Session: BMB Education and Professional Development
(R2734) Structural Modeling and 3-D Printing of the PTF1-L Heterotrimeric Complex: Understanding the Structure and Function of a Ptf1a P191T Hypomorphic Mutation that Causes Pancreatic Aplasia in Children
Director Biomed Program Thomas Jefferson High School Dallas, Texas, United States
Ward Coats (Thomas Jefferson High School)
Biomedical Research students at Thomas Jefferson High School in Dallas, Texas participate in an ongoing research project to study the structure and function of the PTF1 transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a common E-box binding protein (E12/47, E2.2, or TCF12), Ptf1a and either Rbpj or Rbpjl. The Rbpj form of the complex (PTF1-J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl-form (PTF1-L) is required for the formation of mature acinar cells of the exocrine pancreas. Children born with a Ptf1a P191T substitution lack a functional pancreas, are severely diabetic, require insulin therapy and in some cases, exocrine enzyme therapy for their survival. Proline-191 is located within the highly conserved basic helix-loop-helix (bHLH) domain of Ptf1a, which is essential for dimerization with a common E-box binding protein. Structural modeling and energy minimization of PTF1-L(Ptf1a/E12/Rbpjl) and PTF1-L(Ptf1a(P191T)/E12/Rbpjl) heterotrimeric complexes using Pymol and MDweb software revealed that proline-191 terminates helix-1, and that substitution of a threonine at this position might alter the helical structure at the end of helix-1, leading to changes in hydrogen bonding between Arg-187 in helix-1 and the loop C-alpha backbone, and mispositioning of loop residue Arg-191 whose side chain is forming an interaction with deoxycytidine in the E-Box. The culmination of these structural changes in the Ptf1a mutant protein can in part explain the observed 75% decrease in transactivation activity of the mutant compared to the wild type Ptf1a protein measured in transient cell transfection assays. Using 3D printing technology, the Hillcrest Biomedical Research Team has generated a model of the PTF1-L heterotrimeric complex. Determination of the structures for the PTF1-L and PTF1-L-Ptf1a(P191T) complexes will elucidate the mechanism for the strong cooperative DNA binding of the PTF1-L complex and will provide insights into the structural changes that are introduced by the Ptf1a P191T substitution that cause pancreatic aplasia in children born with this mutation.