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

Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, K(hyd), is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; M(r) 3 kDa) of the squash family and rCMTI-V (Mr ~ 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different K(hyd) values-2.4 (CMTI-III) and 9 (CMTI-V)-at 25 °C. The reactive-site peptide bond (P₁-P₁') is that between Arg⁵ and Ile⁶ in CMTI-III, and that between Lys⁴⁴ and Asp⁴⁵ in CMTI-V. The order parameters (S²) of backbone NHs of uniformly ¹⁵N-labeled rCMTI-III and rCMTI-III* were determined from measurements of ¹⁵N spin-lattice and spin- spin relaxation rates, and {¹H}-¹⁵N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI- III (<S²> = 0.71) and rCMTI-III* (<S²> = 0.63) are more flexible than those of rCMTI-V (<S²> = 0.83) and rCMTI-V* (<S²> = 0.85). The binding loop residues, P₄-P₁, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P₁'-P₄' residues, on the other hand, are associated with <S²> values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (P(n) residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys³-Cys²⁰ crosslink. In contrast, the newly formed N-terminal (P(n)' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P₁' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the P(n) residues and, surprisingly, increased flexibility of the P(n)' residues seem to facilitate the resynthesis of the P₁-P₁' bond, leading to a lower K(hyd) value.