The fold of a helical membrane protein is largely determined by interactions between membrane-imbedded helices. To elucidate recurring helix-helix interaction motifs, we dissected the crystallographic structures of membrane proteins into a library of interacting helical pairs. The pairs were clustered according to their three-dimensional similarity (rmsd <1.5 Å), allowing 90% of the library to be assigned to clusters consisting of at least five members. Surprisingly, three quarters of the helical pairs belong to one of five tightly clustered motifs whose structural features can be understood in terms of simple principles of helix-helix packing. Thus, the universe of common transmembrane helix-pairing motifs is relatively simple. The largest cluster, which comprises 29% of the library members, consists of an antiparallel motif with left-handed packing angles, and it is frequently stabilized by packing of small side chains occurring every seven residues in the sequence. Righthanded parallel and antiparallel structures show a similar tendency to segregate small residues to the helix-helix interface but spaced at four-residue intervals. Position-specific sequence propensities were derived for the most populated motifs. These structural and sequential motifs should be quite useful for the design and structural prediction of membrane proteins.helix-helix packing ͉ protein design ͉ structure prediction H elical transmembrane (TM) proteins are a major class of membrane proteins that are critically involved in functionally rich processes, including bioenergetics, signal transduction, ion transmission, and catalysis. The mechanisms by which TM proteins fold into native structures are beginning to be understood from a confluence of structural and biochemical studies (1-3). The determinants of a membrane protein's fold can be understood by dissecting its structure into pairs of interacting helices, which, together with the connecting loops and extramembrane domains, comprise the overall structure. Along these lines, various workers have examined the geometric characteristics of helix-helix interactions in membrane proteins (4) as well as the features in the amino acid sequences that predispose the helices to adopt these geometries. One outstanding success has been the recognition of the GX 3 G motif, in which Gly (or other small residues) spaced four residues apart mediate a close approach of TM helices (5-7). This motif was first observed in the TM domain of glycophorin A (GpA) (5) and has subsequently been found in both water-soluble (8) and membrane proteins (9). In the classical GpA GX 3 G motif, the helices cross with a right-handed crossing angle of Ϸ40°. In a different TM motif stabilized by ''knobs-into-holes'' packing (10), the helices cross with a smaller left-handed crossing angle.Other surveys of helix-helix packing in membrane proteins have focused on distributions of the interhelical angles, distances, and the composition of the side chains packed at the interface. The helix-helix interfaces tend to be well pa...

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