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Identification of a Ca2+-binding domain in the rubella virus nonstructural protease - PubMed

Identification of a Ca2+-binding domain in the rubella virus nonstructural protease

Yubin Zhou et al. J Virol. 2007 Jul.

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 Ca(2+)-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 Ca(2+)-coordinating loop into a non-Ca(2+)-binding scaffold protein, CD2. The grafted EF-loop bound to Ca(2+) and its trivalent analogs Tb(3+) and La(3+) with K(d)s of 214, 47, and 14 microM, respectively. Mutations (D1210A and D1217A) of two of the potential Ca(2+)-coordinating ligands in the EF-loop led to the elimination of Tb(3+) binding. Inductive coupled plasma mass spectrometry was used to confirm the presence of Ca(2+) ([Ca(2+)]/[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 Ca(2+) binding induced local conformational changes and increased thermal stability (Delta T(m) = 4.1 degrees C). The infectivity of an RUB infectious cDNA clone containing the mutations D1210A/D1217A was decreased by approximately 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 degrees C, and the mutant NS protease was temperature sensitive at 39 degrees C, confirming that the Ca(2+)-binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions.

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Figures

FIG. 1.
FIG. 1.

Genomic organization of RUB and identification of a potential EF-hand Ca2+-binding motif. (A) Schematic representation of the RUB genome organization and domain locations. M, methyltransferase; G, proline-rich hinge; P, protease; H, helicase; R, RNA-dependent RNA polymerase; C, capsid protein; E1 and E2, envelope glycoproteins. The X domain represents a conserved domain with unknown function that is also found in NSP3 from alphaviruses and belongs to the Appr-1-processing enzyme family. The protease contains at least one Zn2+-binding site as well as a putative EF-hand Ca2+-binding motif (box with diagonal pattern). It has been proposed that the residues C1152 and H1273 constitute the catalytic dyad (stars). The minimal metal-binding domain RUBCa (aa 1143 to 1252) is bacterially expressed and used in this study. (B) Sequence alignment results of putative EF-hand Ca2+-binding motif RUBCa in the protease domain with calmodulin and calcyphosine. The motif remained conserved in both clades I (genotypes RV1a, 1B, 1C, 1D, and 1E) and II (genotypes RV2A, 2B, and 2c) of RUB. Boldface residues represent the potential Ca2+-coordinating residues. NCBI or GenBank accession numbers are the following: CaM_EF1 and CaM_EF2 (calmodulin, P62158); CAYP_hum and CAYP_rab (calcyphosine from human, Q13938; rabbit, P41150). The genomic sequence of the eight different genotypes of RUB was sequenced in our laboratory (unpublished data). (C) Homology modeling of RUB NS protease. The leader protease of FMDV (pdb code 1qmy, chain A), a papain-like cysteine protease with a high-resolution structure available, is chosen as the template for homology modeling. The active site consists of C1152 and H1273 (shown as sticks). The predicted EF-hand Ca2+-binding motif, located at the opposite side, is highlighted in blue with the calcium ion shown as a cyan sphere. The cysteine (green) and tryptophan (magenta) residues in the protease domain are shown as sticks. The cleavage site G1301-G1302 is highlighted in black.

FIG. 2.
FIG. 2.

Grafting the predicted EF-hand Ca2+-binding loop into CD2.D1 and formation of metal-protein complex. (A) Model structure of the engineered protein CD2.RUBCa. The Ca2+-binding loop (black) from the RUB NS protease (aa 1206 to 1217), with two glycines on the left and three glycines on the right, rendering flexibility, is grafted to the loop which connects strands C′′ and D. The model structure was built using the automated comparative protein-modeling server SWISS-MODEL. Aromatic residues Trp (black) are shown as sticks. The Ca2+ ion is shown as a sphere. Intrinsic Trp fluorescence emission spectra (B) and far-UV CD spectra (C) of CD2 (open circle) and engineered protein CD2.RUBCa (closed circle) are compared to examine the perturbation of insertion on the scaffold protein CD2.D1. Buffers consists of 10 mM Tris, 10 mM KCl, pH 7.4. (D) Electrospray mass spectra of CD2 with a grafted EF-loop from RUB NS protease (P) in the presence of a fivefold molar excess of TbCl3 (+156).

FIG. 3.
FIG. 3.

Obtaining metal-binding affinity using aromatic residue-sensitized Tb3+-FRET and 1D 1H NMR. (A) Tb3+ fluorescence enhancement at 545 nm due to resonance energy transfer (excited at 282 nm) as a function of protein concentrations of CD2 (open diamond), CD2.RUBCa (closed circle), and its mutant D5A/D12A (open circle). (B) Tb3+ titration of CD2.RUBCa. Normalized fluorescence intensity was plotted as a function of the Tb3+ concentration. The inset shows the Ca2+ competition titration curve of CD2.RUBCa (1.5 μM) preincubated with 40 μM Tb3+ in 20 mM PIPES, 10 mM KCl, pH 6.8. (C) La3+ titration of CD2.RUBCa monitored by 1D 1H NMR. Some resonances (arrows) at amide regions of 1D 1H NMR spectra of CD2.RUBCa (0.2 mM) shift with the increased concentrations of La3+ (from bottom to top: 0, 39.2, 113.2, 214.3, 442.2, 360.7, 605.1, 929.1, and 1,411.2 μM, respectively) in 20 mM PIPES, 10 mM KCl, at pH 7.4. (D) The chemical shift change at different resonant regions as a function of the concentration of La3+. ppm, parts per million.

FIG. 4.
FIG. 4.

Metal selectivity of the engineered protein CD2.RUBCa. (A) Metal competition assay. The addition of 1.5 μM CD2.RUBCa to free Tb3+ (40 μM) solution resulted in an increase of fluorescence intensity at 545 nm by over 20-fold due to the binding of Tb3+ to the protein and the resultant FRET. K+ (100 mM), 10 mM Mg2+, 1 mM Ca2+, and 0.1 mM La3+ were subsequently added to individually prepared solutions containing 40 μM Tb3+ and 1.5 μM CD2.RUBCa. (B) Amide region of 1D 1H NMR spectrum of CD2.RUBCa with sequential addition of 100 mM K+, 10 mM Mg2+, and 1 mM Ca2+. Resonances exhibiting changes are indicated by arrows. ppm, parts per million.

FIG. 5.
FIG. 5.

Metal ion titration of the minimal metal-binding domain RUBCa monitored by aromatic residue-sensitized Tb3+ fluorescence (A) and intrinsic Trp fluorescence (B). (A) Normalized Tb3+ fluorescence spectra of RUBCa with increasing concentrations of Tb3+ (from bottom to top: 0, 1.0, 4.0, 9.9, 14.8, 19.6, and 14.4 μM, respectively). The inset shows the Tb3+ fluorescence enhancement at 545 nm due to energy transfer as a function of the concentration of Tb3+. (B) Intrinsic Trp fluorescence emission spectra of RUBCa (2.5 μM) with increasing concentrations of Ca2+ (from top to bottom: 0, 49.8, 291.3, 566.0, 740.7, and 909.1 μM, respectively). The inset shows the intrinsic Trp fluorescence intensity plotted as a function of the concentration of Ca2+. An average dissociation constant of 316 μM was obtained by assuming a 1:1 binding model. The excitation wavelength was set at 282 nm. All the buffers used in metal titration consist of 20 mM PIPES, 10 mM KCl, pH 6.8.

FIG. 6.
FIG. 6.

Ca2+-induced conformational changes and thermal unfolding of the putative Ca2+-binding domain RUBCa. (A) Far-UV CD spectra of RUBCa with 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle) in 10 mM Tris-HCl, 100 mM KCl. The inset shows normalized CD signal at 222 nm plotted as a function of increasing temperature (5 to 90°C) in the presence of 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle). (B) Fluorescence emission spectra of 40 μM ANS (open square) and ANS-RUBCa complex with 1 mM EGTA (open circle) or 1 mM Ca2+ (closed circle). The excitation wavelength was set at 390 nm. The buffer consisted of 10 mM Tris-HCl, 10 mM KCl (pH 7.4).

FIG. 7.
FIG. 7.

Effects of mutations of the potential Ca2+ coordination ligands on RUB replication. Transcripts from the wt infectious cDNA clone, Robo502, or Robo502AA containing the D1210A and D1217A mutations in the Ca2+-binding loop were used to transfect Vero cells. Culture fluid from the transfection plate (P0) was harvested on day 7 posttransfection and passaged twice in Vero cells (P1 and P2). The virus titer in the P0, P1, and P2 culture fluids was determined by plaque assay in triplicate (A). Open bar, Robo502; black bar, Robo502AA. (B) Representative plaques at each passage. (C) To check for the generation of revertants in the Robo502AA population, four plaques were picked from terminal plaque assay dilution plates from P1 (left) culture fluid, and after one round of amplification in Vero cells, the sequence of the metal-binding domain in the NSP was determined, as shown in comparison to the wt sequence.

FIG. 8.
FIG. 8.

Replicon RNA synthesis and P200 cleavage in transfected Vero cells. Vero cells were transfected with transcripts from RUBrep/GFP or RUBrepAA/GFP containing mutations D5A and D12A (A) or from RUBrep-HA/GFP or RUBrepAA-HA/GFP, which express an HA-epitope-tagged P150 (B). (A) Total cell RNA was extracted 1 to 4 days posttransfection, and replicon plus-strand RNA species (G, genomic; SG, subgenomic) were resolved by Northern blotting following agarose gel electrophoresis. (B) Transfections were performed at 35°C or 39°C. Six hours posttransfection, cells were lysed and the P200 precursor and P150 product were resolved by Western blotting probed with anti-HA antibodies following SDS-PAGE (the other product, P90, does not appear because it does not contain the HA epitope). Cells transfected with a replicon containing a C1152S catalytic site mutation (RUBrep-NS*-HA/GFP) that cannot mediate P200 cleavage served as an uncleaved control.

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