Structural aspects of the inhibition of DNase and rRNase colicins by their immunity proteins

Highly Discriminating Protein–Protein Interaction Specificities in the Context of a Conserved Binding Energy Hotspot

“…Structural comparisons of DNase -Im protein complexes with the free proteins (E7 and E9) indicate that neither the immunity protein nor the enzyme undergoes major structural change on complex formation. 23,34 Hence, the first-order changes in DNase fluorescence that accompany binding likely Figure 5. Correlating in vitro E9 DNase -immunity protein binding affinities with in vivo biological protection against colicin E9.…”

“…55 Random amino acids were introduced into six selected positions (residues 31,33,34,38,41,42 in Im2) by incorporating an NNC/G codon in the PCR primer, where N represents a mixture of A, C, G and T. The full-length final round PCR product was purified from an agarose gel, digested with Sfi I and Not I, and ligated into the pre-digested pCAN-TAB6 vector. The ligation mixture was introduced into TG-1 cells by electroporation on a Bio-Rad Gene Pulser.…”

The PD-(D/E)XK superfamily revisited: identification of new members among proteins involved in DNA metabolism and functional predictions for domains of (hitherto) unknown function

“…The N-terminus reveals a strongly hydrophobic stretch of residues predicted to form a transmembrane helix, which could be either used as a leader peptide to guide the translocation of the nuclease domains through the membrane or it could anchor it to the membrane. Programs for the prediction of transmembrane protein topology HMMTOP [48], TMAP [49], and TMPRED [50] predicted that the nuclease domain has a cytosolic orientation. We speculate that COG4741 members could be released to the environment as toxic agents against other cells, like endonuclease colicins (review: [51]), or be used to guard the cell against the uptake of foreign DNA and/or to cleave the encountered nucleic acids to produce (oligo)nucleotides that can be used by the host.…”

The role of the N-terminal loop in the function of the colicin E7 nuclease domain

“…In order to exert cell killing activity, ColE7 has to get across both the outer and the inner cell membrane, facilitated by the receptor-binding and translocation domains [3,4]. The host cell itself is protected by the simultaneously expressed immunity protein Im7 blocking the DNA-binding site [5,6] of the NColE7 domain due to tight interactions based on charge-complementarity [7][8][9][10][11][12].…”