Solution Structure and Backbone Dynamics of Recombinant Cucurbita maxima Trypsin Inhibitor-V Determined by NMR Spectroscopy^,

Abstract

The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other inhibitors of the potato I family--eglin c [Peng, J. W., & Wagner, G. (1992) Biochemistry 31, 8571-8586] and barley chymotrypsin inhibitor 2 [CI-2; Shaw, G. L., Davis, B., Keeler, J., & Fersht, A. R. (1995) Biochemistry 34, 2225-2233]. The Cys3-Cys48 linkage found only in rCMTI-V appears to somewhat reduce the N-terminal flexibility; likewise, the C-terminal of rCMTI-V, being part of a beta-sheet, appears to be more rigid.

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family

“…Therefore, we use the structures of intact and modified forms of the natural protein (CMTI-I; Bode et al, 1989;Holak et al, 1989a;Krishnamoorthi et al, 1992aKrishnamoorthi et al, , 1992b for the recombinant versions as well. It was observed previously that the extra N-terminal Gly residue did not affect the structures of rCMTI-V (Liu et al, 1996a) and rCMTI-V* (Liu et al, 1996b). The entire molecule of rCMTI-III* appears to have undergone tertiary structural changes, as evidenced by 'H, "N, and "C chemical-shift changes of its backbone and side-chain atoms (Krishnamoorthi et al, 1992a(Krishnamoorthi et al, , 1992b(Krishnamoorthi et al, .…”

“…Dynamical studies of rCMTI-V and rCMTI-V* (Liu et al, 1996a(Liu et al, , 1996b have shown that the newly formed C terminal (P, segment) in rCMTI-V* becomes more flexible, whereas the newly formed N terminal (PL fragment) remains as rigid as in the intact form. The reduced binding affinity of CMTI-V* relative to that of CMTI-V (Khyd = K:/Kd 9) may be attributed to the increased flexibility of the binding loop fragment, because the other regions of the inhibitor do not undergo any significant structural or dynamical changes.…”

“…lA), a potato I family member, and characterized differences in the structural, functional, thermodynamic, and dynamic properties of both intact and modified inhibitor ( Krishnamoorthi et al, 1990;Cai et al, 1995aCai et al, , 1995bCai et al, , 1996Wen et al, 1995;Liu et al, 1996aLiu et al, , 1996b. The Khyd of CMTI-V is -9 at 25 "C, pH 6.8, and, as expected, the modified form shows a reduction in its inhibitory activity toward trypsin and factor m a , a serine proteinase involved in blood coagulation (Cai et al, 1995b).…”

“…1. Ribbon representations of the three-dimensional structures of (A) CMTI-V (Liu et al, 1996a) and (B) CMTI-I (Bode et al, 1989). showing the binding loop anchors to the protein scaffold.…”

“…1A; Cai et al, 1995a;Liu et al, 1996a), the reactive-site loop (residues 40-49) is held in position by two hydrogen bonds-the Arg'" side chain anchoring the PL segment and the Args2 side chain holding in position the P,, region. The C y~~-C y s~~ crosslink provides additional support for the PL region.…”