Solution structure of the DNA-binding domain of TraM.

The solution structure of the DNA-binding domain of the TraM protein, an essential component of the DNA transfer machinery of the conjugative resistance plasmid R1, is presented. The structure has been determined using homonuclear 2-dimensional NMR spectroscopy as well as 15N labeled heteronuclear 2- and 3-dimensional NMR spectroscopy. It turns out that the solution structure of the DNA binding domain of the TraM protein is globular and dominantly helical. The very first amino acids of the N-terminus are unstructured.

Multisite mutagenesis of interleukin 5 differentiates sites for receptor recognition and receptor activation.

Multisite mutagenesis of single-chain and monomeric forms of human interleukin 5 (IL-5) was performed to investigate mechanistic features of receptor activation and the possibility of differentiating sites of activation from those for receptor interaction. The normally dimeric human IL-5 contains two domains, each containing a four-helix bundle. IL-5 has previously been re-engineered into the monomeric, one-domain GM1 form by introducing an eight-residue linker between the third and fourth helices. In this study, we tested a combination of mutations in a single-chain IL-5 (scIL-5) construct, [(89)SLRGG(92),W(110)/(89)AAAAA(92), A(110)]scIL-5. This mutein was found to retain substantial IL-5 receptor alpha-chain binding but with selectively suppressed proliferation of the IL-5-dependent cell line TF-1.28. This result confirms recent findings that IL-5 receptor alpha-chain recognition can be supported by the (89)SLRGG(92) epitope and that, in contrast, Glu110 is important in receptor activation. On the basis of this result, two mutants of GM1 were constructed with the intent to retain receptor alpha-chain binding while modifying receptor activation epitopes. In the first, [(88)SLRGG(92),W(110)]GM1, the wild-type CD-loop sequence (89)EERRR(92) was converted to the mimotope (89)SLRGG(92), and Glu110 to Trp. In the second, [A(13), A(110)]GM1, wild-type Glu13, and Glu110 were both mutated to Ala. GM1 and mutants were expressed in high yield in Escherichia coli, purified under denaturing conditions from inclusion bodies, and refolded. Monomers were screened for binding to shIL-5Ralpha-Fc using optical biosensor and ELISA and for bioactivity by proliferation of TF-1.28 cells. Both [(88)SLRGG(92),W(110)]GM1 and [A(13),A(110)]GM1 were found to interact with the shIL-5Ralpha-Fc, with affinities of 69-585 nM, 2-15-fold weaker than that of the original GM1. The mutants also were able to compete with IL-5 for binding to shIL-5Ralpha in an ELISA. In contrast, both mutants exhibited a disproportionately decreased capacity to stimulate TF-1. 28 cell proliferation. [A(13),A(110)]GM1 bioactivity was 160-fold lower than that of GM1, while that for the [(88)SLRGG(92),W(110)]GM1 mutant was 2600-fold lower. The largely retained IL-5 receptor alpha-chain binding affinities versus relatively suppressed bioactivities of [A(13),A(110)]GM1 and [(88)SLRGG(92),W(110)]GM1 variants, in particular the latter, point to the existence of separable IL-5 epitopes for receptor binding and activation and establish the potential to design smaller IL-5 mimetic antagonists.

Conformational changes of gp120 in epitopes near the CCR5 binding site are induced by CD4 and a CD4 miniprotein mimetic.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Conformational behaviour of the TraM headpiece.

The structure of the headpiece of the TraM protein was investigated in different solvents. The very first 22 amino acids which alternate in their hydrophilic and hydrophobic character formed a helical structure in the presence of a membrane mimetic. In water alone the structure was flexible with a small amount of helicity according to circular dichroism measurements, whereas a loop structure was observed in dimethyl sulphoxide.