Colin Macdonald

The structural and energetic basis for high selectivity in a high-affinity protein-protein interaction

High-affinity, high-selectivity protein-protein interactions that are critical for cell survival present an evolutionary paradox: How does selectivity evolve when acquired mutations risk a lethal loss of high-affinity binding? A detailed understanding of selectivity in such complexes requires structural information on weak, noncognate complexes which can be difficult to obtain due to their transient and dynamic nature. Using NMR-based docking as a guide, we deployed a disulfide-trapping strategy on a noncognate complex between the colicin E9 endonuclease (E9 DNase) and immunity protein 2 (Im2), which is seven orders of magnitude weaker binding than the cognate femtomolar E9 DNase-Im9 interaction. The 1.77 Å crystal structure of the E9 DNase-Im2 complex reveals an entirely noncovalent interface where the intersubunit disulfide merely supports the crystal lattice. In combination with computational alanine scanning of interfacial residues, the structure reveals that the driving force for binding is so strong that a severely unfavorable specificity contact is tolerated at the interface and as a result the complex becomes weakened through “frustration.” As well as rationalizing past mutational and thermodynamic data, comparing our noncognate structure with previous cognate complexes highlights the importance of loop regions in developing selectivity and accentuates the multiple roles of buried water molecules that stabilize, ameliorate, or aggravate interfacial contacts. The study provides direct support for dual-recognition in colicin DNase-Im protein complexes and shows that weakened noncognate complexes are primed for high-affinity binding, which can be achieved by economical mutation of a limited number of residues at the interface.

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.

Biophysical Features of Bacillithiol, the Glutathione Surrogate of Bacillus subtilis and other Firmicutes

Bacillithiol (BSH) is the major low‐molecular‐weight (LMW) thiol in many low‐G+C Gram‐positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers. Here, BSH has been characterised and compared with other LMW thiols in terms of its thiol pKa, redox potential and thiol–disulfide exchange reactivity. Both the thiol pKa and the standard thiol redox potential of BSH are shown to be significantly lower than those of glutathione whereas the reactivities of the two compounds in thiol–disulfide reactions are comparable. The cellular concentration of BSH in Bacillus subtilis varied over different growth phases and reached up to 5 mM, which is significantly greater than previously observed from single measurements taken during mid‐exponential growth. These results demonstrate that the biophysical characteristics of BSH are distinctively different from those of GSH and that its cellular concentrations can reach levels much higher than previously reported.

NMR detection of slow conformational dynamics in an endonuclease toxin.

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid15 N- and 13C/15N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with Cα and Cβ chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D1 H-1H-15N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 °C with a forward rate constant of 1.61 ± 0.5 s-1 and a backward rate constant of 1.05 ± 0.5 s-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743–13750 and 13751–13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI, is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form. The possibility that cis–trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA ⇋ NB interconversion to be identified, Pro65 and Pro73. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.

Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.

Transformation of a methyleneamide ligand at molybdenum: electrochemical oxidation to a cyanide, reactions with elemental oxygen, sulphur or selenium and X-ray crystal structures of trans-[Mo(CN)Cl(dppe)2]·MeOH and trans-[Mo(NCS)Cl(dppe)2]; electroreduction of the cyanide to an aminocarbyne, trans-[Mo(CNH2)Cl(dppe)2](dppe = Ph2PCH2CH2PPh2)

Deprotonation of the methylimide trans-[Mo(NMe)Cl(dppe)2]+(dppe = Ph2PCH2CH2PPh2) gives the reactive methyleneamide trans-[Mo(NCH2)Cl(dppe)2]A; oxidation of A at a platinum anode or by iodine gives trans-[Mo(CN)Cl(dppe)2]B, the structure of which has been determined by X-ray crystallography. Carbon-13 labelling studies suggest that the rearrangement of the MoNC framework to MoCN is intramolecular. Compound A reacts with chalcogens to give heterocumulene complexes trans-[Mo(NCX)Cl(dppe)2]C(X = O, S or Se), and the X-ray crystal structure of the sulphur derivative shows that the NCS ligand is N-bonded as an isothiocyanate. The electrochemistry of compound B is extensive: reduction under N2 gives trans-[Mo(N2)(CN)(dppe)2]– and in the presence of phenol affords the isolable aminocarbyne trans-[Mo(CNH2)Cl(dppe)2].

Reversible DNA i-motif to hairpin switching induced by copper(II) cations

i-Motif forming DNA sequences have previously been used for many different nanotechnological applications, but all have used changes in pH to fold the DNA. Here it is shown that Cu(ii) cations can be used to re-fold i-motifs into hairpin structures, without changing the pH.

Increased dynamics in the 40–57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation

Thrombocytopenia 4 is an inherited autosomal dominant thrombocytopenia, which occurs due to mutations in the human gene for cytochrome c that results in enhanced mitochondrial apoptotic activity. The Gly41Ser mutation was the first to be reported. Here we report stopped-flow kinetic studies of azide binding to human ferricytochrome c and its Gly41Ser variant, together with backbone amide H/D exchange and 15N-relaxation dynamics using NMR spectroscopy, to show that alternative conformations are kinetically and thermodynamically more readily accessible for the Gly41Ser variant than for the wild-type protein. Our work reveals a direct conformational link between the 40–57 Ω-loop in which residue 41 resides and the dynamical properties of the axial ligand to the heme iron, Met80, such that the replacement of glycine by serine promotes the dissociation of the Met80 ligand, thereby increasing the population of a peroxidase active state, which is a key non-native conformational state in apoptosis.

Structural Evidence That Colicin A Protein Binds to a Novel Binding Site of TolA Protein in Escherichia coli Periplasm

Background: Colicins interact with Tol proteins in the periplasm to facilitate their killing of E. coli cells. Results: The N terminus of colicin A interacts with the C terminus of TolA through β-strand addition. Conclusion: Colicin A interacts with TolA at a novel binding site to promote cell killing. Significance: TolA is integral to cell entry of colicin A, providing information to refine current models of colicin translocation. The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action. We have determined the first structure of the C-terminal domain of TolA (TolAIII) bound to an N-terminal ColA polypeptide (TA53–107). The interface region of the TA53–107-TolAIII complex consists of polar contacts linking residues Arg-92 to Arg-96 of ColA with residues Leu-375–Pro-380 of TolA, which constitutes a β-strand addition commonly seen in more promiscuous protein-protein contacts. The interface region also includes three cation-π interactions (Tyr-58–Lys-368, Tyr-90–Lys-379, Phe-94–Lys-396), which have not been observed in any other colicin-Tol protein complex. Mutagenesis of the interface residues of ColA or TolA revealed that the effect on the interaction was cumulative; single mutations of either partner had no effect on ColA activity, whereas mutations of three or more residues significantly reduced ColA activity. Mutagenesis of the aromatic ring component of the cation-π interacting residues showed Tyr-58 of ColA to be essential for the stability of complex formation. TA53–107 binds on the opposite side of TolAIII to that used by g3p, ColN, or TolB, illustrating the flexible nature of TolA as a periplasmic hub protein.

Protonation of bis[1,2-bis(diethylphosphino)ethane]bis(dinitrogen)-molybdenum and -tungsten with fluoroboric acid–diethyl ether (1/1) in benzene; crystal and molecular structure of bis[1,2- bis(diethylphosphino)ethane]fluoro[hydrazido(2–)]tungsten tetrafluoroborate

The reaction of HBF4·Et2O with [W(N2)2(depe)2] in benzene can, depending upon the conditions, give rise to [WF(NNH2)(depe)2]BF4, [WF(NNH3)(depe)2][BF4]2, or [WH(F)(NNH2)(depe)2][BF4]2. The compounds are to some extent interconvertible. The structures have been completely elucidated using 31P and 15N n.m.r. spectroscopy, and the X-ray crystal structure of [WF(NNH2)(depe)2]BF4 has been determined. This reveals no unusual bond distances, but an extensive system of hydrogen bonding. The parallel reaction of [Mo(N2)2(depe)2] yields only one product, tentatively formulated as [Mo(BF4)(NNH2)(depe)2]BF4. The relevance of these observations to the mechanism of the protonation of complexed dinitrogen is discussed.