Download presentation
Presentation is loading. Please wait.
Published byTobias Byron Adams Modified over 5 years ago
1
Crystal Structure of PU.1/IRF-4/DNA Ternary Complex
Carlos R. Escalante, Abraham L. Brass, Jagan M.R. Pongubala, Ella Shatova, Leyi Shen, Harinder Singh, Aneel K. Aggarwal Molecular Cell Volume 10, Issue 5, Pages (November 2002) DOI: /S (02)
2
Figure 1 Ets and IRF Family Members
(A) The sequences of mouse (m) PU.1 and human (h) Spi-B Ets domains aligned with representative members (mEts-1, hSAP-1α, mGABPα, mFli-1, mElf-1, mERF, mER81, mTel-1, and hE4TF) of other subfamilies of Ets proteins (Graves and Petersen, 1998). Shown above the sequences are the locations of α helices, β strands, and loops in PU.1. The residues at the PU.1-IRF-4 interface—mediating direct protein-protein interactions—are highlighted in red. (B) The sequences of mIRF-4 (Pip) and mIRF-8 (ICSBP) DNA binding domains aligned with other IRF family members. Shown above the sequences are the locations of α helices, β strands, and loops in IRF-4. The residues at the PU.1-IRF-4 heterodimer interface are highlighted in red (c.f., Figure 3A). Molecular Cell , DOI: ( /S (02) )
3
Figure 2 Protein and DNA Structure
(A) An overview of the PU.1/IRF-4/DNA ternary complex. The PU.1 Ets domain (red) and IRF-4 DNA binding domain (blue) are accommodated on opposite faces of the DNA (gold). The residues involved in direct protein-protein interactions are drawn at the back of the DNA. The figure was generated with program MOLMOL (Koradi et al., 1996). (B) PU.1 and IRF-4 bend the DNA into an “S shape.” PU.1 binds primarily to the top half of the DNA, while IRF-4 binds primarily to the bottom half (c.f., Figure 2A). The DNA was analyzed with the program arcFit (Slickers et al., 1998). (C) A continuous van der Waals surface extends between PU.1 and IRF-4. The surface, computed with program GRASP (Nicholls et al., 1991), is colored according to electrostatic properties, where red to blue spans −10 to +10 kT in electrostatic potential. The view corresponds to a 180° rotation about the DNA axis, compared to that in (A). Molecular Cell , DOI: ( /S (02) )
4
Figure 3 Protein-Protein Interactions
(A) A detailed view of the interactions between the PU.1 Ets domain (red) and the IRF-4 DNA binding domain (blue). Arg222 and Lys 223 in PU.1 reach across the DNA minor groove to form salt links with Asp117 in IRF-4. The Arg222 side chain makes hydrophobic contacts with Val111 and Leu116 in IRF-4. In addition, a spine of six water molecules (green circles) traverse the minor groove, connecting Lys219 in PU.1 to His56 in IRF-4 through a network of hydrogen bonds (blue dotted lines). (B) IRF-2 modeled in place of IRF-4 to illustrate the basis of anticooperativity between PU.1 and IRF-1. We selected IRF-2 for the modeling because its structure is more accurately determined than that of IRF-1 (Escalante et al., 1998; Fuji et al., 1999). Asn100 and Lys101 in IRF-1 are equivalent to Lys100 and Lys101 in IRF-2. Lys101 in IRF-1 would be sterically and electrostatically replused by Lys223 in PU.1, in the way shown here for Lys101 in IRF-2. (C) The conformation of Arg222 in PU.1 is compared in the absence (−IRF-4) and in the presence (+IRF-4) of IRF-4. The −IRF-4 conformation is derived from the structure of PU.1/DNA binary complex (Kodandapani et al., 1996). The DNA is drawn from the binary complex. Molecular Cell , DOI: ( /S (02) )
5
Figure 4 Mutagenesis (A) Mutation of residues at the PU.1/IRF-4 heterodimer interface followed by gel electrophoresis using the λB composite site. DNA binding reactions carried out in the presence of varying concentrations of wild-type PU.1 (aa 160–272) or PU.1 mut (R222A, K223A) Ets domains in the absence or presence of IRF-4 DBD (aa 19–139). (B) DNA binding reactions carried out in the presence of varying concentrations of wild-type IRF-4 (aa 19–139) or IRF-4 mut (V111A, L116A, D117A) in the absence or presence of PU.1 Ets domain. Molecular Cell , DOI: ( /S (02) )
6
Figure 5 DNA-Protein Interactions
(A) Sketches summarizing the protein-DNA contacts. PU.1 residues and the Ets core (GGAA) are highlighted in red, while IRF-4 and the “traditional” IRF core (GAAA) are highlighted in blue. Direct hydrogen bonds to DNA bases in the major groove and the sugar-phosphate backbone are shown as solid lines, and water-mediated contacts as dashed lines. Shown alongside are contacts to the Ets core in the minor groove, from His56 belonging to IRF-4. (B) A view along PU.1 DNA-recognition helix (α3), showing residues Gln228, Arg232, Arg235, and Asn236 interacting with bases in the DNA major groove. (C) A view along the IRF-4 DNA recognition helix showing residues Arg98, Cys99, Asn102, and Lys103 interacting with bases in the DNA major groove. Note that Lys103 donates a hydrogen bond to a guanine outside of the traditional GAAA core sequence. Hydrogen bonds are shown as dashed lines. Molecular Cell , DOI: ( /S (02) )
Similar presentations
© 2024 SlidePlayer.com. Inc.
All rights reserved.