Molecular Recognition in Helix-Loop-Helix and Helix-Loop-Helix-Leucine Zipper Domains

Abstract

Helix-loop-helix (HLH) and helix-loop-helix-leucine zipper (HLHZip) are dimerization domains that mediate selective pairing among members of a large transcription factor family involved in cell fate determination. To investigate the molecular rules underlying recognition specificity and to isolate molecules interfering with cell proliferation and differentiation control, we assembled two molecular repertoires obtained by directed randomization of the binding surface in these two domains. For this strategy we selected the Heb HLH and Max Zip regions as molecular scaffolds for the randomization process and displayed the two resulting molecular repertoires on phage capsids. By affinity selection, many domains were isolated that bound to the proteins Mad, Rox, MyoD, and Id2 with different levels of affinity. Although several residues along an extended surface within each domain appeared to contribute to dimerization, some key residues critically involved in molecular recognition could be identified. Furthermore, a number of charged residues appeared to act as switch points facilitating partner exchange. By successfully selecting ligands for four of four HLH or HLHZip proteins, we have shown that the repertoires assembled are rather general and possibly contain elements that bind with sufficient affinity to any natural HLH or HLHZip molecule. Thus they represent a valuable source of ligands that could be used as reagents for molecular dissection of functional regulatory pathways.

The Arabidopsis Basic/Helix-Loop-Helix Transcription Factor Family[W]

“…The amino acid sequence in this region presumably dictates the specificity of the interaction, but relatively little is known about how the specificity is defined (Ciarapica et al, 2003). There is evidence, however, for a variety of interaction patterns among different family members in nonplant systems, including apparent obligate homodimerization or heterodimerization, or the dual capacity for both homodimerization and heterodimerization (Grandori et al, 2000;Baudino and Cleveland, 2001;Ciarapica et al, 2003;Levens, 2003).…”

“…bHLH proteins are well known to dimerize, but the critical molecular determinants involved are not well defined (Shirakata et al, 1993;Littlewood and Evan, 1998;Ciarapica et al, 2003). On the other hand, the Leu residue at position 27 in our alignment has been shown to be structurally necessary for dimer formation in the mammalian Max protein (Brownlie et al, 1997).…”

“…Therefore, it is notable that this is the only invariant residue in all 147 AtbHLH proteins ( Figure 1, Table 1), consistent with a similar essential function in plant bHLH protein dimerization. Current information indicates that dimerization specificity is affected by multiple parameters, including hydrophobic interfaces, interactions between charged amino acids in the HLH region, and partner availability, but no complete explanation for partner recognition specificity has been documented (Ciarapica et al, 2003). Thus, although empirically it seems logical that bHLH proteins most closely related in sequence in the HLH region are the most likely to form heterodimers, there has been no systematic investigation of this possibility to date.…”

“…The amino acid sequence in this region presumably dictates the specificity of the interaction, but relatively little is known about how the specificity is defined (Ciarapica et al, 2003). There is evidence, however, for a variety of interaction patterns among different family members in nonplant systems, including apparent obligate homodimerization or heterodimerization, or the dual capacity for both homodimerization and heterodimerization (Grandori et al, 2000;Baudino and Cleveland, 2001;Ciarapica et al, 2003;Levens, 2003). Clearly, the capacity to heterodimerize with other family members immediately expands the diversity of possible intermolecular interactions, potentially creating new functional activities, such as recognition of new hybrid DNA binding sites, and directing the convergence of separate signaling pathways to the same promoter via the pairing of domains that recognize distinct signaling molecules (Grandori et al, 2000;Massari and Murre, 2000).…”

“…This mechanism involves the generation of a high degree of complexity and diversity in transcriptional regulatory activity through variation in the DNA sequence motif recognized by individual bHLH proteins, the capacity to combinatorially amplify the spectrum of possible specific protein-DNA interactions, through selective heterodimerization between bHLH proteins with different DNA sequence recognition specificity, and the capacity to interact with a network of transcriptional coactivators, corepressors, and signaling molecules through selective protein-protein interactions (Grandori et al, 2000;Baudino and Cleveland, 2001;Ciarapica et al, 2003;Levens, 2003).…”