Amino acid signature enables proteins to recognize modified tRNA

Abstract

Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

Figure 1

Human modified and unmodified ASL^Lys3_UUU. (A) Human ASL^Lys3_UUU with all naturally occurring modifications (mcm⁵s²U₃₄, ms²t⁶A₃₇, and Ψ₃₉). The construct for this study was not modified at position 39. (B) The unmodified hASL^Lys3_UUU used in this study.

Figure 2

Search algorithm flow strategy. An initial peptide sequence is chosen (in this instance peptide P6). Random numbers were generated to determine whether to mutate one amino acid or not (“No” or “Yes”). If yes, then one amino acid from the sequence was randomly changed to an amino acid from the same residue category (Table 1). If no, then two amino acids from the sequence were randomly exchanged regardless of the residue category. The SCMF algorithm was then used to determine the lowest-energy rotamer combination. The MC algorithm was used to accept or reject the newly generated peptide sequence based on the calculation of binding free energy (ΔG¹_binding).

Figure 3

Self-consistent mean field (SCMF) procedure. A trial exchange between two amino acids is implemented. The conformational probability matrix P⁰ is set initially so that all possible rotamers at any one site have equal probabilities. The effective potential experienced by each rotamer at each site is calculated, and the Boltzmann law is used to determine new conformational probabilities of the rotamers for each amino acid and hence a new conformational probability matrix P¹. If the absolute error between P⁰ and P¹ is less than 10^–3, the rotamer combination with the highest conformational probability is selected from P¹ to repack the side chains. Otherwise, the conformational matrix P is updated by employing a self-consistent iteration until the absolute error falls below a certain tolerance.

Figure 4

Fluorescence of chemically synthesized peptides effected by modified and unmodified hASL^Lys3_UUU. An initial fluorescent signal (FS₀) of peptide alone (1.5 μM) was obtained. Then, a 2-fold excess of ASL was added to each peptide, and the fluorescent signal (FS₁) was monitored. The percent change (100·(FS₁/FS₀)) is graphed for each of the assayed peptides. Dark gray bars represent the percent change in fluorescence in the presence of the modified hASL^Lys3_UUU, and light gray bars represent the percent change in the presence of the unmodified hASL^Lys3_UUU. Sequences for P1–P38 are presented in Table 3

Figure 5

Peptide P27 binds the modified hASL^Lys3_UUU with high affinity and specificity. (A) The computed equilibrium binding structure of the modified hASL^Lys3_UUU bound by P27. The peptide backbone is in gold, and the ribose-phosphodiester backbone of the hASL^Lys3_UUU is colored in green. (B) Enlargement of the interaction demonstrating the specificity achieved in the binding of the two modifications by the amino acids R₁ (red), F₇ (light green), W₁₁ (light purple), and R₁₂ (dark green). The peptide backbone is in gold and the side chains in color. The modifications ms²t⁶A₃₇ (purple) and mcm⁵s²U₃₄ (blue) are bound by amino acids at the beginning, middle, and end of the peptide. The ribose-phosphodiester backbone of the hASL^Lys3_UUU is not shown. The table characterizes the contributions of different binding modes: ΔG_Binding, Gibbs free energy of binding; BE_w/o GBSUR, binding energy without GBSUR; VDW, van der Waals energy; ELE, electrostatic energy; EGB, polar solvation energy based on the generalized Born (implicit solvent) model; and GBSUR, nonpolar solvation energy, which is the product of the solvent-accessible surface area of the solute molecules and the interfacial tension between the solute and solvent. (C) Individual contributions of each amino acid to the VDW, ELE + EGB, and GBSUR. The amino acids are colored as in B. (D) Individual contributions of each nucleoside to the VDW, ELE + EGB, and GBSUR. The nucleosides engaged in the interaction with P27 are those of the anticodon loop, particularly the modified nucleosides at U₃₄ and A₃₇. The modified nucleosides are colored as in B.