A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity-protein specificity

Abstract

We report the first detailed comparison of two immunity proteins which, in conjunction with recent protein engineering data, begins to explain how these structurally similar proteins are able to bind and inhibit the endonuclease domain of colicin E9 (E9 DNase) with affinities that differ by 12 orders of magnitude. In the present work, we have determined the X-ray structure of the Escherichia coli colicin E7 immunity protein Im7 to 2.0 A resolution by molecular replacement, using as a trial model the recently determined NMR solution structure of Im9. Whereas the two proteins adopt similar four-helix structures, subtle structural differences, in particular involving a conserved tyrosine residue critical for E9 DNase binding, and the identity of key residues in the specificity helix, lie at the heart of their markedly different ability to bind the E9 DNase. Two other crystal structures were reported recently for Im7; in one, Im7 was a monomer and was very similar to the structure reported here, whereas in the other it was a dimer to which functional significance was assigned. Since this previous work suggested that Im7 could exist either as a monomer or a dimer, we used analytical ultracentrifugation to investigate this question further. Under a variety of solution conditions, we found that Im7 only ever exists in solution as a monomer, even up to protein concentrations of 15 mg/ml, casting doubt on the functional significance of the crystallographically observed dimer. This work provides a structural framework with which we can understand immunity-protein specificity, and in addition we believe it to be the first successfully refined crystal structure solved by molecular replacement using an NMR trial model with less than 100% sequence identity.

Measurement of histidine pKavalues and tautomer populations in invisible protein states

“…Notably, neither H40 nor H47 exhibit the 4:1 ratio in the native conformation of the protein, with H40 predominantly in the rare δ tautomer form and H47 exclusively in the « state, suggesting that both histidines are in hydrogen-bonded environments. Indeed, crystal structures of Im7 show that H47 stacks on W75 and is hydrogen bonded to the backbone amide of D49, whereas H40 becomes buried by the N terminus in the native state, potentially forming a hydrogen bond with the backbone carbonyl of K4 (77,78). Thus, significant rearrangements of the histidine side chains must accompany the I-to-N transition, leading to burial of the exposed H40 and H47 side chains.…”

NMR trial models: experiences with the colicin immunity protein Im7 and the p85α C-terminal SH2–peptide complex

“…The structure and its determination are described in detail by Dennis et al (1998); relevant details are summarized here. Crystals were either I222 or I2 1 2 1 2 1 (the translation function worked only in I222; see below), with unit-cell parameters a = 45.1, b = 50.6, c = 75.2 A Ê , = = = 90 .…”

“…The structure is a distorted four-helical bundle, as described in detail in Dennis et al (1998), where it is compared with the structure of the related Im9 (Osborne et al, 1996) to account for differences in binding speci®cities to their cognate colicin DNase domains. It is worth mentioning that subsequent studies on the crystal structures of the complex between Im7 with the DNase domain of colicin E7 (Ko et al, 1999) as well as the complex of Im9 with the DNase domain of colicin E9 have shown that the immunity protein remarkably does not bind at the highly conserved DNase active site.…”

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].…”