Differential modulation of binding loop flexibility and stability by Arg(50) and Arg(52) in Cucurbita maxima trypsin inhibitor-V deduced by trypsin-catalyzed hydrolysis and NMR spectroscopy

The side chains of Arg(50) and Arg(52) in Cucurbita maxima trypsin inhibitor-V (CMTI-V) anchor the binding loop to the scaffold region [Cai, M., Gong, Y., Kao, J. L.-F., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The consequences of these hydrogen-bonding and electrostatic interactions on the conformational flexibility and stability of the binding loop were evaluated by trypsin-catalyzed hydrolysis of CMTI-V mutants, in which each of the two arginines was individually replaced with Ala, Lys, or Gln by genetic engineering methods. All the mutants exhibited significantly increased vulnerability to the protease attack at many sites, including the reactive-site (Lys(44)-Asp(45) peptide bond), with the R50 mutants showing much more pronounced effects than the R52 counterparts. For CMTI-V and the mutants studied, a qualitative correlation was inferred between binding loop flexibility and retention time on a reverse-phase high-pressure liquid chromatography C-18 column. The R50 mutants were found to be more flexible than the corresponding R52 versions. These results demonstrate that Arg(50) contributes more to the stability and function of CMTI-V. The differing strengths of the hydrogen bonds made by Arg(50) and Arg(52) were characterized by determining the internal dynamics of their side chains at pH 5.0 and 2.5: N-15 NMR longitudinal and transverse relaxation rates and N-15-H-1 nuclear Overhauser effect (NOE) enhancements were measured for the main-chain and side-chain NH groups in N-15-labeled recombinant CMTI-V (rCMTI-V) and the model-free parameters [Lipari, G., & Szabo, A. (1982) J. Am. Chern. Sec. 104, 4546-4559; 4559-4570] were calculated. At both pH 5.0 and 2.5, the arginines at positions 26, 47, 58, and 66 are found to be highly mobile, as the calculated general order parameters, S-2 values, of their NepsilonH groups fall in the range 0.03-0.18. The corresponding values for Arg(50) and Arg(52) are 0.73 and 0.63, respectively, at pH 5.0, thus confirming that the two arginines are rigid and hydrogen-bonded. At pH 2.5, these hydrogen bonds are still retained with Arg(50) appearing to be more restrained (S-2 = 0.71) than Arg(52) (S-2 = 0.56). This is consistent with a greater contribution by Arg(50) to th, conformational stability of the reactive-site loop in CMTI-V. The results also indicate that the Arg(50) and Arg(52) Side chains are not hydrogen-bonded to carboxylate groups, which would be protonated at pH 2.5 and, hence, unavailable for hydrogen-bonding interactions. The overall folding of rCMTI-V appears not to be significantly affected by the pH change, as indicated by comparisons of H-1 and N-15 chemical shifts, sequential NOE cross-peaks, and S-2 values of the backbone atoms, and the conserved side-chain dynamics of Trp(9) and Trp(54) residues that are involved in hydrophobic and hydrogen-bonding interactions with others in the protein core and the binding loop, respectively.