A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication

The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn2+-binding ligands. This minidomain bound to Zn2+ with a stoichiometry of ≈0.7 and an apparent dissociation constant of <500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Zn2+ binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cys1175, Cys1178, Cys1225 and Cys1227 were required for the binding of Zn2+. Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)48CXC Zn2+-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication.

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Identification of a Ca2+-Binding Domain in the Rubella Virus Nonstructural Protease

Journal of Virology ◽

10.1128/jvi.00605-07 ◽

2007 ◽

Vol 81 (14) ◽

pp. 7517-7528 ◽

Cited By ~ 18

Author(s):

Yubin Zhou ◽

Wen-Pin Tzeng ◽

Wei Yang ◽

Yumei Zhou ◽

Yiming Ye ◽

...

Keyword(s):

Conformational Changes ◽

Rubella Virus ◽

Metal Binding ◽

Nonstructural Protein ◽

Binding Domain ◽

Binding Motif ◽

Temperature Sensitive ◽

Reading Frame ◽

Metal Binding Domain ◽

Ef Hand

ABSTRACT The rubella virus (RUB) nonstructural protein (NS) open reading frame (ORF) encodes a polypeptide precursor that is proteolytically self cleaved into two replicase components involved in viral RNA replication. A putative EF-hand Ca2+-binding motif that was conserved across different genotypes of RUB was predicted within the nonstructural protease that cleaves the precursor by using bioinformatics tools. To probe the metal-binding properties of this motif, we used an established grafting approach and engineered the 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2. The grafted EF-loop bound to Ca2+ and its trivalent analogs Tb3+ and La3+ with K d s of 214, 47, and 14 μM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca2+-coordinating ligands in the EF-loop led to the elimination of Tb3+ binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca2+ ([Ca2+]/[protein] = 0.7 ± 0.2) in an NS protease minimal metal-binding domain, RUBCa, that spans the EF-hand motif. Conformational studies on RUBCa revealed that Ca2+ binding induced local conformational changes and increased thermal stability (ΔT m = 4.1°C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by ∼20-fold in comparison to the wild-type (wt) clone, and these mutations rapidly reverted to the wt sequence. The NS protease containing these mutations was less efficient at precursor cleavage than the wt NS protease at 35°C, and the mutant NS protease was temperature sensitive at 39°C, confirming that the Ca2+-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.

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Copper chaperone Atox1 interacts with the metal-binding domain of Wilson's disease protein in cisplatin detoxification

Biochemical Journal ◽

10.1042/bj20121656 ◽

2013 ◽

Vol 454 (1) ◽

pp. 147-156 ◽

Cited By ~ 40

Author(s):

Nataliya V. Dolgova ◽

Sergiy Nokhrin ◽

Corey H. Yu ◽

Graham N. George ◽

Oleg Y. Dmitriev

Keyword(s):

Metal Binding ◽

Wilson’S Disease ◽

Wilson's Disease ◽

Binding Domain ◽

Binding Motif ◽

Copper Chaperone ◽

Copper Binding ◽

Copper Transporters ◽

Metal Binding Domain ◽

Disease Protein

Human copper transporters ATP7B (Wilson's disease protein) and ATP7A (Menkes' disease protein) have been implicated in tumour resistance to cisplatin, a widely used anticancer drug. Cisplatin binds to the copper-binding sites in the N-terminal domain of ATP7B, and this binding may be an essential step of cisplatin detoxification involving copper ATPases. In the present study, we demonstrate that cisplatin and a related platinum drug carboplatin produce the same adduct following reaction with MBD2 [metal-binding domain (repeat) 2], where platinum is bound to the side chains of the cysteine residues in the CxxC copper-binding motif. This suggests the same mechanism for detoxification of both drugs by ATP7B. Platinum can also be transferred to MBD2 from copper chaperone Atox1, which was shown previously to bind cisplatin. Binding of the free cisplatin and reaction with the cisplatin-loaded Atox1 produce the same protein-bound platinum intermediate. Transfer of platinum along the copper-transport pathways in the cell may serve as a mechanism of drug delivery to its target in the cell nucleus, and explain tumour-cell resistance to cisplatin associated with the overexpression of copper transporters ATP7B and ATP7A.

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The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

10.1101/389460 ◽

2018 ◽

Author(s):

Mohammed Jamshad ◽

Timothy J. Knowles ◽

Scott A. White ◽

Douglas G. Ward ◽

Fiyaz Mohammed ◽

...

Keyword(s):

Conformational Changes ◽

Metal Binding ◽

Cytoplasmic Membrane ◽

Protein Recognition ◽

X Ray ◽

Ribosome Binding ◽

X Ray Crystallography ◽

Substrate Protein ◽

Metal Binding Domain

AbstractIn bacteria, the translocation of a subset of proteins across the cytoplasmic membrane by the Sec machinery requires SecA. Although SecA can recognise nascent polypeptides, the mechanism of cotranslational substrate protein recognition is not known. Here, we investigated the role of the C-terminal tail (CTT) of SecA, which consists of a flexible linker (FLD) and a small metal-binding domain (MBD), in its interaction with nascent polypeptides. Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the entire CTT or the MBD alone had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function. Autophotocrosslinking, mass spectrometry, x-ray crystallography and small-angle x-ray scattering experiments provided insight into the CTT-mediated conformational changes in SecA. Finally, photocrosslinking experiments indicated that binding of SecA to substrate protein affected its interaction with the ribosome. Taken together, our results suggest a mechanism for substrate protein recognition.Impact StatementSecA is an evolutionarily conserved ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. We investigated how SecA recognises its substrate proteins at the ribosome as they are still being synthesised (i.e. cotranslationally).

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Structural insights into how GTP-dependent conformational changes in a metallochaperone UreG facilitate urease maturation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712658114 ◽

2017 ◽

Vol 114 (51) ◽

pp. E10890-E10898 ◽

Cited By ~ 18

Author(s):

Man Hon Yuen ◽

Yu Hang Fong ◽

Yap Shing Nim ◽

Pak Ho Lau ◽

Kam-Bo Wong

Keyword(s):

Conformational Changes ◽

Metal Binding ◽

Sole Source ◽

Planar Geometry ◽

Binding Motif ◽

Nickel Ion ◽

Gtp Hydrolysis ◽

Nickel Ions ◽

Gtp Binding

The ability of metallochaperones to allosterically regulate the binding/release of metal ions and to switch protein-binding partners along the metal delivery pathway is essential to the metallation of the metalloenzymes. Urease, catalyzing the hydrolysis of urea into ammonia and carbon dioxide, contains two nickel ions bound by a carbamylated lysine in its active site. Delivery of nickel ions for urease maturation is dependent on GTP hydrolysis and is assisted by four urease accessory proteins UreE, UreF, UreG, and UreH(UreD). Here, we determined the crystal structure of the UreG dimer from Klebsiella pneumoniae in complex with nickel and GMPPNP, a nonhydrolyzable analog of GTP. Comparison with the structure of the GDP-bound Helicobacter pylori UreG (HpUreG) in the UreG2F2H2 complex reveals large conformational changes in the G2 region and residues near the 66CPH68 metal-binding motif. Upon GTP binding, the side chains of Cys66 and His68 from each of the UreG protomers rotate toward each other to coordinate a nickel ion in a square-planar geometry. Mutagenesis studies on HpUreG support the conformational changes induced by GTP binding as essential to dimerization of UreG, GTPase activity, in vitro urease activation, and the switching of UreG from the UreG2F2H2 complex to form the UreE2G2 complex with the UreE dimer. The nickel-charged UreE dimer, providing the sole source of nickel, and the UreG2F2H2 complex could activate urease in vitro in the presence of GTP. Based on our results, we propose a mechanism of how conformational changes of UreG during the GTP hydrolysis/binding cycle facilitate urease maturation.

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Conformational changes in CLIP-170 regulate its binding to microtubules and dynactin localization

The Journal of Cell Biology ◽

10.1083/jcb.200402082 ◽

2004 ◽

Vol 166 (7) ◽

pp. 1003-1014 ◽

Cited By ~ 124

Author(s):

Gideon Lansbergen ◽

Yulia Komarova ◽

Mauro Modesti ◽

Claire Wyman ◽

Casper C. Hoogenraad ◽

...

Keyword(s):

Conformational Changes ◽

Metal Binding ◽

Intramolecular Interaction ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Scanning Force Microscopy ◽

Binding Motif ◽

Force Microscopy ◽

Scanning Force ◽

Metal Binding Motif

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

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Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite

Journal of Bacteriology ◽

10.1128/jb.186.6.1861-1868.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1861-1868 ◽

Cited By ~ 14

Author(s):

Lingyun Song ◽

Jonathan Caguiat ◽

Zhongrui Li ◽

Jacob Shokes ◽

Robert A. Scott ◽

...

Keyword(s):

Transcriptional Activation ◽

Metal Binding ◽

Coiled Coil ◽

Mercury Resistance ◽

Binding Motif ◽

Single Chain ◽

Metal Binding Domain ◽

Α Helix

ABSTRACT The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

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Characterization of the Zinc Binding Activity of the Rubella Virus Nonstructural Protease

Journal of Virology ◽

10.1128/jvi.74.13.5949-5956.2000 ◽

2000 ◽

Vol 74 (13) ◽

pp. 5949-5956 ◽

Cited By ~ 23

Author(s):

Xin Liu ◽

Jenny Yang ◽

A. Mohamad Ghazi ◽

Teryl K. Frey

Keyword(s):

Fusion Protein ◽

Rubella Virus ◽

Protease Activity ◽

Cleavage Site ◽

Metal Ion ◽

Catalytic Site ◽

Zinc Binding ◽

Binding Motif ◽

Protease Domain ◽

Binding Studies

ABSTRACT The rubella virus (RUB) nonstructural (NS) protein (NSP) ORF encodes a protease that cleaves the NSP precursor (240 kDa) at a single site to produce two products. A cleavage site mutation was introduced into a RUB infectious cDNA clone and found to be lethal, demonstrating that cleavage of the NSP precursor is necessary for RUB replication. Based on computer alignments, the RUB NS protease was predicted to be a papain-like cysteine protease (PCP) with the residues Cys1152 and His1273 as the catalytic dyad; however, the RUB NS protease was recently found to require divalent cations such as Zn, Co, and Cd for activity (X. Liu, S. L. Ropp, R. J. Jackson, and T. K. Frey, J. Virol. 72:4463–4466, 1998). To analyze the function of metal cation binding in protease activity, Zn binding studies were performed using the minimal NS protease domain within the NSP ORF. When expressed as a maltose binding protein (MBP) fusion protein by bacteria, the NS protease exhibited activity both in the bacteria and in vitro following purification when denatured and refolded in the presence of Zn. Atomic absorption analysis detected 1.6 mol of Zn bound per mol of protein refolded in this manner. Expression of individual domains within the protease as MBP fusions and analysis by a Zn65 binding assay revealed two Zn binding domains: one located at a predicted metal binding motif beginning at Cys1175 and the other one close to the cleavage site. Mutagenesis studies showed that Cys1175 and Cys1178 in the first domain and Cys1227 and His1273, the His in the predicted catalytic site, in the second domain are essential for zinc binding. All of these residues are also necessary for the protease activity, as were several other Cys residues not involved in Zn binding. Far-UV circular dichroism (CD) analysis of the MBP-NS protease fusion protein showed that the protease domain contained a large amount of alpha-helical structure, which is consistent with the results of secondary-structural prediction. Both far-UV–CD and fluorescence studies suggested that Zn did not exert a major effect on the overall structure of the fusion protein. Finally, protease inhibitor assays found that the protease activity can be blocked by both metal ion chelators and the metalloprotease inhibitor captopril. In conjunction with the finding that the previously predicted catalytic site, His1273, is essential for zinc binding, this suggests that the RUB NS protease is actually a novel virus metalloprotease rather than a PCP.

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The C-terminal tail of the bacterial translocation ATPase SecA modulates its activity

eLife ◽

10.7554/elife.48385 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Mohammed Jamshad ◽

Timothy J Knowles ◽

Scott A White ◽

Douglas G Ward ◽

Fiyaz Mohammed ◽

...

Keyword(s):

Conformational Changes ◽

Metal Binding ◽

Ribosome Binding ◽

Substrate Protein ◽

Metal Binding Domain ◽

Opposing Effects ◽

Substrate Proteins ◽

Binding Substrate

In bacteria, the translocation of proteins across the cytoplasmic membrane by the Sec machinery requires the ATPase SecA. SecA binds ribosomes and recognises nascent substrate proteins, but the molecular mechanism of nascent substrate recognition is unknown. We investigated the role of the C-terminal tail (CTT) of SecA in nascent polypeptide recognition. The CTT consists of a flexible linker (FLD) and a small metal-binding domain (MBD). Phylogenetic analysis and ribosome binding experiments indicated that the MBD interacts with 70S ribosomes. Disruption of the MBD only or the entire CTT had opposing effects on ribosome binding, substrate-protein binding, ATPase activity and in vivo function, suggesting that the CTT influences the conformation of SecA. Site-specific crosslinking indicated that F399 in SecA contacts ribosomal protein uL29, and binding to nascent chains disrupts this interaction. Structural studies provided insight into the CTT-mediated conformational changes in SecA. Our results suggest a mechanism for nascent substrate protein recognition.

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Manipulating the Conformation of 3,2′:6′,3″-Terpyridine in [Cu2(μ-OAc)4(3,2′:6′,3″-tpy)]n 1D-Polymers

Chemistry ◽

10.3390/chemistry3010015 ◽

2021 ◽

Vol 3 (1) ◽

pp. 182-198

Author(s):

Dalila Rocco ◽

Samantha Novak ◽

Alessandro Prescimone ◽

Edwin C. Constable ◽

Catherine E. Housecroft

Keyword(s):

Single Crystal ◽

Crystal Structures ◽

Metal Binding ◽

Polymer Chains ◽

X Ray Diffraction ◽

Face To Face ◽

Single Crystal Structures ◽

Π Stacking ◽

Metal Binding Domain ◽

Packing Interactions

We report the preparation and characterization of 4′-([1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (1), 4′-(4′-fluoro-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (2), 4′-(4′-chloro-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (3), 4′-(4′-bromo-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (4), and 4′-(4′-methyl-[1,1′-biphenyl]-4-yl)-3,2′:6′,3″-terpyridine (5), and their reactions with copper(II) acetate. Single-crystal structures of the [Cu2(μ-OAc)4L]n 1D-coordination polymers with L = 1–5 have been determined, and powder X-ray diffraction confirms that the single crystal structures are representative of the bulk samples. [Cu2(μ-OAc)4(1)]n and [Cu2(μ-OAc)4(2)]n are isostructural, and zigzag polymer chains are present which engage in π-stacking interactions between [1,1′-biphenyl]pyridine units. 1D-chains nest into one another to give 2D-sheets; replacing the peripheral H in 1 by an F substituent in 2 has no effect on the solid-state structure, indicating that bifurcated contacts (H...H for 1 or H...F for 2) are only secondary packing interactions. Upon going from [Cu2(μ-OAc)4(1)]n and [Cu2(μ-OAc)4(2)]n to [Cu2(μ-OAc)4(3)]n, [Cu2(μ-OAc)4(4)]n, and [Cu2(μ-OAc)4(5)]n·nMeOH, the increased steric demands of the Cl, Br, or Me substituent induces a switch in the conformation of the 3,2′:6′,3″-tpy metal-binding domain, and a concomitant change in dominant packing interactions to py–py and py–biphenyl face-to-face π-stacking. The study underlines how the 3,2′:6′,3″-tpy domain can adapt to different steric demands of substituents through its conformational flexibility.

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