Genetic Selection for and Molecular Dynamic Modeling of a Protein Transmembrane Domain Multimerization Motif from a Random Escherichia coli Genomic Library Jennifer A. Leeds 1 , Dana Boyd 1 , Damon R. Huber 2 , G. Koji Sonoda 3 Hieu T. Luu 1 , Donald M. Engelman 3 and Jon Beckwith 1 * 1 Department of Microbiology and Molecular Genetics Harvard Medical School Boston, MA 02115, USA 2 Program in Biological and Biomedical Sciences, Harvard Medical School, Boston MA 02115, USA 3 Department of Molecular Biophysics and Biochemistry Yale University, New Haven CT 06520, USA In order to identify new transmembrane helix packing motifs in naturally occurring proteins, we have selected transmembrane domains from a library of random Escherichia coli genomic DNA fragments and screened them for homomultimerization via their abilities to dimerize the bacterio- phage l cI repressor DNA-binding domain. Sequences were isolated using a modi®ed l cI headpiece dimerization assay system, which was shown previously to measure transmembrane helix-helix association in the E. coli inner membrane. Screening resulted in the identi®cation of sev- eral novel sequences that appear to mediate helix-helix interactions. One sequence, representing the predicted sixth transmembrane domain (TM6) of the E. coli protein YjiO, was chosen for further analysis. Using site- directed mutagenesis and molecular dynamics, a small set of models for YjiO TM6 multimerization interface interactions were generated. This work demonstrates the utility of combining in vivo genetic tools with computational systems for understanding membrane protein structure and assembly. # 2001 Academic Press Keywords: library; transmembrane; Escherichia coli; multimerization; lambda repressor *Corresponding author Introduction Integral membrane proteins play essential roles in numerous cellular functions, including cell div- ision, intra and inter-cellular signaling, and trans- port of macromolecules. 1±4 Mutations affecting structure and assembly of membrane proteins often lead to cell toxicity in microbes and pathol- ogies such as cystic ®brosis and Alzheimer's dis- ease in humans. 5,6 With the advent of modern genomics, integral membrane proteins have gained much attention. For example, they are often ident- i®ed from genome-wide viability screens for new drug targets. Yet compared to soluble proteins, suf®cient tools generally do not exist to map the interactions that are required for tertiary structure and oligomeric assembly of integral membrane proteins. In depth structural studies of several model inte- gral membrane proteins such as the human eryth- rocyte protein glycophorin A (GpA) and phospholamban have de®ned some basic rules of membrane protein structure and assembly. Integral membrane proteins often depend on their trans- membrane (TM) segments for assembly into func- tional structures (for reviews, see 7,8 ). TM domains are generally thought to adopt an alpha-helical sec- ondary structure, with tertiary structure being determined by interhelical interactions between these preformed domains. 9 The types of helix-helix interactions that can occur among integral mem- brane proteins are thought to consist primarily of van der Waals interactions, 10 ± 12 with polar interactions sometimes helping to drive the Present addresses: G. K. Sonoda, DoubleTwist, 2001 Broadway, Oakland, CA 94612, USA; H. T. Luu, Ursinus College, Collegeville, PA 19426, USA. Abbreviations used: TM, transmembrane; l, bacteriophage lambda; GpA, glycophorin A; pfu, plaque-forming units; ES, export signal; E.O.P, ef®ciency of plating; CTD, C-terminal domain; MBP, maltose- binding protein; ss, signal sequence; ORF, open reading frame. E-mail address of the corresponding author: jbeckwith@hms.harvard.edu doi:10.1006/jmbi.2001.5007 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 313, 181±195 0022-2836/01/010181±15 $35.00/0 # 2001 Academic Press