Small Residues Inhibit Homo-Dimerization of the Human Carbonic Anhydrase XII Transmembrane Domain.

Florian Cymer,Dirk Schneider

doi:10.3390/membranes11070512

Abstract

Amino acids with small side chains and motifs of small residues in a distance of four are rather abundant in human single-span transmembrane helices. While interaction of such helices appears to be common, the role of the small residues in mediating and/or stabilizing transmembrane helix oligomers remains mostly elusive. Yet, the mere existence of (small)xxx(small) motifs in transmembrane helices is frequently used to model dimeric TM helix structures. The single transmembrane helix of the human carbonic anhydrases XII contains a large number of amino acids with small side chains, and critical involvement of these small amino acids in dimerization of the transmembrane domain has been suggested. Using the GALLEX assay, we show here that the transmembrane domain indeed forms a strong transmembrane helix oligomer within a biological membrane. However, single or multiple mutations of small residue(s) to isoleucine almost always increased, rather than decreased, the interaction propensities. Reduction of helix flexibility and of protein–lipid contacts caused by a reduced lipid accessible surface area likely results in stabilization of helix–helix interactions within the membrane.

Highlights

While studied to a great extent in recent decades, the principles guiding folding of α-helical membrane proteins and proper assembly on individual α-helices are still only rudimentarily understood
The human carbonic anhydrase (CA) XII is a bitopic membrane protein located in the plasma membrane of different tissues
As the catalytically active domains are dimeric in solution, and the TM domains are rich in small residues and containxxx(small) motifs, a dimeric TM helix structure was assumed and modeled using the glycophorin A (GpA) dimer structure as a template [39], without experimental proof

Summary

Introduction

While studied to a great extent in recent decades, the principles guiding folding of α-helical membrane proteins and proper assembly on individual α-helices are still only rudimentarily understood. The human GpA TM helix became a paradigm for studying sequence specificity in TM helix oligomerization. The sequence LIxxGVxxGVxxT drives the interaction of the GpA TM helix [1], and especially the motif of two Gly residues in a distance of four (the GxxxG-motif) has been identified to be key for dimerization [1,2,3]. In an in vivo screen selecting sequences driving strong homo-dimerization of a random library of TM helices, the GxxxG-motif appeared [4], which indicated a more generalized importance of this motif in mediating TM helix interactions. Further studies have indicated that other residues with small side-chains (Ala, Ser, Cys) can mediate sequence-specific TM helix dimerization (discussed in [6,7]), and the motif is better described as “(small)xxx(small)”

Methods

Results

Conclusion