Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain

Accepted Manuscript Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain Rachit Shah, Tsui-Fen Chou, Kimberly M. Maize, Alexander Strom, Barry C. Finzel, Carston R. Wagner PII:

S0006-291X(17)31469-9

DOI:

10.1016/j.bbrc.2017.07.111

Reference:

YBBRC 38217

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 9 July 2017 Revised Date:

18 July 2017

Accepted Date: 20 July 2017

Please cite this article as: R. Shah, T.-F. Chou, K.M. Maize, A. Strom, B.C. Finzel, C.R. Wagner, Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.07.111. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Graphical Abstract (figure):

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Inhibition by Divalent Metal Ions of Human Histidine Triad Nucleotide Binding Protein1

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(hHint1), a Regulator of Opioid Analgesia and Neuropathic Pain

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Rachit Shah†, Tsui-Fen Chou#†, Kimberly M. Maize, Alexander Strom, Barry C. Finzel and Carston R. Wagner*

Departments of Medicinal Chemistry, University of Minnesota,

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Minneapolis, Minnesota 55455, USA

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*Address correspondence to:

C. R. Wagner, University of Minnesota, Dept. Medicinal

Chemistry, 8-174 Weaver Densford Hall, 308 Harvard St. S.E., Minneapolis, MN 55455. Phone: 612-625-2614. E-mail: [email protected] #

Present address: Harbor-UCLA Medical Center and Los Angeles Biomedical Research

Institute, Division of Medical Genetics, Department of Pediatrics, 1124 W. Carson Street, Torrance, California 90502, United States.

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†

Both authors contributed equally

Keywords: Histidine triad nucleotide binding protein1 (Hint1), Phosphoramidase, human Hint1,

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Escherichia coli (E. coli) Hint.

Abbreviations

hHint1, human histidine triad nucleotide binding protein 1; CNS, Central nervous system;

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EDTA, Ethylene diamino tetraacetic acid; LC-MS/MS, Liquid chromatography tandem mass

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spectrometry; CD, Circular dichroism.

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Abstract Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for

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the management of pain. However the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the

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effect of divalent metal ions (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on the structural integrity and catalytic activity of hHint1. With the exception of Mg2+, all the divalent ions inhibited

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hHint1, the rank of order was found to be Cu2+ >Zn2+ >Cd2+ ≥Ni2+ >Mn2+ based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu2+ is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time and concentration dependent inhibition, with the rate of inactivation highly dependent on

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alterations of the C-terminus. With the exception of Cu2+, restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears

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to be structurally sensitive to irreversible inactivation by copper, which may be of

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neurotoxicological and pharmacological significance.

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1. Introduction: Trace elements and metal ions play an important role in maintaining homeostasis in a wide range of cellular processes. The break down of this homeostasis has been shown to significantly alter a

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wide range of physiological processes, especially in the central nervous system (CNS). Alteration of physiological levels of zinc ions can modulate the activity of a wide variety of ion channels, including N-methyl-D-aspartate (NMDAR) and nicotinic acetylcholine receptors

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(nAChRs).1, 2 In addition, an increase in the concentration of zinc ions in glutaminergic synapses has been associated with epilepsy, schizophrenia, autism and Alzheimer’s (AD).3-5 Similarly,

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increased levels of copper have been found in patients suffering from schizophrenia and Alzheimer’s disease (AD)6, 7, while the neurotoxicity of manganese appears to be associated with alterations in the glutamate-GABA pathway.8

Recently, Histidine triad nucleotide binding protein 1 (Hint1) was found to be associated

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with a number of G-protein coupled receptors (GPCR) in the CNS.9, 10 Histidine triad nucleotide binding proteins (Hints) are ubiquitous purine nucleoside phosphoramidases and acyl-AMP hydrolases that have been found in all kingdoms of life.11 Hints are members of the most ancient

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sub-family of the histidine triad protein superfamily (HIT), which are defined by the highly conserved sequence motif, His-X-His-X-His-XX, where X is a hydrophobic residue (Fig. 1 A). E. coli contains only one Hint gene (echinT); whereas three human Hint genes (HINT1,

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HINT2, HINT3) have been identified.14-16 Although possessing highly similar sequences overall, hHint1 and echinT have distinct substrate specificities that have been attributed to dissimilarity in their C-terminal sequences (Supplemental Table S1).17

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Human Hint1 has been found to be a potential regulator of transcription activation complex

formation18,

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apoptosis20,

neurotransmitter

receptor

modulation21-23,

cell

differentiation and tumorigenicity 24-27, tRNA synthetase amino acid adenylation 28-31 and axonal

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neuropathy.32, 33 In addition, a number of cellular and in vivo studies have implicated hHint1 in the regulation of NMDAR, the mu-opioid receptor (MOR), as well as the dopaminergic and cannabinoid signaling pathways.34,

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Neuropharmacological studies have demonstrated that

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HINT1-/- mice display increased morphine analgesia and amphetamine sensitivity, as well as reduced levels of anxiety and nicotine dependence.36-39 Consistent with these findings, we have

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recently demonstrated that an active site targeted small molecule inhibitor of hHint1 enhances opioid analgesia and reduced allodynia in a mouse model.40 The nucleotide-binding site of hHint1 includes multiple histidines that could plausibly interact with divalent cations. (Fig. 1 B, C). The interaction of Hint1 with MOR signaling pathway has been proposed to be governed by

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the release of the intracellular zinc ions.41 Nevertheless, the effect of zinc or other divalent ions on catalysis has not been studied. Hence, we have elected to investigate the effect of divalent

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cations (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on hHint1 enzymatic activity and structure.

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2. Materials and Methods: 2.1 Time- and Concentration- dependent Inactivation of Hints’ Activity by Divalent Cations (ZnCl2, CuCl2, NiCl2, CdCl2, MnCl2) – Incubation mixtures (200 µL) contained the

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indicated concentrations of metal ions and hHint1 (0.1 µM), echinT (0.25 µM) or the chimera (1 µM) in HEPES buffer (20 mM, pH 7.2) at 23 °C. The phosphoramidase activity was measured by withdrawing aliquots (10 µL) at 15 s intervals over a time frame of 15 to 60 s and transferring

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them to the substrate solution containing TpAd (590 µL, 2.03 µM for hHint1 and chimera, or

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30.5 µM for echinT), at 25 °C. The detailed development of a series of fluorogenic substrate such as TpAd has been described previously.42 The fluorescence spectra were monitored over a 4 min interval at 25 °C. The percentage of activity remaining after incubation with metal ion was calculated with respect to controls that contained HEPES buffer, but not metal ions. Inactivation data were plotted as ln % remaining activity ([E]/[E]t) versus time (t) (Scheme 1, eq 1) and the

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observed inactivation rate constant (kobs) was obtained from the slope of the line. The secondorder rate constant (KI/ kinact) was obtained by double reciprocal plot (eq. 3) and was derived from the slope of the plot (eq. 4). Measurements were carried out in duplicate with variances

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provided from the mean.

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IC50 values were calculated from fitting the percentage of the remaining activity (%RA) after 60s incubation with various concentrations of metals to a Langmuir equation [%RA =100/(1 + [Metal]/IC50)] by non-linear regression analysis using JUMP IN program. The result was expressed a mean ± standard error.

2.2 Chelation of Metal Ions by EDTA—The inhibitory effect of metal ions on Hint activity in the presence or absence of EDTA was evaluated by withdrawing aliquots of enzyme metal

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mixtures, then adding them to a substrate solution containing EDTA (4.2 mM or 45 mM). The phosphoramidase activity was measured as described above.

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2.3 Circular Dichroism (CD) Spectroscopy— CD spectra of proteins were obtained at 23 °C with a J710 spectropolarimeter (Jasco). Proteins at concentrations of 10 µM in sodium phosphate buffer (10 mM, pH 7.2) with or without metal ions were analyzed in a quartz cuvette with path-

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length of 1 mm, and spectra were accumulated and averaged over nine scans. Subtraction of

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buffer background from the protein spectrum was performed by using excel program.

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3. Results 3.1 Copper (II) displays time dependent and irreversible inactivation of hHint1

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The hHint1 active site contains three conserved histidines (His-51, His-112, and His-114) in close proximity that might be available for metal binding. (Fig. 1 B, C) To explore the potential role of divalent metal ions, we began by monitoring the effect of CdCl2, CuCl2, MgCl2, MnCl2, NiCl2, and ZnCl2 on the hydrolysis of the model substrate tryptamine adenosine

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phosphoramidate (TpAd) by hHint1. We have previously found that an hHint1/echinT chimera

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made by replacing the hHint1 C-terminus with the echinT terminus switches the substrate specificity between the two enzymes, so we also chose to explore the effect of divalent metal ions on echinT and the chimera. (See supplemental Table S1). As represented in Fig. 2 for NiCl2 with echinT and CuCl2 with hHint1, all the proteins exhibited a loss of activity in the presence of MnCl2, ZnCl2, CuCl2, NiCl2, or CdCl2. No inhibition was observed in the presence of MgCl2 at

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concentrations as high as 2 mM (data not shown)

Both Zn2+and Cu2+ exhibit a significant pseudo-first-order, concentration- and time-

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dependent loss of activity in hHint1. The second-order rate constants (kinact /KI) were calculated according to Scheme 1 and obtained from the slopes of the plots, as shown in Fig. 2C, D (Table

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1). The rank order of inhibition by divalent metal ions for all the proteins was found to be Cu2+ >Zn2+ > Cd2+ ≥Ni2+ >>Mn2+. With the exception of Cu2+, the rate of inhibition (kinact /KI) by all the metal ions was found to be faster for echinT than hHint1. Excepting NiCl2 and CdCl2, the rate of inhibition for the chimera was also shown to be more comparable to echinT (Table 1). Thus, the composition of the C-terminus importantly modulates the observed rate of inhibition. The trends observed in the rate of inhibition were found to be consistent with the steady-state IC50

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values for most of the metals (except CdCl2) (Table 2); regardless of the enzyme, CuCl2 exhibited the greatest inhibitory effect.

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To investigate the reversibility of divalent metal ion mediated inhibition, we carried out inhibition studies in the presence of the chelating agent EDTA. Incubation of the enzymes with Zn2+, Cd(II) or Ni(II) (Supplemental Information, Fig. S1) followed by treatment with excess

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EDTA resulted in full recovery of the catalytic activity of hHint1, echinT and the chimera. While all the Hints were most susceptible to inhibition by Cu2+ (Supplemental Information, Fig. S2),

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hHint1 is distinctive in that the Cu2+-inhibition appears irreversible; hHint1 activity could not be restored even in the presence of EDTA as high as 45 mM (10X) (Supplemental Information, Fig. S3A). In contrast, the chimera exhibited a greatly reduced susceptibility to Cu(II) inhibition at the lowest dose (10 µM), while at higher concentrations (30 µM), only partial recovery of

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activity could be observed after the treatment with EDTA. (Supplemental information, Fig. S3).

3.2 Interaction of Cu(II) with His-112 appears to significantly destabilize the secondary

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structure of hHint1

We next wanted to investigate the impact of metal ion binding on the secondary structure

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of hHint1. Consequently, CD spectra of the Hint proteins treated or not treated with metal ions were obtained in the far-UV region (190~260 nm). The result was expressed as the mean residue ellipticities (MRE). As can be seen in Fig. 3A, treatment of hHint1 (yellow diamond) with ZnCl2 resulted in minor changes in hHint1, while treatment with CuCl2 led to unfolding of the protein. Thus, the interaction of Cu(II) with His-112 appears to significantly destabilize the secondary structure hHint1 leading to irreversible inactivation. In contrast, CD spectra of echinT treated

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with Cu(II) revealed only minor secondary structure perturbations. (Supplemental Fig. S4) Consistent with the previous results, we found that treatment of the chimera with Cu(II) resulted in complete loss of the proteins secondary structure (Supplemental information: Fig. S4C).

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Incubation of wild type hHint1 with 10-fold excess of Cu2+ resulted in the formation of visible aggregates within few minutes. A comparison of the protein solution with and without copper under the visible microscope clearly showed aggregate formation (Supplemental information

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Fig. S5).

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3.3 X-ray crystal structure of hHint1 in complex with Cu(II)

To determine if specific sites exist for copper binding, we performed soaks of crystals of wild-type hHint1 and generated a new crystal structure (PDB ID 5EMT). Diffraction data to 1.50 Å resolution were collected at a wavelength (1.37 Å) near the copper absorption edge, and

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data were processed to exploit anomalous differences. Sulfur and Copper atoms are clearly visible in anomalous difference electron density maps from this structure (Supplemental Figure S6). Peaks attributable to copper are found coordinated between His-112 and His-114 in the

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catalytic site, and between His-59 and His-110. A third copper ion appears coordinated to His122. These three copper sites appear in both chain A and chain B (Fig. 4), although the

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anomalous difference density for the His-112/His-114 copper in chain B is weak—this site is partially occluded by a symmetry mate. Another copper is found near the His-59/His-110 copper in chain A only, and binding here is likely influenced by crystal packing.

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4. Discussion Although the precise biological function of Hint1 catalytic activity has yet to be determined, our investigation provides insight into the effect of divalent cations (Mg2+, Mn2+,

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Ni2+, Zn2+, Cu2+, and Cd2+) on the activity of Hint proteins. Magnesium ion (Mg2+) was found not to affect either the kcat or Km values of hHint1, echinT or the chimera (data not shown). Manganese (Mn2+), which is often used as a substitute for Mg2+ had a minor effect; the second-

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order inactivation rate constant for echinT was found to be 10-fold greater than the value for hHint1 and 2-fold greater than for the chimera. This result may reflect the greater affinity of

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Mn(II) for amines than Mg(II). Very little inhibition of hHint1 by Ni(II) was observed, while echinT and the chimera were found to be 20-fold and 2-fold more sensitive to inactivation, respectively, than hHint1. From crystal structure analyses, the predominant coordination number is six for Mn2+ and Mg2+, while for Zn2+ coordination numbers ranging from four, five, and six

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have been observed.43 However, the exchange rate of coordinating groups to Zn2+ is much slower, than that for Mn2+ and Mg2+, possibly leading to enhanced inhibition. Time dependent inhibition studies for all the metal ions were consistent with a

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mechanism of inactivation proceeding through initial formation of a reversible complex as indicated by the x-axis intercept on the double reciprocal plot of inhibition (Fig 2), followed by a

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second step that is likely dependent on more stable binding to the active site. With the exception of Cu2+, inhibition of each proteins could be reversed by the addition of excess EDTA and did not result is significant alterations in secondary structure. Of all the metals, Cu2+ was found to have the highest rate of inactivation and the lowest IC50 values for hHint1 and echinT. However, in contrast to echinT, inactivation of hHint1 by Cu2+ was found to be irreversible.

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Typically, copper prefers to coordinate with histidines, methionine, cysteine, asparagine and aspartate. Interestingly, by taking advantage of the length and nature of amino acid residues, copper-binding proteins display unique structural properties in their motifs, which govern their

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coordinating and oxidative ability. For example, Cu1+ bound to the bacterial copper trafficking factor, CusF, is coordinated to two methionines and a histidine.44 While in case of prion proteins, the HGGGW motif binds Cu2+ by coordinating with the flexible backbone amide residues of

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glycine, while interacting with the indole moiety of a tryptophan via a water molecule.45

In our structure (PDB ID 5EMT), the active site copper is coordinated by two histidines

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(His-112 and His-114), and a water atom (chain A) or just the two histidines (chain B). The other copper binding sites are similarly low in coordination partners. Our crystal structure also represents likely initial binding mode of copper and other divalent cations. Given our finding that Cu2+ treatment results in irreversible inactivation of hHint1, the

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largely unaltered structural integrity of the observed binding site is somewhat surprising. Constraints on protein conformation imposed by crystal packing and nearly equimolar ratio of effective protein concentration to ligand in crystals may stabilize the enzyme to denaturation in

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this study. Consequently, the copper coordination in this structure likely reflects initial reversible

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metal binding of copper similar to that possible by other inhibitory metal ions.

In contrast to the other test metals, the loss of hHint1 activity after incubation with Cu2+

was characterized by loss of hHint1 structural integrity upon chelation with histidine. Given the near identity of the active sites, the ability to restore the activity of echinT and the chimera by treatment with EDTA in a time dependent manner strongly suggests that the C-terminus is a contributing factor in determining the loss of hHint1 structural integrity upon incubation with

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Cu2+. The ability, therefore, of the chimera to resist the loss of structural integrity when treated with copper, suggests that the C-terminus uniquely facilitates copper induced destabilization of hHint1. We also show that upon incubation of only ten fold excess of copper; we observe

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formation of visible aggregates under the microscope within minutes of incubation. Consistent with these results, we speculate that the primary mechanism of irreversible activation by Cu2+ is

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due to loss in secondary structure and formation of hHint1 aggregates.

Recent studies have shown that hHint1 is a determinant of the quality of the interaction

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between GPCR-NMDAR.10 For example, MOR agonists decouple hHint1 from NMDAR and promote negative feedback through increased NMDAR responsiveness.34 In contrast, CB1 agonists maintain the hHint1-NMDAR interaction necessary to restrain NMDAR promoted excitotoxicity and preserve cell viability.10, 35 NMDAR hypo function has been associated with

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the development of psychotic and schizophrenic symptoms, while hyper activation with neurotoxicity.46 Furthermore, the role of hHint1 in the regulation of the dopaminergic pathway has been documented with studies in knock out mice indicating increased sensitivity to

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amphetamine and decreased nicotine dependence.37, 39 Therefore, alterations in hHint1 function could potentially participate in triggering or exacerbating a number of pharmacological

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symptoms. Indeed, alteration in function or aberrant expression of hHint1 has been found to be associated in brain tissues of clinical patients suffering from schizophrenia.47 In addition, altered levels of copper have been found in brain tissue of Parkinson’s disease and Alzheimer’s disease patients.6, 7, 48-50 Consequently, given the sensitivity of hHint1 to irreversible denaturation by copper, further studies of the capacity of metal ions to alter the cellular and physiological

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function of hHint1 should shed light on the participation of this unique and multifunctional protein on neurological function and disorders.

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Acknowledgment

The authors are also grateful to Jay Nix of the Molecular Biology Consortium of ALS Beamline 4.2.2 for crystallographic data collection and processing. KMM was supported by the

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University of Minnesota Frieda Martha Kunze Fellowship and the American Foundation for Pharmaceutical Education. Funding from the University of Minnesota Endowment is gratefully

acknowledged.

Supporting Information

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acknowledged. Funding from the University of Minnesota Foundation is gratefully

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The supporting information is available free of charge on the journal website.

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[44] Loftin, I. R., Franke, S., Roberts, S. A., Weichsel, A., Héroux, A., Montfort, W. R., Rensing, C., and McEvoy, M. M. Biochemistry (2005) 44, 10533-10540. [45] Millhauser, G. L. Acc Chem Res. (2004) 37, 79-85. [46] Sánchez-Blázquez, P., Rodríguez-Muñoz, M., and Garzón, J. Front Pharmacol (2014) 4, 169. [47] Varadarajulu, J., Schmitt, A., Falkai, P., Alsaif, M., Turck, C. W., and Martins-de-Souza, D. Eur Arch Psychiatry Clin Neurosci (2012) 262, 167-172. [48] Desai, V., and Kaler, S. G. (2008) Role of copper in human neurological disorders, Am J Clin Nutr 88, 855S-858S. [49] Ventriglia, M., Bucossi, S., Panetta, V., and Squitti, R. J Alzheimers Dis (2012) 30, 981984. [50] Zhao, H. W., Lin, J., Wang, X. B., Cheng, X., Wang, J. Y., Hu, B. L., Zhang, Y., Zhang, X., and Zhu, J. H. PLoS One (2013) 8, e83060.

A

B

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C

Fig. 1. X-ray Crystallographic Structure of hHint1. (A) Overall dimer structure with bound AMPCP (PDB: 1AV5); (B) active site region in one monomer with bound AMPCP (PDB:

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1AV5) and (C) active site with bound AMP, histidine 112 of hHint1 is the nucleophilic residue

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that forms covalent P-N bond with its substrate (PDB: 1KPF) [1]

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D

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C

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B

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A

Fig. 2. Time- and Concentration-Dependent Inactivation. (A) echinT by NiCl2 and (B) hHint1 by CuCl2. (C and D) The double reciprocal plot of observed inactivation rate constant (1/kobs) was plotted as a function of the metal concentration (eq 3). The y-intercept on the linear fit gave the

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inverse of the rate of inactivation. The second order inactivation rate constant (kinact /KI) was

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derived from the reciprocal of the slope (eq 4).

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Hint + M2+

kinact

Ln ([E]/[E]t) = - (kobs) × t

M2+

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eq 3

eq 4

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1/ kobs = (KI/ kinact) × 1/[I] + 1/ kinact

eq 2

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eq 1

kobs = kinact × [I]/(KI + [I])

KI/ kinact = 1/slope

Hint

Scheme 1. Metal ion dependent inactivation of Hint and equations to calculate the second order

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rate of inactivation.

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Table 1. Comparison of Second Order Inactivation Rate Constants (kinact/KI) of MnCl2,

kinact /KI (s-1 M-1) Metal Ion

hHint1

MnCl2

0.0082 ± 0.000001 0.06 ± 0.003 10 ± 0.4

Chimera

0.096 ± 0.0006

289 ± 3

17 ± 4

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NiCl2

echinT

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NiCl2, ZnCl2, CuCl2, and CdCl2. for hHint1, echinT, and the Chimera.

247 ± 7

1812 ± 82

1021 ± 54

CuCl2

12853 ± 241

6847 ± 294

2024 ± 28

CdCl2

86 ± 5

265 ± 8

108 ± 15

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ZnCl2

echinT, and the Chimera.

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Table 2. Comparison of IC50 (µ µM) of MnCl2, NiCl2, ZnCl2, CuCl2, and CdCl2. for hHint1,

IC50 (µM) echinT

Chimera

1076000 ± 24200

137000 ± 24000

84000 ± 10000

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MnCl2

hHint1

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Metal Ion

NiCl2

1488 ± 158

33 ± 4

460 ± 94

ZnCl2

47 ± 7

6.0 ± 0.7

7.3 ± 1.4

CuCl2

1.1 ± 0.1

0.67 ± 0.03

2.5 ± 0.5

CdCl2

121 ± 26

41 ± 3

53 ± 14

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Fig. 3. Circular Dichroism Spectra of the hHint1 proteins. Circular Dichroism Spectra were collected from 190 to 260 nm at 23 oC with protein concentration of 10 µM. (A) Wild-type

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hHint1 (black) and hHint1 incubated with ZnCl2 (0.15mM, red), ZnCl2 (1.5mM, blue), CuCl2 (0.15 mM, light blue), and CuCl2 (1.5 mM, purple) at 23 oC for 5 min. (B) H112G-hHint1

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(black) and H112G-hHint1 incubated with CuCl2 (1.5 mM, red) at 23 oC for 5 min

Fig. 4. Coordination of copper in hHint1. The anomalous difference density is shown contoured at 4σ. (A) A copper ion is coordinated by A/His59, A/His110, the carbonyl of A/Gly14 and a water molecule. A second copper binds near this site. The unmarked density corresponds to a symmetry-related position of this ion. (B) A copper ion is coordinated by A/His112, A/His114, and a water molecule. (C) A copper ion is coordinated by A/His122 and a water molecule.

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The Susceptibility of the NMDA and Mu Opioid Receptor Regulator, Human Histidine Triad Nucleotide Binding Protein1 (hHint1), to Divalent Metal Ion Inhibition

and Carston R. Wagner*

•

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Highlights

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Rachit Shah†, Tsui-Fen Chou#†, Kimberly M. Maize, Alexander Strom, Barry C. Finzel

hHint1 is an integral part of zinc mediated protein assemblies involved in the

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regulation NMDA receptor. Nevertheless, the effect of divalent metal ions on its enzymatic activity is unknown. •

hHint1 exhibited time dependent inhibition in the presence of divalent metal ions.

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Inhibition was found to be reversible for all the divalent metal ions except for

•

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Copper (II).

Copper (II) induces secondary structural changes and leads to complete unfolding

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of hHint1.