Probing the H-protein-induced Conformational Change and the Function of the N-terminal Region of Escherichia coliT-protein of the Glycine Cleavage System by Limited Proteolysis

• Published in: The Journal of biological chemistry Vol. 278; no. 12; pp. 10067 - 10072
• Main Authors: Kazuko Okamura-Ikeda, Naomi Kameoka, Kazuko Fujiwara, Yutaro Motokawa
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 01.03.2003
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210853200

T-protein, a component of the glycine cleavage system, catalyzes a tetrahydrofolate-dependent reaction. Previously, we reported a conformational change of Escherichia coli T-protein upon interacting with E. coli H-protein (EH), showing an important role for the N-terminal region of the T-protein in the interaction. To further investigate the T-protein catalysis, the wild type (ET) and mutants were subjected to limited proteolysis. ET was favorably cleaved at Lys 81 , Lys 154 , Lys 288 , and Lys 360 by lysylendopeptidase and the cleavages at Lys 81 and Lys 288 were strongly prevented by EH. Although ET was highly resistant to trypsinolysis, the mutant with an N-terminal 7-residue deletion (ETΔ7) was quite susceptible and instantly cleaved at Arg 16 accompanied by the rapid degradation of the resulting C-terminal fragment, indicating that the cleavage at Arg 16 is the trigger for the C-terminal fragmentation. EH showed no protection from the N-terminal cleavage, although substantial protection from the C-terminal fragmentation was observed. The replacement of Leu 6 of ET with alanine resulted in a similar sensitivity to trypsin as ETΔ7. These results suggest that the N-terminal region of ET functions as a molecular “hasp” to hold ET in the compact form required for the proper association with EH. Leu 6 seems to play a central role in the hasp function. Interestingly, Lys 360 of ET was susceptible to proteolysis even after the stabilization of the entire molecule of ET by EH, indicating its location at the surface of the ET-EH complex. Together with the buried position of Lys 81 in the complex and previous results on folate binding sites, these results suggest the formation of a folate-binding cavity via the interaction of ET with EH. The polyglutamyl tail of the folate substrate may be inserted into the bosom of the cavity leaving the pteridine ring near the entrance of the cavity in the context of the catalytic reaction.