Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic n-terminal mutations

TY - JOUR

T1 - Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic n-terminal mutations

AU - Németh, Eszter

AU - Körtvélyesi, Tamás

AU - Thulstrup, Peter Waaben

AU - Christensen, Hans Erik Mølager

AU - Kožíšek, Milan

AU - Nagata, Kyosuke

AU - Czene, Anikó

AU - Gyurcsik, Béla

PY - 2014

Y1 - 2014

N2 - The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7=KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK>KGNG ∼ GGNK> GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.

AB - The nuclease domain of colicin E7 (NColE7) promotes the nonspecific cleavage of nucleic acids at its C-terminal HNH motif. Interestingly, the deletion of four N-terminal residues (446-449 NColE7=KRNK) resulted in complete loss of the enzyme activity. R447A mutation was reported to decrease the nuclease activity, but a detailed analysis of the role of the highly positive and flexible N-terminus is still missing. Here, we present the study of four mutants, with a decreased activity in the following order: NColE7 >> KGNK>KGNG ∼ GGNK> GGNG. At the same time, the folding, the metal-ion, and the DNA-binding affinity were unaffected by the mutations as revealed by linear and circular dichroism spectroscopy, isothermal calorimetric titrations, and gel mobility shift experiments. Semiempirical quantum chemical calculations and molecular dynamics simulations revealed that K446, K449, and/or the N-terminal amino group are able to approach the active centre in the absence of the other positively charged residues. The results suggested a complex role of the N-terminus in the catalytic process that could be exploited in the design of a controlled nuclease.

KW - DNA cleavage

KW - Flow linear dichroism

KW - Isothermal calorimetry

KW - Positively charged N-terminal residues

U2 - 10.1002/pro.2497

DO - 10.1002/pro.2497

M3 - Journal article

C2 - 24895333

AN - SCOPUS:84904734052

SN - 0961-8368

VL - 23

SP - 1113

EP - 1122

JO - Protein Science

JF - Protein Science

IS - 8

ER -