Investigation of Homeodomain Membrane Translocation Properties: Insights from the Structure Determination of Engrailed-2 Homeodomain in Aqueous and Membrane-Mimetic Environments

Abstract

In addition to their well-known DNA-binding properties, homeodomains have the ability to efficiently translocate across biological membranes through still poorly-characterized mechanisms. To date, most biophysical studies addressing the mechanisms of internalization have focused on small synthetic peptides rather than full-length globular homeodomains. In this work, we characterized the conformational properties of chicken Engrailed 2 homeodomain (En2HD) in aqueous solution and in membrane mimetic environments using circular dichroism, Trp fluorescence, and NMR spectroscopy. En2HD adopts a well-defined three-helical bundle fold in aqueous solution. The Trp-48 residue, which is critical for internalization, is fully buried in the hydrophobic core. Circular dichroism and fluorescence reveal that a conformational transition occurs in anionic lipid vesicles and in micelles. En2HD loses its native three-dimensional structure in micellar environments but, remarkably, near-native helical secondary structures are maintained. Long-range interactions could be detected using site-directed spin labels, indicating that the three helices do not adopt extended orientations. Noncovalent paramagnetic probes yielded information about helix positioning and unveiled the burial of critical aromatic and basic residues within the micelles. Our results suggest that electrostatic interactions with membranes may be determinant in inducing a conformational change enabling Trp-48 to insert into membranes.

Bidirectional transfer of Engrailed homeoprotein across the plasma membrane requires PIP2

“…We have no evidence of multimer formation in the case of EN2, which possess only one cysteine. However, in the presence of negatively charged lipid bicelles, the conformation of EN2 protein analyzed by NRM is shifted, promoting the exposure of the hydrophobic core of the homeodomain (Carlier et al, 2013). The interplay between electrostatic and hydrophobic (supported by the reduced affinity of the EN2 ww>KK mutant) interactions with PIP 2 might act as a switch for EN2 to shuttle between the two environments.…”

Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides

“…The use of gadodiamide, a paramagnetic MRI contrast agent, has been considered for several years as one of the most accurate approaches to probe the immersion depth of polypeptides within micelles [50][51][52]. Gadodiamide is a water-soluble Gd (III) complex that enhances T 1 relaxation rates of protons in a distance-dependent manner.…”

Altered staining patterns and expression level of Engrailed-2 in Benign prostatic hyperplasia and Prostate Cancer predict Prostatic disease progression

“…Structurely, the full length of EN2 protein has 333aa, including three alpha helices,with the helix 1 and 2 at the N end binding DNA, and helix 3 at C end,mainly mediating the exocrine and internalization of EN2 protein [12,19]. We produced a monoclonal antibody targeting Helix 3 in EN2,and conducted immunohistochemical staining with this homemade antibody to detect EN2 expression patterns in 25 BPH and 25 PC cases.…”