Statistical analysis of amino acid patterns in transmembrane helices: The GxxxG motif occurs frequently and in association with beta-branched residues at neighboring positions

To find motifs that mediate helix-helix interactions in membrane proteins, we have analyzed frequently occurring combinations of residues in a databas e of transmembrane domains. Our analysis was performed with a novel formali sm, which we call TMSTAT, for exactly calculating the expectancies of all p airs and triplets of residues in individual sequences, taking into account differential sequence composition and the substantial effect of finite leng th in short segments. We found that the number of significantly over and un der-represented pairs and triplets was much greater than the random expecta tion. Isoleucine, glycine and valine were the most common residues in these extreme cases. The main theme observed is patterns of small residues (Gly, Ala and Ser) at i and i + 4 found in association with large aliphatic resi dues (Ile, Val and Leu) at neighboring positions (i.e. i +/- 1 and i +/- 2) . The most over-represented pair is formed by two glycine residues at i and i + 4 (GxxxG,31.6% above expectation, p<1x10(-33)) and it is strongly asso ciated with the neighboring beta-branched residues lie and Val. In fact, th e GxxxG pair has been described as part of the strong interaction motif in the glycophorin A transmembrane dimer, in which the pair is associated with two Val residues (GVxxGV). GxxxG is also the major motif identified using TOXCAT, an in vivo selection system for transmembrane oligomerization motif s. In conjunction with these experimental observations, our results highlig ht the importance of the GxxxG + beta-branched motif in transmembrane helix -helix interactions. In addition, the special role for the beta-branched re sidues lie and Val suggested here is consistent with the hypothesis that re sidues with constrained rotameric freedom in helical conformation might red uce the entropic cost of folding in transmembrane proteins. Additional mate rial is available at http://engelman.csb.yale. edu/tmstat and http://bioinf o.mbb.yale.edu/tmstat. (C) 2000 Academic Press.