄-MTα and Co^II₄-MTα species existing in water. Furthermore, the Fe^II₄-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe^II₄-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.     

Binding mode of SARS-CoV-2 fusion peptide to human cellular membrane

Infection of human cells by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) relies on its binding to a specific receptor and subsequent fusion of the viral and host cell membranes. The fusion peptide (FP), a short peptide segment in the spike protein, plays a central role in the initial penetration of the virus into the host cell membrane, followed by the fusion of the two membranes. Here, we use an array of molecular dynamics simulations that take advantage of the highly mobile membrane mimetic model to investigate the interaction of the SARS-CoV2 FP with a lipid bilayer representing mammalian cellular membranes at an atomic level and to characterize the membrane-bound form of the peptide. Six independent systems were generated by changing the initial positioning and orientation of the FP with respect to the membrane, and each system was simulated in five independent replicas, each for 300 ns. In 73% of the simulations, the FP reaches a stable, membrane-bound configuration, in which the peptide deeply penetrated into the membrane. Clustering of the results reveals three major membrane-binding modes (binding modes 1–3), in which binding mode 1 populates over half of the data points. Taking into account the sequence conservation among the viral FPs and the results of mutagenesis studies establishing the role of specific residues in the helical portion of the FP in membrane association, the significant depth of penetration of the whole peptide, and the dense population of the respective cluster, we propose that the most deeply inserted membrane-bound form (binding mode 1) represents more closely the biologically relevant form. Analysis of FP-lipid interactions shows the involvement of specific residues, previously described as the “fusion-active core residues,” in membrane binding. Taken together, the results shed light on a key step involved in SARS-CoV2 infection, with potential implications in designing novel inhibitors.     

Fluorescent muscarinic EGFP-hM1 chimeric receptors: design, ligand binding and functional properties

We describe the construction, expression and characterization of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal part of the muscarinic hM1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-terminal end of the receptor. Also correct protein folding and targeting to plasma membrane is obtained upon addition of a signal peptide promoting amino-terminal domain translocation through the membrane. Ligand binding properties of--and activation of the calcium release response by--the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-labelled receptors are valuable model systems for further functional, biochemical and structural studies.     

The cadmium binding domains in the metallothionein isoform Cd7-MT10 from Mytilus galloprovincialis revealed by NMR spectroscopy

The metal–thiolate connectivity of recombinant Cd₇-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of ¹⁵N T ₁ and T ₂ relaxation times and steady state {¹H}–¹⁵N heteronuclear NOEs. The ¹¹³Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of ¹¹³Cd NMR signals. The complete assignment of cysteine Hα and Hβ proton resonances and the analysis of 2D ¹¹³Cd–¹¹³Cd COSY and ¹H–¹¹³Cd HMQC type spectra allowed us to identify a four metal–thiolate cluster (α-domain) and a three metal–thiolate cluster (β-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the α and β domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the α-domain of mussel Cd₇-MT10 is of the form M₄S₁₂ instead of M₄S₁₁, and has a novel topology. The β-domain has a metal–thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the β-domain displays a more disordered conformation than the α-domain. It is concluded that in mussel Cd₇-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal–thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.