Design of a mimic HPV transcriptional regulator E2 that increase specific DNA binding affinity

D. E. WETZLER; M. J. COMIN; M. GALLO; K. KRAJEWSKI; D. O. CICERO

Salta, Argentina

Congreso; 3rd Latin American Protein Society Meeting; 2010

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:IT; mso-fareast-language:IT;} a:link, span.MsoHyperlink {color:blue; text-decoration:underline; text-underline:single;} a:visited, span.MsoHyperlinkFollowed {color:purple; text-decoration:underline; text-underline:single;} ins {mso-style-type:export-only; text-decoration:none;} span.msoDel {mso-style-type:export-only; mso-style-name:""; display:none; color:red;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> The human papillomavirus (HPV) is a main responsible of the cervical cancer. The E2 protein acts as transcriptional modulator which loss of function leads to E7 and E6 oncoproteins up- regulation. The C-terminal domain of E2 is a symmetric dimmer that interacts with the target DNA through an a-helix. A peptide corresponding to the DNA recognition helix of HPV-16 E2 displays only residual structure and binds DNA with lower affinity than the full-length E2. There is a great deal of interest in obtaining small synthetic mimics of transcription factors capable of reproducing their specificity and affinity DNA-binding properties. In an attempt to promote the DNA binding of the E2 isolated peptide, we have designed and synthesized a conjugate compound of the E2 a-helix peptide and a derivative of the antibiotic distamycin (Dst) carrying a primary amino function in the linker between both moieties. This strategy involves simultaneous minor- and major-groove interactions. The conjugate was completely characterized by NMR spectroscopy, which additionally shows that the coupling of the E2 peptide with Dst does not affect the structural properties of the peptide. DNA melting experiments indicate that the conjugate improves significantly the affinity of the peptide for specific DNA. Circular dichroism experiments show that stoichiometric amounts of specific DNA increase the helical structurepuopulation of the design peptide. Circular dichroism titration and gel shift experiments show that the conjugate peptide increaseenhances binding constant 15-fold, maintaining its specificity. These results confirm that the peptide-Dst compoundconjugates are is a promising tool to obtain compounds that bind the E2 target DNA-sequences with remarkable affinity and are encouraging for the design of E2 conjugates with alternative linkers that can bind DNA with higher specificity and affinity.