Molecular recognition in helix-loop-helix and helix-loop-helix-leucine zipper domains. Design of repertoires and selection of high affinity ligands for natural proteins.
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 lambda 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.
Ciarapica
Roberta
R
Istituto di Biologia and Patologia Molecolari Consiglio Nazionale delle Ricerche, Università La Sapienza, 00185 Roma, Italy.
Rosati
Jessica
J
Cesareni
Gianni
G
Nasi
Sergio
S
D000595:Amino Acid Sequence; D000818:Animals; D010582:Bacteriophage lambda; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D006801:Humans; D051796:Inhibitor of Differentiation Proteins; D016350:Leucine Zippers / Q000235:genetics*; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000737:chemistry / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics; D010215:Papio; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins; D014157:Transcription Factors
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051796:Inhibitor of Differentiation Proteins; D008024:Ligands; C499330:MAX protein, human; C079439:MXD1 protein, human; C499479:Mad protein, mouse; C067850:Myc associated factor X; D017570:MyoD Protein; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C068422:Max protein, mouse; C075640:ID3 protein, human; D005982:Glutathione Transferase