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Abstract
Human cytomegalovirus encodes an unusual protein kinase UL97 which can phosphorylate exogenous substrates, including histone H2B and nucleoside analogs such as ganciclovir. The previous result interestingly showed that the peptides phosphorylated by UL97 have K/R at the 5 positions (P+5) downstream from the pSer. To confirm the importance of the basic residue in the position, we used two peptide libraries, 4S4K (MAXXXXSXXXXKXANNN) and 4S6N (MAXXXXSXXXXXXNNN). The activity of phosphorylation by UL97 was higher in the peptide library 4S4K than 4S6N, suggesting the importance of basic residue at P+5 position. The screening with a peptide library 4S4K showed slight tendencies for N in the P+1 and P+2, M in the P+2, K in the P+4 and P+6 positions and several amino acids in the other positions. This result will give information to develop an optimal peptide for screening a novel UL97 inhibitor.
References
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Figure 1.
Time courses and relative activities of two peptide libraries by UL97. Two peptide libraries, 4S6N (Met-Ala-X-X-X-X-Ser-X-X-X-X-X-X-A-N-N-N) (❍) and 4S4K (Met-Ala-X-X-X-X-Ser-X-X-X-X-K-X-A-N-N-N) (), were phosphorylated by GST-UL97 in the presence of ATP, with aliquots at the indicated time. After separating the [γ-32P] ATP from the peptides on a DEAE-sephacel column, the radioactivities were measured by using liquid scintillation counter (LSC).
Figure 2.
Quantitative separation of non-phosphorylated and phosphorylated peptides. A peptide library (Met-Ala-X-X-X-X-Ser-X-X-X-X-K-X-A-N-N-N) was phosphorylated by GST-UL97. After separating the [γ-32P] ATP from the peptides on a DEAE-sephacel column, the peptide mixture was loaded on a column of ferric-IDA beads. The column was eluted with 3 ml of Buffer A (50 mM MES, 1 M NaCl, pH 5.5) (Arrow 1), 2 ml of water (Arrow 2), 2 ml of 0.1% (NH4)OAc pH 9.5 (Arrow 3) and 2 ml of 0.1% (NH4)OAc pH 11.5 (Arrow 4), 4 ml of 100 mM EDTA, pH 8.0 (Arrow 5); Solutions were added at the positions indicated by the arrows. All elutions were at 0.5 ml/min and 1 ml fractions were collected. The radioactivities at each fraction were expressed as c.p.m.
Figure 3.
Substrate specificity of UL97 detected by the degenerate peptide library. The phosphopeptides produced by phosphorylating the serine-kinase substrate library with UL97 were sequenced. Each panel indicates the relative abundance of the 15 amino acids at a given cycle of sequencing. For example, (a) is cycle 3, the first degenerate position in the library mixture. Cycle 7 (not shown) is the site of phosphorylation (phosphoserine) and cycle 12 (not shown) is the site of lysine. Thus, (a-d) indicate amino acid preferences at positions –4, –3, –2, –1 amino terminal to the phosphorylation site, respectively, and (e-i) indicate, respectively preferences at positons +1, +2, +3, +4, +6 carboxy-terminal to the phosphorylation site. Value at y-axis indicates the relative amount of each amino acid at each cycle. The single-letter amino-acid code is used: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; Y, Tyr.
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