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

Jianhua Liu, Yuxi Gong, Om Prakash, Lisa Wen, Insuk Lee, Jenq Kuen Huang, Ramaswamy Krishnamoorthi

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Abstract

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.

Original languageEnglish
Pages (from-to)132-141
Number of pages10
JournalProtein Science
Volume7
Issue number1
DOIs
Publication statusPublished - 1998 Jan

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Cucurbita
Serine Proteinase Inhibitors
Serine Proteases
Solanum tuberosum
Protein Binding
Catalytic Domain
Nuclear magnetic resonance
Proteins
Scaffolds
Hydrolysis
Cucurbita maxima CMTI protein
Peptides
Enzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

@article{bc191504a4ed4847835a53ea7e7ca602,
title = "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",
abstract = "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.",
author = "Jianhua Liu and Yuxi Gong and Om Prakash and Lisa Wen and Insuk Lee and Huang, {Jenq Kuen} and Ramaswamy Krishnamoorthi",
year = "1998",
month = "1",
doi = "10.1002/pro.5560070114",
language = "English",
volume = "7",
pages = "132--141",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Cold Spring Harbor Laboratory Press",
number = "1",

}

TY - JOUR

T1 - NMR studies of internal dynamics of serine proteinase protein inhibitors

T2 - 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

AU - Liu, Jianhua

AU - Gong, Yuxi

AU - Prakash, Om

AU - Wen, Lisa

AU - Lee, Insuk

AU - Huang, Jenq Kuen

AU - Krishnamoorthi, Ramaswamy

PY - 1998/1

Y1 - 1998/1

N2 - 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.

AB - 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.

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