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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Understanding and Modulation of the Folding of a Helix-Loop-Helix Dimerization DomainMichael Beisswenger 1 , Sara Pellegrino 2 , Roberto Fanelli 2 ,Nicola Ferri 3 , Maria L. Gelmi 2 , and Chiara Cabrele 11 Fakultät für Chemie und Biochemie, Ruhr-Universität Bochum, Bochum, 44801,Germany; 2 DISMAB-Sez. di Chimica Organica, Università degli Studi di Milano,Milan, 20133, Italy; 3 Dip. di Scienze Farmacologiche, Università degli Studi di Milano,20133, Milan, ItalyIntroductionThe helix-loop-helix (HLH) domain is the characteristic dimerization domain of the HLHtranscription factors family [1]. This domain triggers the formation of protein dimers thatdisplay a parallel four-helix bundle [2]. This type of protein-protein interaction is essentialfor the biological function of the HLH proteins. For example, the Id proteins are HLHtranscription factors that inhibit the action of other HLH factors by sequestering them ininactive heterodimers [3]. By using this mechanism, the Id proteins promote cellproliferation and inhibit cell differentiation during development and tumorigenesis. We areinterested in the inhibition of Id protein dimerization with their endogenous partners (i.e.E47 and MyoD). Our work focuses on the identification of the structural prerequisites forthe correct HLH folding, as well as of short peptide sequences targeting the Id HLHdomain and altering its folding/dimerization properties.Results and DiscussionStudies based on amino-acid substitutions [4] and backbone modifications [5,6] haveunderlined the role of some primary-structure elements on the secondary and tertiarystructures adopted by a synthetic Id HLH peptide. Side-chain packing and backbonedirection are both crucial for the optimal fold. For example, two O-acyl-isopeptide analogsof the Id1 HLH domain (residues 66-106) bearing the backbone and side-chainmodification at one of the two helix-loop junctions are disordered at pH 4-5, whereas thenative Id1 HLH peptide is structured (though less than at pH 7). This indicates that thechemical changes at the junctions destabilize both the tertiary and secondary structures.However, these are fully restored upon conversion of the O-acyl-isopeptide analogs into thenative peptide at pH 7 [5,6].Further, we have evaluated the ability of short peptides to interact with the Id1 HLHdomain and prepared some peptidic constructs that have been tested on vascular smoothmuscle cells expressing the Id1 protein. Two constructs (2a and 2b) based on covalentdimers of the Id1 fragment 91-101 have been shown to be active in the micromolar range,reducing cell growth and migration, stimulating cell differentiation and considerablydecreasing the cellular level of the Id1 protein [7] (Figure 1). The covalent linkage isrepresented by the building block N-benzoyl cis-3-carboxy-cyclopentylglycine [8] in theFig. 1. Effect on the Id1 fragment 91-101 as monomer (1) or covalent dimer (2a and2b) on the proliferation of vascular smooth-muscle cells.600