Topology of the disulfide bonds in the antiviral lectin scytovirin

Abstract

The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the "cystine clusters" [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584). Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require considerable precautions when being fragmented for the purpose of disulfide assignment.

Figure 1

Amino acid sequence, putative location of disulfides, and theoretical tryptic digests of scytovirin. (A) Amino acid sequence, with the two domains shown in separate lines. The three possible topologies of disulfide pairing are marked by thin lines. The positively charged residues that determine the location of tryptic cleavage of SVN are highlighted. Mode A: The topology corresponding to previous disulfide assignment by mass spectrometry and NMR., Mode B: The topology consistent with the crystallographic results. Mode C: The remaining possible topology, not reported to date. (B) Peptides resulting from tryptic digestion of SVN. Peptide numbers (in parentheses) correspond to Tables I and II. Cystines are colored red and disulfide bonds are shown with lines. Fragments that are not involved in reshuffling at pH 8.0 are highlighted in gray. All other fragments are highlighted in green, blue, and yellow. The reshuffled pattern at pH 8.0 is shown in the bottom two panels. The theoretical monoisotopic masses are listed for each identified peptide.

Figure 2

Peptide maps of SVN. (A) RP-HPLC peptide map of SVN at pH 6. The peptides absorbing at 214 nm were manually collected and analyzed by MS and N-terminally sequenced (see Material and Methods for conditions). The structures of the peptides in each fraction are indicated (see Table I for more details). Cleaved peptide bonds are spaced. (B) RP-HPLC peptide map of SVN at pH 8 obtained under chromatographic conditions optimized to separate the newly emerging species of m/z = 1318.6 (fraction 3) and m/z = 1553.6 (fraction 7). Note that tryptic cleavage at pH 6 was much slower compared to pH 8 and that the pH 6 peptide map represents the ∼50% time point of total SVN cleavage. Fraction 6 contains partially separated QCD derivative of a fragment isolated in fraction 4. Heterodimers of m/z = 2511.0 and m/z = 2719.1 were present as minor components in the chromatogram. Insets indicate the full scale of absorbance. Peptide numbers correspond to Figure 1(B) and Tables I and II.

Figure 3

HPLC analysis of SVN and its fragments. (A) Synthetic scytovirin (SD1A: Cys20–Cys26/Cys32–Cys38 in blue; SD1B: Cys20–Cys32/Cys26–Cys38 in red) and recombinant rSD1 (in green) analyzed at 40°C by reversed-phase HPLC. (B) Trypsin-digested rSD1 (green), synthetic SD1A (blue), and synthetic SD1B (red) analyzed by LC-MS. The determined molecular masses (Da) of major fragments are shown, and the 4943.1 Da peak is intact SD1B. The y-axis value corresponds to percent total ion count relative to the most abundant peak. See Materials and Methods for chromatographic and mass spectrometric conditions.

Figure 4

Monitoring of disulfide interchange of heterotrimeric cluster of m/z = 2529.0 by RP-HPLC peptide mapping. (A) Time course of tryptic digestion of rSD1 at pH 8 documenting appearance of a new species of m/z = 1318.6. Asterisk designated peak appeared with time but did not yield any peptide signal on MS. (B) pH dependence of tryptic digestion (3 h, 37°C) at pH 6, pH 8, and pH 10. Heterodimer of m/z = 2511.0 was not present in the chromatograms.

Figure 5

Conversion of purified heterotrimeric tryptic peptide clusters of SVN monitored by MALDI-TOF MS. Purified peptides were incubated at pH 8 and aliquots of the reaction mixture were desalted and analyzed (see Materials and Methods for details). (A) Representative mass spectra of purified fragment of m/z = 2737.1 at τ = 0 and τ = 16 h at pH 8. The m/z = 2720.105 species is the pGu derivative of the m/z = 2737.136 species. (B) Stability plot for m/z = 2529.0. (C) Stability plot for m/z = 2737.1.

Figure 6

Stability plot for purified heterodimeric and heterotrimeric species obtained from SVN domain 1 (A) and SVN domain 2 (B). The conditions were the same as described in legend for Figure 5.