Sequence motifs, polar interactions and conformational changes in helical membrane proteins A Rachael Curran and Donald M Engelman  The a helices of transmembrane proteins interact to form higher order structures. These interactions are frequently mediated by packing motifs (such as GxxxG) and polar residues. Recent structural data have revealed that small sidechains are able to both stabilize helical membrane proteins and allow conformational changes in the structure. The strong interactions involving polar sidechains often contribute to protein misfolding or malfunction. Addresses Department of Molecular Biophysics and Biochemistry, Yale University, PO Box 208114, New Haven, CT 06520-8114, USA  e-mail: donald.engelman@yale.edu Current Opinion in Structural Biology 2003, 13:412–417 This review comes from a themed issue on Membranes Edited by Eric Gouaux and Stephen H White 0959-440X/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0959-440X(03)00102-7 Abbreviations bR bacteriorhodopsin CFTR cystic fibrosis conductance regulator GpA glycophorin A MCP major coat protein MscL mechanosensitive channel of large conductance MscS mechanosensitive channel of small conductance PDGF bR platelet-derived growth factor b receptor TM transmembrane Introduction Transmembrane (TM) proteins, which represent 20–30% of all open reading frames in sequenced genomes, form the functional basis of biological compartmentalization at the cellular level. However, to date, there are only 50 unique high-resolution structures of membrane proteins com- pared with thousands of their water-soluble counterparts (see http://blanco.biomol.uci.edu/Membrane_Proteins_ xtal.html). Excepting b-barrel structures, TM helix inter- actions are a dominant theme. Several different approaches have been successful in furthering our understanding of how helices interact in TM proteins. Here, we examine key features that have become apparent: sequence motifs, polar interactions and conformational changes. Membrane-spanning domains are composed mainly of a helices, whose associations within the cellular membrane are generally governed by electrostatic and van der Waals interactions. Other factors, such as ligand binding [1,2] and the folding of extramembranous loops [3,4], also contribute to the packing of TM helices. The folding of helical TM proteins may include two energetically distinct stages. The first stage sees the formation and insertion of a helices into the membrane and, in the second stage, these preformed helices interact within the cellular membrane. A further step, whereby addi- tional structure re-enters the membrane, may be subse- quent to the first two. The free energy change associated with helix–helix interaction in detergent micelles has recently been investigated [5]. Analysis of the helical packing within membranes has revealed that the most favored packing angle is left handed, at approximately 208 [6] (compared to right- handed at 358 for water-soluble proteins), although right-handed contacts do occur. Left-handed packing increases the interfacial area between the helices. The general packing of a helices in membrane proteins has been described by the ‘knobs-into-holes’ packing model first described for soluble coiled coils [7]. However, it has become clear that there is much more detail that needs to be considered if we are to develop a predictive under- standing of helix interactions in membrane proteins. Of specific interest is the role of sequence motifs and polar residues. Here, we discuss their involvement in mediat- ing helix–helix interactions in membrane proteins and the functional nature of such contacts. Sequence motifs Many concepts of how helices interact in membranes come from work on the TM domain of glycophorin A (GpA). GpA is a dimeric protein expressed on the surface of erythrocytes. Saturation mutagenesis studies have defined the dimeric interface of the TM domain as consisting of a right-handed seven-residue motif — LIxxGVxxGVxxT [8]. This interface was later confirmed by NMR studies [9]. More recently, general sequence motifs have emerged as important mediators of interac- tions between TM helices. Studies of the TM domain of the major coat protein (MCP) from bacteriophage also highlighted a GxxxG dimerization motif [10,11]. The GxxxG (GG 4 ) motif found in GpA and MCP has been shown to mediate strong helix association for several different TM domain sequences in a genetic screen of the inner membrane of Escherichia coli [12]. This same motif was highlighted in a survey of pairwise interactions as being the most statistically significant over-represented pair of amino acids in single-pass TM helices [13]. Other over-represented pairs include II 4 , GA 4 and IG 1 . The 412 Current Opinion in Structural Biology 2003, 13:412–417 www.current-opinion.com