RUN-CBFβ Interaction in C. elegans: Computational Prediction and Experimental Veriication http://www.jbsdonline.com Abstract The Runt domain proteins are eukaryotic transcription factors that regulate major develop- mental pathways. All members of this family contain a highly-conserved sequence-spe- ciic DNA binding domain: the Runt domain (RD). Structural and biochemical studies have shown that the Runt domain undergoes a conformational transition upon binding to DNA and that this process is regulated by an unrelated partner protein CBFβ that enhances the DNA binding afinity of RD. Most of the reported studies on the Runt domain transcription fac- tors were performed on proteins from mammals and Drosophila whereas very little has been known about the C. elegans RD protein, RUN, which provides the simplest model system for understanding the function of this class of transcription factors. We performed computational studies on RD domains from various species including C. elegans, Drosophila, and human, using the atom-atom contact surface area scoring method. The scoring analysis indicates that the DNA binding regulation of the C. elegans RD protein (CeRD) occurs via its interac- tion with a CBFβ-like partner, as found for the human proteins, whereas a different mode of regulation may occur in the Drosophila system. Sequence, secondary structure and fold analyses of a putative CBFβ protein identiied in the C. elegans genome, CeCBFβ, sharing a 22% identity with the human protein, predict a similar structure of this protein to that of the human CBFβ protein. We produced the C. elegans proteins CeRD and CeCBFβ in bacteria and conirmed their physical interaction as well as cross interactions with the corresponding human proteins. We also conirmed the structural similarity of CBFβ and CeCBFβ by circu- lar dichroism analysis. The combined results suggest that a similar mechanism of regulation operates for the human and the C. elegans RD proteins despite the low sequence identity between their CBFβ proteins and the evolutionary distance between the two systems. Keywords: Runt domain; CBFβ; Contact surface area; Energetic scores; Allosteric transition. Introduction The Runt domain (RD) proteins belong to a small group of transcription factors with vital roles in developmental pathways ranging from sex determination and segmentation in Drosophila to osteogenesis and hematopoiesis in mammals (1- 3). These proteins known also as RUNX, following the Nomenclature Committee of the Human Genome Organization (HUGO) were previously known as CBFα, AML, or PEBP2α (4). The RD proteins have been found in different organisms including human, mouse, purple sea urchin, frog, zebra ish, fruit ly, and nematode. All members of this family share a conserved 128 amino acid region called the Runt domain (RD) (Fig. 1) (5). RD conducts two central functions of these proteins: (i) It binds to a consensus DNA sequence, PyGPyGGTPy (Py-pyrimidine) (6), by which it regulates transcription of target genes; (ii) It interacts with an unrelated partner Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 24, Issue Number 4, (2007) ©Adenine Press (2007) Oded Suad 1,§ Eran Eyal 2,§ Immanuel Blumenzweig 1 Naama Kessler 1 Ditsa Levanon 3 Yoram Groner 3 Zippora Shakked 1,* 1 Department of Structural Biology Weizmann Institute of Science Rehovot 76100, Israel 2 Department of Plant Sciences Weizmann Institute of Science Rehovot 76100, Israel 3 Department of Molecular Genetics Weizmann Institute of Science Rehovot 76100, Israel § These authors contributed equally to this work. 343 * Phone: 972-8-9342672 Fax: 972-8-9344154 Email: zippi.shakked@weizmann.ac.il Abbreviations: RD, Runt domain; hRD, Human Runt domain; CeRD, C. elegans Runt domain; CBFβ, Core-binding factor beta; CeCBFβ, C. elegans core-binding factor beta; MBP, Maltose-binding protein.