Structural Modeling of the PTF1-J and PTF1-L Heterotrimeric Transcription Factor Complexes

1. Conclusions
We have generated structural models for the PTF1-J and
PTF1-L heterotrimeric transcription factor complexes bound
to their consensus DNA binding sequence. The models
provide the orientation of the Ptf1a/E12 heterodimer relative
to the Rbpj and Rbpjl proteins and identify the protein
surface areas at the heterotrimeric interface.
We have generated a structural model of the C2 sequence of
Ptf1a bound to the Beta-trefoil domain of mammalian Rbpj
and Rbpjl.
In our models the C2 region of Ptf1a binds to the BTDs in an
extended polypeptide conformation and maintains the main
chain and tryptophan, proline, hydrophobic pocket
interactions that are present in the Ram domain of the Notch-
IC Rbpj structure.
ZDock trials using the C2 region of Ptf1a based on the
structure of the Ram domain of Notch-IC bound to Rbpj
generated 1325 docking structures ranked according to their
binding energy. Inspection of these ligand receptor models
identified solutions 3, 9 and 11 as having ligand interactions
consistent with our model for the C2 region of Ptf1a bound
to the Beta-trefoil domain of mammalian Rbpj.
Abstract
Students in Biomedical Research Studies at Hillcrest High School in Dallas,
Texas participate in an ongoing research program at the University of Texas
Southwestern Medical Center to study the structure and function of a
mammalian 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, HEB,
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. PTF1-L is involved in an auto-regulatory loop for the maintenance
of transcription of both Ptf1a and Rbpjl genes. We have generated structural
models for the PTF1-J and PTF1-L heterotrimeric transcription factor
complexes bound to their consensus DNA binding sequences. The models
provide the orientation of the Ptf1a/E12 heterodimer relative to the Rbpj and
Rbpjl proteins and identify the protein surface areas at the heterotrimeric
interface. Determination of the structures for the PTF1-J and PTF1-L
complexes will elucidate the structural motifs required for the protein-protein
interactions found in the trimeric complex, as well as the mechanism and the
structural determinants for the strong cooperative DNA binding of the
complexes.
Kelly Stein1
, Alix Kohrs1
, Lauren Schuller1
, Andrea Marshall1
, Raymond MacDonald2
, Ward Coats1
1
Hillcrest High School, Dallas, Texas
2
Department of Molecular Biology, University of Texas: Southwestern Medical Center, Dallas, Texas
Generation of Models for RBP-L, Ptf1a and E12
by the Swiss-Model Program
Input: Amino acid sequences for RBP-L, PTF1a, and E12 in fasta format
Swiss-Model Automatic Modeling Mode
Output: Structure Models for RBP-L, PTF1a and E12
Results
Model Amino Acids Reference Molecule PDB ID
Rbpjl 45 – 476 Human Rbpj 2F8X
Ptfa 164- 221 Heterodimer E47 /NeuroD1 2QL2
E12 550- 606 Heterodimer E47/NeuroD1 2QL2
Sequences for mouse RBbpjl, Pft1a and E12 were used as search templates in the Swiss-Model Automatic
Modeling Program. The Pymol modeling program was then used to build the PTF1 heterotrimeric complex
on a DNA molecule containing the Ptf1 consensus binding sequence. The DNA structure model containing
the PTF1-J consensus binding sequence was generated by the 3D-DART web server.
ZDOCK Structure Solutions
Docking Trial of the Model for the Ptf1a C2 Domain with the Beta-
trefoil Domain of Rbpj and Rbpjl
Input: Ligands Molecules (Structural Model for the Ptf1a C2 Domain) PBD file
Receptor Molecule (Beta-trefoil Domain of Rbpj or Rbpjl) PDB files
ZDOCK Server (PDB file: generate a list of contact and noncontact residues)
ZDOCK searches all possible binding modes in the translational and rotational space
between the two proteins and evaluates each by an energy scoring function.
Output: Ligand file: (ZDOCK may alter the conformation of the binding ligand)
Receptor file: (ZDOCK may alter the conformation of the receptor)
List of the top 2000 prediction structures
Model of the PTF1-J Trimeric Complex
Binding Interactions for E12, RBP and Ptf1a
Structural Modeling of the PTF1-J and PTF1-L Heterotrimeric Transcription Factor Complexes
Abstract [LB171]
Current Directions
•Generate bacterial expression systems for Rbpj, Rbpjl, Ptf1a and
E12.
•Optimize protein expression levels for the engineered constructs by
varying temperature, time of induction with IPTG and bacterial
strains.
•Perform Nickel NTA affinity purification.
•Determine crystallization conditions for the PTF1-J and PTF1-L
heterotrimeric complexes
References
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bHLH complex, Genes Dev. 2007 Oct 15;21(20):2629-43.
Toshihiko Masui, Qiaoming Long, Thomas M. Beres, Mark A. Magnuson, and Raymond J. MacDonald, Early pancreatic development requires the vertebrate
Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex, Genes Dev. 2007 October 15; 21(20):2629–2643.
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Model of the PTF1-J Trimeric Complex
Model of the PTF1-J Trimeric Complex
Figure 1a: Side view of the ribbon diagram of the PTF1-J
heterotrimeric transcription factor complex; E12 is shown in
green, Ptf1a is shown in red and mammalian Rbpj is shown in
multiple colors representing the three domains of the protein.
Figure 1b: Side view of the electrostatic surface potential
representation of the PTF1-J heterotrimeric transcription
factor complex; blue represents areas with a positive surface
potential, red represents areas with a negative surface
potential and white represents areas with a neutral surface
potential.
Figure 2a: Top view of the ribbon diagram of the PTF1-J
heterotrimeric transcription factor complex; E12 is shown in
green, Ptf1a is shown in red and mammalian Rbpj is shown in
multiple colors representing the three domains of the protein.
Figure 2b: Top view of the electrostatic surface potential
representation of the PTF1-J heterotrimeric transcription factor
complex; blue represents areas with a positive surface
potential, red represents areas with a negative surface potential
and white represents areas with a neutral surface potential.
Figure 3a: Top view rotated 90 degrees counter clockwise of
the ribbon diagram of the PTF1-J heterotrimeric transcription
factor complex; E12 is shown in green, Ptf1a is shown in red
and mammalian Rbpj is shown in multiple colors representing
the three domains of the protein.
Figure 3a: Top view rotated 90 degrees counter clockwise of
the electrostatic surface potential representation of the Rbpj
transcription factor; blue represents areas with a positive
surface potential, red represents areas with a negative surface
potential and white represents areas with a neutral surface
potential.
Figure 4a: End view of the ribbon diagram of the Rbpj
transcription factor; Rbpj is shown in multiple colors
representing the three domains of the protein.
Figure 4b: End view of the electrostatic surface potential
representation of the Rbpj transcription factor; blue represents
areas with a positive surface potential, red represents areas
with a negative surface potential and white represents areas
with a neutral surface potential.
Figure 5a: End view of the ribbon diagram of the E12/Ptf1a
heterodimer transcription factor complex; E12 is shown in
green and Ptf1a is shown in red.
Figure 5b: End view of the electrostatic surface potential
representation of the E12/PTF1a heterodimer transcription
factor complex; blue represents areas with a positive surface
potential, red represents areas with a negative surface potential
and white represents areas with a neutral surface potential.
Figure 6a: Ribbon diagram of the PTF1-J heterotrimeric
transcription factor complex; E12 is shown in green, Ptf1a is
shown in red, the modeled interaction of the C2 sequence of
Ptf1a with the beta-trefoil domain of Rbpj is shown in red and
Rbpj is shown in multiple colors representing the three
domains of the protein.
Figure 6b: Ribbon diagram of the PTF1-L heterotrimeric
transcription factor complex; E12 is shown in green, Ptf1a is
shown in red, the modeled interaction of the C2 sequence of
Ptf1a with the beta-trefoil domain of Rbpjl is shown in red and
Rbpl is shown in multiple colors representing the three
domains of the protein.
Figure 7: Alpha carbon trace of the ZDOCK structure solutions of
the C2 sequence of Ptf1a bound to the beta-trefoil domain of
mammalian Rbpj shown in blue.