Humans Are In The Domain
Protein Sci. 2019 Jun; 28(6): 1151–1156.
Zinc finger domain of the human DTX protein adopts a unique Band fold
Kazuhide Miyamoto
1 Department of Pharmaceutical Wellness Care, Faculty of Pharmaceutical Sciences, Himeji Dokkyo Academy, Himeji, Hyogo, Nihon
Yuma Fujiwara
1 Department of Pharmaceutical Health Intendance, Kinesthesia of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo, Japan
Kazuki Saito
1 Section of Pharmaceutical Health Intendance, Faculty of Pharmaceutical Sciences, Himeji Dokkyo Academy, Himeji, Hyogo, Japan
Received 2019 Jan 18; Accepted 2019 Mar 27.
Abstract
The Deltex (DTX) family is involved in ubiquitination and acts every bit Notch signaling modifiers for controlling cell fate determination. DTX promotes the development of the ubiquitin chain via its Ring finger (DTX_RING). In this study, the solution construction of DTX_RING was determined using nuclear magnetic resonance (NMR). Moreover, by experiments with a metallochromic indicator, nosotros spectrophotometrically estimated the stoichiometry of zinc ions and found that DTX_RING possesses zinc‐binding capabilities. The Simple Modular Architecture Inquiry Tool database predicted the structure of DTX_RING every bit a typical Band finger. All the same, the actual DTX_RING structure adopts a novel RING fold with a unique topology distinct from other Ring fingers. We unveiled the position and the range of the DTX_RING active site at the atomic level. Artificial Band fingers (ARFs) are made by grafting active sites of the Band fingers onto cross‐brace structure motifs. Therefore, the present structural analysis could exist useful for designing a novel ARF.
Keywords: Band finger, E3 enzyme, NMR construction, artificial Band finger, DTX, ubiquitination
Short abstract
PDB Code(southward): 6IR0
Introduction
Cross‐braced motifs are mutual to various poly peptide domains, such every bit RING,1 PHD,2 FYVE,3 and ZZ4 fingers, which bind to zinc atoms with eight ligands of Cys and/or His. As well this shared motif, each unique construction plays an important role for its molecular office.4 RING fingers are involved in protein ubiquitination,5 which is a cascade reaction consisting of ubiquitin (Ub)‐activating (E1), Ub‐conjugating (E2), and Ub‐ligating (E3) enzymes. Most Ring fingers function equally E3 enzymes and catalyze the transfer of Ub from E2 enzymes to the substrate lysines.half dozen, 7 The canonical Band fingers adopt a ββαβ foldeight and possess a functional α region (agile site) for E2‐binding.five, 9, x Although some Ring fingers, similar RING2 of Band‐in‐betwixt‐RING, are involved in protein ubiquitination, they do not have the active site for E2‐bounden.xi By contrast, PHD, FYVE, and ZZ fingers are not involved in protein ubiquitination.ii, 3, 12 For instance, the PHD finger from the human Williams‐Beuren Syndrome Transcription Gene protein specifically binds to phosphatidylinositol 3‐phosphate and phosphatidylinositol v‐phosphate.xiii
Previously, we developed a method for making an bogus RING finger (ARF) that specifically binds to E2 enzymes and assumes the role of a substrate. Such ARFs were engineered by grafting agile sites of Band fingers into other cross‐brace zinc motifs (e.one thousand., PHD fingers).xiv, 15, xvi The ubiquitination organisation of ARF uses a novel and concise machinery without substrates, and its utilize enables the simplified detection of E2 activity during ubiquitination.17, 18 Various cancers are associated with E2 enzymes, such as leukemia,19 breast cancer,17, twenty and gastric cancer.21 E2 activities are considered to be potential cancer biomarkers.21 Thus, the ARF methodology may open a screening method for E2 enzymes, leading to novel cancer diagnostic techniques. In guild to extend the ARF method to various RING fingers, the identification of appropriate positions for grafting active sites is desirable. The Deltex (DTX) family functions every bit an E3 enzyme via its Band finger and acts as Notch signaling modifiers for controlling cell fate conclusion.22 The alternative Notch bespeak through the DTX poly peptide is an oncogenic gene in malignant glioma.23 The expression level of the DTX poly peptide is associated with prolonged survival in cancer patients.
In this report, nosotros have determined the solution structure of the Band finger domain of the DTX protein (DTX_RING) using nuclear magnetic resonance (NMR). The Simple Modular Architecture Research Tool (SMART)24 database predicted the domain structure of DTX_RING equally a RING finger. Yet, NMR structure determination revealed that the actual DTX_RING adopts a novel RING fold with a unique topology. The determined DTX_RING structure was compared with the typical RING fold.
Results and Discussion
The DTX_RING domain binds ii zinc atoms
The man DTX_RING was prepared by the standard peptide synthesis method.25 The amino acid sequences for the human DTX_RING and its homologs were shown in Figure 1(a). Cysteine modification using p‐hydroxymercuribenzoic acid (PHMB) and iv‐(2‐pyridylazo) resorcinol (PAR)26, 27 indicated that the DTX_RING peptide has zinc‐bounden capabilities. The absorbance value (A) for the zinc‐PAR complex was 0.28 at 500 nm. The concentration of the DTX_RING peptide was ii.0 μM. The estimated amount of zinc ions binding to DTX_RING was iv.2 μM. Therefore, the zinc:protein stoichiometry was two.1 at 20°C, revealing that the DTX_RING peptide binds to two zinc atoms.
Sequence alignment and structural characteristics of DTX_RING. (a) The sequence alignment of the Ring finger from the homo DTX protein (DTX_RING) and its homologs. ZN1 and ZN2 are the two zinc‐binding sites in a cross‐brace arrangement. Zinc‐binding ligands are indicated in red. Stars show the well‐conserved residues among homologs. The residues provided by the generative REgularized ModeLs of proteINs (GREMLIN) software are boxed. (b) Stereoview drawing traces of the courage atoms for fitting the xx lowest energy structures (residues Asn1‐Thr76). Zinc atoms are shown in magenta. (c) Surface representation and ribbon diagram of DTX_RING, which were shown with the Discovery Studio ii.one software. The Connolly surface was faux with the electrostatic potential (bluish, positive; red, negative). The α1, α2, β1–β2, and β3 regions are shown in greenish, orange, cyan, and xanthous, respectively
Resonance assignments and overall structure
NMR resonances for the 13C/15N‐labeled DTX_RING peptide were assigned using conventional heteronuclear methods.28 The backbone resonance assignments were completely achieved except for the amide protons of Asn1 and Thr76. The Cβ atoms of six Cys residues (14, 17, 47, 55, 71, and 74) were present at betwixt 28.0 and 32.0 ppm, and they were identified as the zinc‐binding ligands.29 Two His49 and His52 residues were in the germination of Nε2‐H tautomers as possible zinc‐bounden ligands because their Cδ2 atoms were present at less than 122 ppm.30 The residues Cys14, Cys17, His52, and Cys55 were clustered to make one of two zinc‐binding sites, supported by the NOE connectivities between their Hβ protons. All the zinc‐binding ligands are conserved among the vi homologs of DTX_RING [Fig. one(a)]. Our information showed that DTX_RING adopts a CthreeH2Ciii‐type zinc arrangement in a cross‐brace fashion. Thus, DTX_RING belongs to the Ring‐Htwo blazon group. To ensure the tetrahedral zinc coordination, the lower and upper constraints were added in a listing of NOE distance restraints. Using the restraints files, the solution construction of DTX_RING was calculated by torsion angle dynamics on the program CYANA31 and by the Smart Minimizer algorithm on the plan Discovery Studio 2.1 (Accelrys Software Inc.).16 A well‐converged NMR structure of the 20 lowest energy structures of DTX_RING is shown in Figure one(b). The statistics of the ensemble construction and distance restraints for DTX_RING are summarized in Table 1. The NMR structures (Thr3‐Glu5, Gly26‐Asp29, Val41‐Leu44, and His49‐Met59) are superimposed over the backbone (N, Cα, and C′) atoms and the non‐hydrogen‐atoms, with rms deviations of 0.40 and 0.76 Å, respectively. The quality of the structures was validated past PROCHECK‐NMR software assay.32 As a result, 91.1% of the well‐ordered region was within the near favored regions and additionally immune regions. The Connolly surface of DTX_RING calculated with Discovery Studio 2.1 software is shown in Figure ane(c). The hydrophobic shallow groove occurred on the surface of the molecule past residues Ile16, Met59, and Pro72. The Asp11, Glu12, Asp13, Glu19, Asp29, and Asp32 residues formed the negatively charged surface, and on the opposite side, Lys34, Lys46, Lys65, and Lys75 contributed to the formation of the highly basic surface of DTX_RING. The hydrophobic shallow groove was surrounded by these charged clusters. The NMR structures indicated that DTX_RING possesses two α‐helices and three β‐strands (α1: Leu53‐Met59, α2: Gly26‐Asp29, β1: Val41‐Leu44, β2: His49‐His52, and β3: Thr3‐Glu5) [Fig. two(a)]. The hydrophobic core for proper folding was formed by the residues Phe51, Leu53, Leu56, and Leu69.
Table 1
Summary of Structure Statistics of DTX_RINGa
| NOE upper distance restraints | |
| Total | 1074 |
| Short‐range (|i −j| = one) | 500 |
| Medium range (1 < |i −j| < v) | 164 |
| Long range (|i −j| ≥ 5) | 410 |
| Dihedral bending restraints (ϕ and ψ) | 32 |
| Constraints for the zinc coordination (upper/lower) | 28/28 |
| CYANA target function value | 0.07 Åtwo |
| Distance constraints violations | |
| Number >0.xv Å | 0 |
| Maximum | 0.14 Å |
| PROCHECK Ramachandran plot analysisb | |
| Residues in most favored regions | 57.two% |
| Residues in additionally allowed regions | 33.nine% |
| Residues in generously allowed regions | vii.1% |
| Residues in disallowed regions | 1.8% |
| RMS divergence to the boilerplate coordinatesb | |
| Backbone atoms | 0.40 Å |
| Heavy atoms | 0.76 Å |
Structural comparisons of DTX_RING and BRCA1_RING. (a) Ribbon diagrams of the lowest free energy structure of DTX_RING with the heavy atoms on the side chains of the residues, which contribute to the formation of the hydrophobic core and the hydrophobic groove. The putative E2‐binding residues are shown in blue. The coloring scheme of the secondary structures is the same as that in Figure ane(c). (b) The RING finger of BRCA1 (BRCA1_RING) with the hydrophobic residues (blueish) for E2‐binding capabilities. The topological comparisons of (c) DTX_RING and (d) BRCA1_RING are illustrated. The dissimilar zinc‐ligands between DTX_RING and BRCA1_RING are shown in ruby
Comparison with other Band domains
DTX_RING belongs to Category II of the Ring group considering of the absenteeism of its residue Trp in the α1 region.x The RING domain of chest cancer Type 1 susceptibility poly peptide, BRCA1_RING (PDB ID: 1JM7), is a typical Band fold of Category II that has α‐helical and antiparallel β‐sheet structures arranged in a cross‐braced zinc‐binding fashion. BRCA1_RING adopts the traditional ββαβ arrangement [Fig. 2(b,d)], whereas DTX_RING possesses a βββα RING fold with an N‐terminal β3, flanked by an additional α2 helix [Fig. two(a,c)]. Thus, our findings showed a unique RING topology for DTX_RING whose overall structure was similar to the typical RING structure. The structure of DTX_RING was predicted to be a typical RING fold by the SMART database search, providing a higher E‐value of viii.0 × x−five. However, the bodily DTX_RING structure possesses a novel RING fold. In the BRCA1_RING domain, the groove formed by Ile26, Leu51, and Pro62 contributes to the E2‐bounden site [Fig. 2(b)].ix The α1 helix is an essential site for the specific E2‐binding in the ubiquitination reaction.x DTX_RING functions as an E3 and has specific activities for poly‐ubiquitin chain elongation with cooperating E2 UbcH5.22 DTX_RING possesses a putative E2‐bounden site (Ile16, Met59, and Pro72), similar to the hydrophobic shallow cleft of BRCA1_RING. Ile16, Met59, and Pro72 are well conserved amongst the homologs [Fig. 1(a)]. The β3 strand of DTX_RING is unlikely to exist involved in functional E2‐binding. A sequence analysis past the Generative REgularized ModeLs of proteINs software33 provided the highly conserved residues for DTX_RING [Fig. 1(a)]. This finding indicated that Ile16, Met59, and Pro72 are the essential residues among the homologs in terms of their conservation and coevolution patterns in the DTX family.33 Thus, it is tempting to speculate that DTX_RING promotes the evolution of the ubiquitination via its hydrophobic groove for E2‐bounden.
Cross‐brace structure for engineering science an ARF
Cross‐braced structures are used to create ARFs, which permit the detection of E2 activities during ubiquitination without substrates. Information technology has been demonstrated that the highly sensitive detection of E2 activities is applicable to the cess of the pathological conditions of acute promyelocytic leukemia‐derived NB4 cells.xix Grafting the helix region (agile site) of the Band finger onto other cantankerous‐brace zinc motifs leads to the creation of ARFs. The present structural study revealed the position and the range of the active site of DTX_RING at the atomic level. This report provides useful information for technology a novel ARF as an artificial E3 ligase of ubiquitination. Nevertheless, more than precise analyses, such equally ubiquitination assays, will be needed to extend the pattern methods of ARFs.
In conclusion, this study is the commencement structural report of the Ring finger domain from the man DTX protein. It was found that the Band finger adopts a novel fold in a cantankerous‐braced fashion. The nowadays structural analysis revealed that DTX_RING may be applied to the creation of an ARF with a specific E2 binding action.
Methods
Peptide synthesis
The sequence of DTX_RING was identified using a SMART database search. The mutant (I36L) was designed to ameliorate water solubility. The xiiiC and 15Northward‐labeled mutant peptide was uniformly synthesized with C‐last amidation by the standard F‐moc solid‐phase method.25 Chemicals for peptide assembly, including amide resin, were obtained from Shimadzu Corp. (Kyoto, Nippon) and Sigma‐Aldrich Co. LLC (St. Louis, MO). After cleavage with trifluoroacetic acid, the peptide was purified by reverse‐phase high operation liquid chromatography (HPLC) equipped with a Shim‐pack C18 column (Shimadzu Corp.). The purity of the peptides was >98%, and the molecular mass was confirmed by matrix‐assisted laser desorption/ionization time‐of‐flying (MALDI‐TOF) MS on a Shimadzu AXIMA‐TOF2. The DTX_RING peptide was dissolved in 0.36 mL of viii One thousand guanidine–HCl, and so it was dialyzed against the degassed solution [20 mM Tris–HCl (pH half-dozen.8), 50 mM NaCl, 1 mM dithiothreitol, and 50 μM ZnCl2] overnight at 4°C using a Slide‐A‐Lyzer Dialysis Cassette (PIERCE).xvi
Stoichiometry of released zinc ions
The concentration of DTX_RING peptide was spectrophotometrically determined by the Bradford method, and bovine serum albumin was used as the standard. Zinc ions released from PHMB were estimated with the metallochromic indicator PAR. Absorbance values (A) for the Zn2+–PAR2 circuitous at 20°C were acquired at 500 nm. The concentration of zinc ions released from DTX_RING was calculated past the equation A = εcl, where the molar absorptivity (ε) is vi.6 × 104 M−1 cm−1, the cell length (l) is i.0 cm, and c represents the molecular concentration. The zinc:poly peptide stoichiometry was estimated based on the corporeality of released zinc ions and the DTX_RING peptide.26, 27
NMR spectroscopy
For NMR assay, the DTX_RING peptide (1 mM) was dissolved in 1H2O/twoH2O (9:ane) in 20 mM Tris‐d 11‐HCl buffer (pH six.nine) (C/D/N Isotopes Inc., Canada) containing 50 mM NaCl, ane mM 1,4‐dl‐dithiothreitol‐d 10, and 50 μM ZnCl2.34, 35 All NMR experiments were carried out at 20°C by the WATERGATE pulse sequence on a Bruker AVANCE 500 MHz equipped with a cryogenic probe and an AVANCE 800 spectrometer.36 The peptide backbone resonance assignments were performed using standard triple resonance experiments.28 Resonances of side chains were assigned using HBHACONH, HCCCONNH, CCCONNH, HCCH‐TOCSY, and HCCH‐COSY spectra. The 3D 15N‐ and 13C‐edited NOESY spectra were recorded with mixing times of 100 ms. Assignments of the effluvious ring were achieved by HCCH‐COSY and thirteenC‐edited NOESY spectra. The spectra were processed using the NMRPipe programme,37 and the NMRView program was used for optimal visualization of the obtained spectra.38
Structure calculation
Tiptop lists for the 15N‐ and 13C‐edited NOESY spectra were made by the height picking and integration functions of NMRView. Assignments of the methyl groups of Val and Leu were stereospecifically assigned when their NOE patterns were distinguished from each other. To build a tetrahedral zinc coordination, distance limits (Zn–Southwardγ, Zn–Cβ, and Sγ–Sγ for Cys and Zn‐Northδ1 and Sγ‐Northwardδ1 for His) were added in the NOE tiptop lists with force constants of 500 kcal mol−1 Å−i.39, 40 Automated NOE cross‐peak assignments and structure calculations with torsion angle dynamics were performed with CYANA 2.one software.31 Dihedral angle restraints were generated using the program TALOS.41 After starting with 100 randomized conformers, structure calculations were carried out through the standard CYANA simulated annealing protocol with 10,000 torsion angle dynamics steps per conformer. Afterwards, the twenty conformers with the everyman CYANA target part values were subjected to energy minimization past the Smart Minimizer algorithm (Max steps 200, RMS gradient 0.01) from the Discovery Studio 2.one software (Accelrys Software Inc.).16 A Ramachandran plot of the obtained structures was validated by PROCHECK‐NMR.32 The Connolly surface of the everyman energy construction was calculated equally solvent contact areas that were traced out by a probe molecule (water) with a radius of 1.4 Å using the Discovery Studio 2.one software. The program MOLMOL42 was utilized to draw the resulting twenty conformers.
Protein Data Bank accession number
The atomic coordinates (code 6IR0) take been deposited in the Protein Data Banking concern, Research Collaboratory for Structural Bioinformatics.
Disharmonize of Interest
The authors declare that they accept no conflict of involvement.
Author Contributions
K.M. designed this study. K.M., Y.F., and Grand.South. performed the experiments. K.M. drafted the principal manuscript and prepared the figures. All authors reviewed the manuscript.
Acknowledgments
The authors would similar to thank Dr. Yoshitsugu Shiro, from the RIKEN SPring‐viii Center, for the NMR instrumentation. This research was supported past a Grant‐in‐Assist for Scientific Research (KAKENHI 17K05942).
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Humans Are In The Domain,
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