Cytoglobin (Cgb) is a member of hemoprotein family with roles in NO metabolism, fibrosis, and tumourigenesis. Similar to other hemoproteins, Cgb structure and functions are markedly influenced by distal key residues. The sixth ligand His81 (E7) is crucial to exogenous ligand binding, heme pocket conformation, and physiological roles of this protein. However, the effects of other key residues on heme pocket and protein biological functions are not well known. In this work, a molecular dynamics (MD) simulation study of two single mutants in CO-ligated Cgb (L46FCgbCO and L46VCgbCO) and two double mutants (L46FH81QCgbCO and L46VH81QCgbCO) was conducted to explore the effects of the key distal residues Leu46(B10) and His81(E7) on Cgb structure and functions. Results indicated that the distal mutation of B10 and E7 affected CgbCO dynamic properties on loop region fluctuation, internal cavity rearrangement, and heme motion. The distal conformation change was reflected by the distal key residues Gln62 (CD3) and Arg84(E10). The hydrogen bond between heme propionates with CD3 or E10 residues were evidently influenced by B10/E7 mutation. Furthermore, heme pocket rearrangement was also observed based on the distal pocket volume and occurrence rate of inner cavities. The mutual effects of B10 and E7 residues on protein conformational rearrangement and other dynamic features were expressed in current MD studies of CgbCO and its distal mutants, suggesting their crucial role in heme pocket stabilization, ligand binding, and Cgb biological functions.

Prion diseases are a group of infectious and fatal neurodegenerative disorders caused by the conformational conversion of a cellular prion protein (PrP) into its abnormal isoform PrPSc. PrP106–126 resembles PrPSc in terms of physicochemical and biological characteristics and is used as a common model for the treatment of prion diseases. Inhibitory effects on fibril formation and neurotoxicity of the prion neuropeptide PrP106–126 have been investigated using metal complexes as potential inhibitors. Nevertheless, the binding mechanism between metal complexes and the peptide remains unclear. The present study is focused on the interaction of PrP106–126 with NAMI-A and NAMI-A-like ruthenium complexes, including KP418, KP1019, and KP1019-2. Results demonstrated that these ruthenium complexes could bind to PrP106–126 in a distinctive binding mode through electrostatic and hydrophobic interactions. NAMI-A-like ruthenium complexes can also effectively inhibit the aggregation and fibril formation of PrP106–126. The complex KP1019 demonstrated the optimal inhibitory ability upon peptide aggregation, and cytotoxicity because of its large aromatic ligand contribution. The studied complexes could also regulate the copper redox chemistry of PrP106–126 and effectually inhibit the formation of reactive oxygen species. Given these findings, ruthenium complexes with relatively low cellular toxicity may be used to develop potential pharmaceutical products against prion diseases.

Fibril formation of amyloid peptides is linked to a number of pathological states. The prion protein (PrP) and amyloid-β (Aβ) are two remarkable examples that are correlated with prion disorders and Alzheimer's disease, respectively. Metal complexes, such as those formed by platinum and ruthenium compounds, can act as inhibitors against peptide aggregation primarily through metal coordination. This study revealed the inhibitory effect of two peroxovanadium complexes, (NH4)[VO(O2)2(bipy)]·4H2O (1) and (NH4)[VO(O2)2(phen)]·2H2O (2), on amyloid fibril formation of PrP106–126 and Aβ1–42via site-specific oxidation of methionine residues, besides direct binding of the complexes with the peptides. Complexes 1 and 2 showed higher anti-amyloidogenic activity on PrP106–126 aggregation than on Aβ1–42, though their regulation on the cytotoxicity induced by the two peptides could not be differentiated. The action efficacy may be attributed to the different molecular structures of the vanadium complex and the peptide sequence. Results reflected that methionine oxidation may be a crucial action mode in inhibiting amyloid fibril formation. This study offers a possible application value for peroxovanadium complexes against amyloid proteins.

Prion diseases belong to a group of infectious, fatal neurodegenerative disorders. The conformational conversion of a cellular prion protein (PrPC) into an abnormal misfolded isoform (PrPSc) is the key event in prion disease pathology. PrP106–126 resembles PrPSc in some physicochemical and biological characteristics, such as apoptosis induction in neurons, fibrillar formation, and mediation of the conversion of native cellular PrPC to PrPSc. Numerous studies have been conducted to explore the inhibiting methods on the aggregation and neurotoxicity of prion neuropeptide PrP106–126. We showed that PrP106–126 aggregation, as assessed by fluorescence assay and atomic force microscopy, is inhibited by platinum complexes cisplatin, carboplatin, and Pt(bpy)Cl2. ESI-MS and NMR assessments of PrP106–126 and its mutant peptides demonstrate that platinum complexes bind to the peptides in coordination and nonbonded interactions, which rely on the ligand properties and the peptide sequence. In peptides, methionine residue is preferred as a potent binding site over histidine residue for the studied platinum complexes, implying a typical thiophile characteristic of platinum. The neurotoxicity induced by PrP106–126 is better inhibited by Pt(bpy)Cl2 and cisplatin. Furthermore, the ligand configuration contributes to both the binding affinity and the inhibition of peptide aggregation. The pursuit of novel platinum candidates that selectively target prion neuropeptide is noteworthy for medicinal inorganic chemistry and chemical biology.

Metal complexes can effectively inhibit the aggregation of amyloid peptides, such as Aβ, human islet amyloid polypeptide, and prion neuropeptide PrP106–126. Gold (Au) complexes exhibited better inhibition against PrP106–126 aggregation, particularly the Au–bipyridyl (bpy) complex; however, the role of different ligand configurations remains unclear. In the present study, three derivants of Au–bpy complexes, namely, [Au(Me2bpy)Cl2]Cl, [Au(t-Bu2bpy)Cl2]Cl, and [Au(Ph2bpy)Cl2]Cl, were investigated to determine their influence on the aggregation and disaggregation of PrP106–126. The steric and aromatic effects of the ligand resulted in enhanced binding affinity. Inhibition was significantly affected by a large ligand. The neurotoxicity of the SH–SY5Y cells induced by PrP106–126 was reduced by the three Au–bpy derivants. However, the disaggregation ability was not in accordance with the results obtained for selected complexes during inhibition, suggesting a different mechanism of interaction between gold complexes and PrP106–126. The key peptide residues contributed to both the inhibition and disaggregation capabilities through the metal coordination and the hydrophobic interaction with the metal complexes. Thus, understanding the aggregation mechanism of the prion peptide would be helpful in designing novel metal-based drugs against amyloid fibril formation.

The toxicity of amyloid-forming proteins can be linked to many degenerative and systemic diseases. Human islet amyloid polypeptide (hIAPP, amylin) has been associated with type II diabetes. Methods for efficient inhibition of amyloid fibril formation are highly clinically important. This study demonstrated the significant inhibitory effects of six vanadium complexes on hIAPP aggregation. Vanadium complexes, such as bis(maltolato)-oxovanadium (BMOV), have been used as insulin-mimetic agents for the treatment of diabetes for many years. Different biophysical methods were applied to investigate the interaction between V complexes and hIAPP. The results indicated that the selected compounds affected the peptide aggregation by different action modes and protected the cells from the cytotoxicity induced by hIAPP. Both the high binding affinity and the ligand spatial effect on inhibiting hIAPP aggregation are significant. Although some of these compounds undergo biotransformation under the conditions of the experiments, and the active species are not identified, it is understood that the effect results from a particular compound and its conversion products. Importantly, our work provided information on the effects of the selected V complexes on hIAPP and demonstrated multiple levels of effects of V complexes against amyloid-related diseases.

Human islet amyloid polypeptide (hIAPP) can be linked to the pathology of type II diabetes. In this study, aromatic ring-containing Ru complexes were found to effectively inhibit the fibril formation of hIAPP and promote the disaggregation of formed fibrils by remarkably changing the β-sheet components.

•Ru complexes of various molecular configurations were employed for this study.•The Ru complexes could bind to PrP106–126 and inhibit its aggregation behavior.•Metal coordination and hydrophobic interaction contributed to the binding affinity.•The Ru-based aromatic complexes displayed better inhibitory effects.•The study paved the way for potential Ru-based metallodrugs against prion diseases.Prion disease is a neurodegenerative disorder that can occur among humans and other animals. The aberrant isoform of prion protein PrPSc has been identified as the infectious agent. The neuropeptide PrP106-126 has been widely used as a suitable model to study the biological and physiochemical properties of PrPSc. PrP106-126 shares several physicochemical and biological properties with PrPSc, including cellular toxicity, fibrillogenesis, and membrane-binding affinity. Ruthenium complexes are commonly employed in anti-cancer studies due to their low cellular toxicity. In this study, six hexacoordinated ruthenium complexes with different molecular configurations were used to investigate their effects on PrP106-126 aggregation inhibition. Results revealed that the interaction between the complexes and the peptide included metal coordination and hydrophobic interaction mainly. Those complexes with aromatic structure displayed better inhibitory effects, although they only had a common binding affinity to PrP106-126. This study provided better understanding on the interaction of metal complexes with PrP106-126 and paved the way for potential Ru-based metallodrugs against prion diseases.Fibrils and spherical like aggregates of PrP106-126 are disaggregated after incubating with ruthenium complexes. Further, the interactions of ruthenium complexes with prion neuropeptide are based on metal coordination and hydrophobic interaction predominantly.

Prion diseases are characterized by conformational conversion of prion protein from a normal cellular form to an abnormal scrapie isoform (PrPSc). PrP106–126 is a prion neuropeptide and an accepted model used to study the characteristics of PrPSc because such a model has biological and physiochemical properties similar to those of PrPSc. Some metal complexes have a strong binding affinity for PrP106–126 and a good inhibitory effect against amyloid fibril formation. However, the effects of the metal ligand configuration on peptide binding and aggregation are not well known. To investigate interaction and peptide aggregation between prion neuropeptides and two gold complexes with different ligand configurations ([Au(bpy)Cl2]PF6 and [Au(dien)Cl]Cl2, where bpy is 2,2′-bipyridine and dien is diethylenetriamine), six prion peptides with either a His111-mutated or a Met109/112-mutated residue were used in this study. The selection of the mutant was based on the corresponding neuropeptide from other species. The results showed that the aromatic gold complex [Au(bpy)Cl2]PF6 exhibits better binding affinity and a better inhibitory effect against peptide aggregation than the tridentate complex [Au(dien)Cl]Cl2. For the sequence–specific PrP106–126 and its mutants, His111 plays the most important role in peptide aggregation and binding affinity. Furthermore, Met112 has a greater effect on the binding affinity than Met109. Compared with the mutated short 14 amino acid peptides, the hydrophobic region of PrP106–126 contributes to both binding affinity and self-aggregation behavior. This work will help to understand and develop potential metallodrugs against amyloid disorder.

Cytoglobin (Cgb) is a hexacoordinate globin that plays various physiological roles, including O2 transport, enzyme activity, and lipid peroxidation. The distal His81(E7) residue acts as the native sixth ligand and is crucial to exogenous ligand binding, distal environment adjustment, and heme pocket stabilization. The role of E7 has been widely studied in myoglobin, neuroglobin, and hemoglobin, but not in Cgb. In this work, the structural dynamic features of CO-ligated Cgb, CgbCO, as well as its three distal mutants H81QCgbCO, H81LCgbCO, and H81VCgbCO, were examined by performing molecular dynamics (MD) simulations. Results revealed that distal mutation significantly affected the dynamic properties of the CD-D-E and EF loop regions of Cgb. Distinct fluctuations and the occurrence of new inner cavities reflected rearrangements of the heme pocket. Distal mutation was found to affect heme motion slightly, indicating a different heme motion mechanism than that for neuroglobin. Some key residues such as E7 and CD3 showed remarkable changes in their dynamics that contributed to heme pocket rearrangement and loop region fluctuations. MD studies of four CgbCO models indicated that the distal E7 residue was a crucial influence on the dynamics of CgbCO in terms of loop fluctuations, cavity rearrangement, and slight heme motion.

Many neurodegenerative disorders are induced by protein conformational change. Prion diseases are characterized by protein conformational conversion from a normal cellular form (PrPC) to an abnormal scrapie isoform (PrPSc). PrP106-126 is an accepted model for studying the characteristics of PrPSc because they share many biological and physiochemical properties. To understand how metal complexes affect the property of the prion peptide, the present work investigated interactions between Pd complexes and PrP106-126 based on our previous research using Pt and Au complexes to target the peptide. The selected compounds (Pd(phen)Cl2, Pd(bipy)Cl2, and Pd(en)Cl2) showed strong binding affinity to PrP106-126 and affected the conformation and aggregation of this active peptide in a different binding mode. Our results indicate that it may be the metal ligand-induced spatial effect rather the binding affinity that contributes to better inhibition on peptide aggregation. This finding would prove valuable in helping design and develop novel metallodrugs against prion diseases.

Cytoglobin (Cgb), the fourth member of the vertebrate heme globin family, is widely expressed in mammalian tissues, and reversibly binds to CO, O2 and other small ligands. The diverse functions of Cgb may include ligand transport, redox reactions and enzymatic catalysis. Recent studies indicate that Cgb is a potential gene medicine for fibrosis and cancer therapy. In the present work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamic characteristics in carboxy and deoxy human Cgb. The simulation results showed that the loop regions and internal cavities were significantly affected through the binding of an exogenous ligand. The AB, GH and EF loops were found to undergo significant rearrangement and this led to distinct cavity adjustments in Xe2, Xe4 and the distal pocket. In addition, solvent accessibility and torsion angle analyses revealed an interactive distal network comprised of His81(E7), Leu46(B10) and Arg84(E10). The MD study of carboxy and deoxy human Cgb revealed that CO-ligated Cgb modulates the protein conformation primarily by loop and cavity rearrangements rather than the heme sliding mechanism found in neuroglobin (Ngb). The significant differences between Cgb and Ngb in the loop and cavity properties are presumably linked to their various biological functions.MD simulations reveal the functionally related structural dynamic characteristics of carboxy and deoxy human cytoglobin in solution.

Neuroglobin, a member of vertebrate globin family, is distributed primarily in the brain and retina. Considerable evidence has accumulated regarding its unique ligand-binding properties, neural-specific distribution, distinct expression regulation, and possible roles in processes such as neuron protection and enzymatic metabolism. Structurally, neuroglobin enjoys unique features, such as bis-histidyl coordination to heme iron in the absence of exogenous ligand, heme orientational heterogeneity, and a heme sliding mechanism accompanying ligand binding. In the present work, molecular dynamics (MD) simulations were employed to reveal functional and structural information in three carboxyl murine neuroglobin mutants with single point mutations F106Y, F106L and F106I, respectively. The MD simulation indicates a remarkable proximal effect on detectable displacement of heme and a larger tunnel in the protein matrix. In addition, the mutation at F106 confers on the CD region a very sensitive mobility in all three model structures. The dynamic features of neuroglobin demonstrate rearrangement of the inner space and highly active loop regions in solution. These imply that the conserved residue at the G5 site plays a key role in the physiological function of this unusual protein.

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in many biological processes such as enzymatic reaction, signal transduction and the mitochondria function. Different from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imidazole of proximal His96(F8) and distal His64(E7) directly bound to the metal ion. In the present work, solution 1H NMR spectroscopy was employed to investigate the electronic structure of heme center of wild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocket were assigned. Two heme orientations resulting from a 180° rotation about the α–γ-meso axis with a population ratio about 2:1 were observed. Then the 1H NMR chemical shifts of the ferriheme methyl groups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle φ is consistent with that from the molecular dynamics simulation but not with those from the crystal data. Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobin reflects the changeability of heme environment for the Ngb family.

In order to understand whether the ameliorating effect on old ages memory disorder by the root of Salvia miltiorhiza is related to the acetylcholinesterase (AChE) inhibition, two main ingredients, salvianolic acid B (1) and rosmarinic acid (2), which were isolated from S. miltiorhiza water extract, were investigated in vitro by NMR relaxation rate in this work. The results showed that the proton selective relaxation rates and the molecular rotational correlation time of proton pairs for compounds 1 and 2 increased significantly by adding of AChE in mixing solution. The study reveals that the two compounds might bind to the enzyme and have AChE inhibitory effect, which could contribute to the ameliorating effect at some extent on old ages memory disorder.

Solution 1H NMR spectroscopy has been carried out to investigate the molecular and electronic structures of the active site in H64Q/V68F double mutant mouse neuroglobin in the cyanomet form. Two heme orientations resulting from a 180° rotation about the α–γ-meso axis were observed with a population ratio about 1:1, and the clearly distinguished B isomer was used to perform the study. Based on the analysis of the dipolar shifts and paramagnetic relaxation constants, the distal Gln64(E7) side chain is obtained to adopt an orientation that may produce hydrogen bond between the NεH1 and the Fe-bound cyanide. The side chain of Phe68(E11) is oriented out of the heme pocket just like that in triple mutant of cyanide complex of sperm whale myoglobin. A 15° rotation of the imidazole ring in axial His96 is observed, which is close to the ϕ angle determined from the crystal structure of NgbCO. The quantitative determinations of the orientation and anisotropies of the paramagnetic susceptibility tensor reveal that cyanide is tilted by 8° from the heme normal which allows for contact to the Gln64(E7) NεH1. The E7 and E11 residues appear to control the direction and the extent of tilt of the bound ligand. Furthermore, the tilt of the ligand has no obvious influence on the heme heterogeneity of cyanide ligation for isomer A/B of the wild type and mutant protein, indicating that factors other than steric effects, such as polarity of heme pocket, impacts on ligand binding affinity.

Conomarphin, a novel conopeptide containing D-amino acid, was identified from the venom of Conus marmoreus and classified into M-superfamily of conotoxin. In this article, we reported the 3D structure of conomarphin at pH 5 determined using 2D 1H NMR method in aqueous solution. Twenty converged structures of this peptide were obtained based on 205 distance constraints, 8 dihedral angle constraints, and 2 hydrogen bond constraints. The root mean square deviation (RMSD) values of the backbone atoms were (0.074±0.029) nm. The refined structure of conomarphin at pH 5 contains a short 310-helix at C-terminal of the peptide. It was also characterized by a loose loop centered at Ala6. Comparison of structural and electrostatic potential between conomarphin at pH 3 and pH 5 were presented. Although the solution structure of conomarphin at pH 5 shared part of the same secondary structure element with the structure of conomarphin at pH 3, it adopted a distinctive backbone conformation with the overall molecule resembling a “flexual arm” when viewed from the front. Structural differences implied that this conopeptide was rather pH sensitive and its bioactivity in vivo might be related to the acidity.

In order to search for better acetylcholinesterase (AchE) inhibitors, the binding properties of AchE with huperizine E, which is a derivative of huperzine A, were investigated with 1H nuclear magnetic resonance (1H NMR) method. The nonselective, selective and double-selective spin-lattice relaxation rates of some protons in huperzine E were acquired in the absence and presence of AchE at a concentration ratio of [ligand]/[protein] = 1: 0.005. The enhancements of selective relaxation rates of these protons were obvious after adding AchE. The molecular motional correlation times of two pairs of protons, H-1a/H-1b and H-2/H-3, in the bound state at T = 298 K were 11.7 and 9.46 ns respectively, while they were 27.7 and 35.2 ps in the free state. All of these show that huperzine E has high binding affinity with AchE.

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in many biological processes such as enzymatic reaction, signal transduction and the mitochondria function. Different from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imidazole of proximal His96(F8) and distal His64(E7) directly bound to the metal ion. In the present work, solution 1H NMR spectroscopy was employed to investigate the electronic structure of heme center of wild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocket were assigned. Two heme orientations resulting from a 180° rotation about the α–γ-meso axis with a population ratio about 2:1 were observed. Then the 1H NMR chemical shifts of the ferriheme methyl groups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle φ is consistent with that from the molecular dynamics simulation but not with those from the crystal data. Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobin reflects the changeability of heme environment for the Ngb family.

Conotoxins are mainly disulfide-rich short peptides active on different ion channels, neurotransmitter receptors or transporters in nervous system, exhibiting highly diversified composition, structures and biological functions. Besides these kinds of conopeptides, some novel cysteine-free conopeptides have also been reported. Conomarphin, a cystine-free 15-residue conopeptide from Conus marmoreus, has been purified and classified into M-superfamily. In addition to its unique characteristic of a D-type phenylalanine at the third residue from the C-terminus, conomarphin has an unusual hydroxyproline residue at position 10. To make an effort to understand the role of hydroxyproline post-translational modification in conomarphin, 1H NMR solution structure of Hyp10Pro variant of conomarphin was resolved and compared with the native conomarphin in the present work. The Hyp10Pro conomarphin has a type II-β-turn near the C-terminus instead of a 310 helix in native conomarphin. The compact loop region in native conomarphin becomes more open when hydroxyproline is displaced by proline. This reveals that hydroxyproline residue is essential for the structure of conomarphin just like D-Phe13. The unusual post-translational modification of conomarphin implies a unique selectivity of hydroxylation in toxin sequence.