In this study, the researchers: 1) Generated structural models of the C2 sequence of Ptf1a bound to the β-trefoil domains of mammalian Rbpj and Rbpjl. 2) Found that the C2 sequence binds in an extended conformation, maintaining interactions seen in the Notch-IC/Rbpj complex. 3) Observed conserved residues in the hydrophobic pocket that maintain binding of proteins with a conserved tryptophan motif.

1. Structural Modeling of the Ptf1a C2 Sequence Bound to Mammalian Rbpj and Rbjl
Nicholas Osborn1
, Zachary Fein1
, Brett Mayberry1
, Raymond MacDonald2
, Ward Coats1
1
Hillcrest High School, Dallas, Texas
2
Department of Molecular Biology, University of Texas: Southwestern Medical Center, Dallas, Texas
Abstract:
Abstract [LB168]
RAM Domain of Notch-IC and Ptf1a C2 Sequence
β-trefoil Domains of Rbpj and Rbpjl
RAM Notch-IC PGNRTRKRRMINASVWMPPMENEEKNRK
Ptf1a C2 EKQLKEQNIIRTAKVWTPEDPRKLNSKS
RAM Motif ΦWΦP
Sequence Alignment of the
β-Trefoil Domains (BTD) of Rbpl and Rbpjl
As a means to introduce biomedical research to high school students, the
MacDonald laboratory at UT Southwestern Medical Center at Dallas has
engaged students from Hillcrest High School in an ongoing research program
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. Tryptophan 298 located in a
conserved sequence (C2) near the C terminus of Ptf1a is required for the
recruitment of Rbpj into the PTF1-J complex. The binding of the RAM domain
of Notch-IC to a hydrophobic pocket in Rbpj involves residues in a conserved
hydrophobic, tryptophan, hydrophobic, proline (ΦWΦP) motif that is also
present in the C2 sequence of Ptf1a suggesting a similar binding interaction for
tryptophan 298. We have modeled the C2 sequence of Ptf1a onto the crystal
structure of the RAM domain of Notch-IC bound to Rbpj to characterize this
binding interaction and to generate structural models for the complex of the C2
sequence of Ptf1a with mammalian Rbpj and Rbpjl.
Complex of the C. elegans Rbpj β-trefoil Domain with the RAM Domain of Notch-IC
Complex of the Mammalian Rbpj β-trefoil Domain with the C2 Sequence of Ptf1a
Complex of the Mammalian Rbpjl β-trefoil Domain with the C2 Sequence of Ptf1a
Salt Bridge Interactions: RAM and C. elegans Rbpj BTD
A/ASP`431/OD1 E/ARG`937/NH2 3.2
A/ASP`446/OD2 E/ARG`938/NH2 2.8
Main Chain Interactions: RAM and C. elegans Rbpj BTD
E/LYS`936/O * A/ASP`446/N 2.9
E/ARG`938/O A A/VAL`444/O 1.8
E/ARG`938/N B A/VAL`444/N 1.9
E/ILE`940/N C A/PHE`442/O 2.0
E/ILE`940/O D A/PHE`442/N 2.2
E/SER`943/O F A/ALA`465/N 1.8
E/TRP`945/H H A/ALA`465/O 1.9
E/PRO`948/O I A/GLN`517/N 1.8
______________________________________________________________________________________________________________________
Main Chain Interactions: Ptf1a C2 and Rbpj BTD
PTF1a_C2//E/GLN`288/O * RBPJ//C/ASP`225/N 1.9
PTF1a_C2//E/ILE`290/N A RBPJ//C/VAL`223/O 2.0
PTF1a_C2//E/ILE`290/O B RBPJ//C/VAL`223/N 1.8
PTF1a_C2//E/ARG`293/N C RBPJ//C/PHE`221/O1.8
PTF1a_C2//E/ARG`293/O D RBPJ//C/PHE`221/N 2.3
PTF1a_C2//E/ARG`293/O E RBPJ//C/GLU`220/N 2.9
PTF1a_C2_E/LYS`296/N F RBPJ//C/GLY`242/O 2.2
PTF1a_C2_E/LYS`296/O G RBPJ//C/ALA`244/N 2.0
PTF1a_C2//E/TRP`298/N H RBPJ//C/ALA`244/O 2.0
PTF1a_C2//E/GLU`301/O I RBPJ //C/GLN`293/N 1.7
______________________________________________________________________________________________________________________
Main Chain Interactions: Ptf1a C2 and Rbpjl BTD
PTF1a_C2//E/GLN`288/O * RBPJL//GLU`264/N 2.4
PTF1a_C2//E/ILE`290/N A RBPJL//PRO`262/O 2.2
PTF1a_C2//E/ARG`293/N C RBPJL//PHE`260/O 1.8
PTF1a_C2//E/ARG`293/O D RBPJL//PHE`260/N 2.1
PTF1a_C2//E/LYS`296/N F RBPJL//THR`281/O 2.0
PTF1a_C2//E/LYS`296/O G RBPJL//THR`283/N 1.9
PTF1a_C2//E/TRP`298/N H RBPJL//THR`283/O 2.1
PTF1a_C2//E/GLU`301/O I RBPJL//ARG`336/N 1.8
Salt Bridge and Main Chain Interactions
The Hydrophobic Pocket of the β-trefoil Domain of Rbpj and Rbpjl
Summary
In the current study we have performed structural modeling of the
C2 sequence of Ptf1a onto the known crystal structure of the RAM
domain of Notch-IC bound to the C. elegans Rbpj protein.
We have generated structural models of the C2 sequence of Ptf1a
bound to the β-trefoil domains of mammalian Rbpj and Rbpjl.
In our models, the C2 sequence of Ptf1a binds to the β-trefoil
domain of mammalian Rbpj and Rbpjl in an extended polypeptide
conformation and maintains the main chain and hydrophobic,
tryptophan, hydrophobic, proline interactions that are present in
the structure of the RAM domain of Notch-IC Rbpj complex.
Superimposition of the β-trefoil domains and inspection of the
hydrophobic pocket reveals that these residues have been
conserved through evolution thus maintaining the ability of Rbpj
and Rbpjl transcription factors to bind proteins that have a
hydrophobic, tryptophan, hydrophobic, proline motif.
References
Thomas M. Beres, Toshihiko Masui, Galvin H. Swift, Ling Shi, R. Michael Henke, and Raymond J. MacDonald, PTF1 Is an
Organ-Specific and Notch-Independent Basic Helix-Loop-Helix Complex Containing the Mammalian Suppressor of Hairless
(RBP-J) or Its Paralogue, RBP-L, Mol Cell Biol. 2006 January; 26(1): 117–130.
Masui T, Long Q, Beres TM, Magnuson MA, MacDonald RJ, Early pancreatic development requires the vertebrate Suppressor of
Hairless (RBPJ) in the PTF1 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.
Wilson JJ, Kovall RA, Crystal structure of the CSL-Notch-Mastermind ternary complex bound to DNA Cell. 2006 Mar
10;124(5):985-96.
Nam Y, Sliz P, Song L, Aster JC, Blacklow SC., Structural basis for cooperativity in recruitment of MAML coactivators to Notch
transcription complexes, Cell. 2006 Mar 10; 124(5):973-83.
Arnold K., Bordoli L., Kopp J., and Schwede T. (2006). The SWISS-MODEL Workspace: A web-based environment for protein
structure homology modelling. Bioinformatics, 22,195-201.
Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T (2009). The SWISS-MODEL Repository and associated resources. Nucleic
Acids Research. 37, D387-D392.
Peitsch, M. C. (1995) Protein modeling by E-mail Bio/Technology 13: 658-660.
Figure 1a: Ribbon diagram of the β-trefoil domain of the C. elegans Rbpj
protein in yellow bound to a stick representation of the Notch-IC domain in
cyan. Residues present in the hydrophobic, tryptophan, hydrophobic,
proline motif are shown in green, blue and cyan respectively.
Figure 2a: Electrostatic surface potential map of the -trefoil domain of the
C. elegans Rbpj protein in red (negative), blue (positive) and white
(neutral) bound to a stick representation of the Notch-IC Domain in cyan.
Residues present in the hydrophobic, tryptophan, hydrophobic, proline
motif are shown in green, blue and cyan respectively.
Figure 3a: Main chain interactions (A, B, C and D, see table 1) between the
C. elegans Rbpj β-trefoil domain in yellow and the Notch-IC domain in
cyan.
Figure 1b: Ribbon diagram of the β-trefoil domain of the mammalian Rbpj
protein in green bound to a stick representation of the C2 sequence of Ptf1a
in red. Residues present in the hydrophobic, tryptophan, hydrophobic,
proline motif are shown in yellow, blue and red respectively.
Figure 2b: Electrostatic surface potential map of the β-trefoil domain of
the mammalian Rbpj protein in red (negative), blue (positive) and white
(neutral) bound to a stick representation of the C2 sequence of Ptf1a in red.
Residues present in the hydrophobic, tryptophan, hydrophobic, proline
motif are shown in yellow, blue and red respectively.
Figure 3b: Main chain interactions (A, B, C and D, see table 1) between
the mammalian Rbpj β-trefoil domain in green and the C2 sequence of
Pft1a in red.
Figure 1c: Ribbon diagram of the β-trefoil domain of the mammalian
Rbpjl protein in blue bound to a stick representation of the C2 sequence of
Ptf1a in red. Residues present in the hydrophobic, tryptophan,
hydrophobic, proline motif are shown in yellow, green and red
respectively.
Figure 2c: Electrostatic surface potential map of the β-trefoil Domain of
the mammalian Rbpjl protein in red (negative), blue (positive) and white
(neutral) bound to a stick representation of the C2 sequence of Ptf1a in red.
Residues present in the hydrophobic, tryptophan, hydrophobic, proline
motif are shown in yellow, green and red respectively.
Figure 3c: Main chain interactions (A, C and D, see table 1) between the
mammalian Rbpj β-trefoil domain in blue and the C2 sequence of Pft1a in
red.
Figure 1d: Alpha carbon trace of the C. elegans
Rbpj β-trefoil domain in yellow and the residues
lining the hydrophobic pocket in green.
Figure 2d: Alpha carbon trace of the mammalian
Rbpj β-trefoil domain in green and the residues
lining the hydrophobic pocket in yellow.
Figure 3d: Alpha carbon trace of the mammalian
Rbpjl β-trefoil domain in blue and the residues
lining the hydrophobic pocket in red.
Figure 4d: Superposition of the alpha carbon trace
of the β-trefoil domains for C. elegans Rbpj
(yellow), mammalian Rbpj (green) and mammalian
Rbpjl (blue). Residues forming the hydrophobic
pockets are green for C. elegans Rbpj, yellow for
mammalian Rpbj and red for mammalian Rpbjl.
Figure 5c: Superposition of the alpha carbon trace of the β-trefoil domains for C. elegans Rbpj
(yellow), mammalian Rbpj (blue) and mammalian Rbpjl (green) bound to the alpha carbon trace
of the Notch-IC domain (cyan), the C2 sequence of Ptf1a-Rbpj (blue) and the C2 sequence of
Ptf1a-Rbpjl (magenta).
Hillcrest Biomedical Research Group: Back row; Dr. Raymond MacDonald, Dr. Ward Coats,
Brett Mayberry, Parker Johnson, Nicholas Osborn. Front row; Regis Guthery, Zachary Fein,
Brandon Boardman, Jessie Degani,