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Entrapment of Water at the Transmembrane Helix−Helix Interface of Quiescin Sulfhydryl Oxidase 2 Christian L. Ried,†,§ Christina Scharnagl,‡ and Dieter Langosch*,† †
Lehrstuhl Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, and Munich Center For Integrated Protein Science (CIPSM), Germany ‡ Fakultät für Physik E14, Technische Universität München, Maximus-von-Imhof-Forum 4, 85354 Freising, Germany S Supporting Information *
by the finding that one of its existing variants, QSOX1A, could be cross-linked to a dimer via the Cys residue of its TMD.14 First, self-interaction of the human QSOX2 TMD was experimentally assessed by the ToxR assay. The ToxR system reports TMD−TMD interaction in the native membrane environment of Escherichia coli.15 The β-galactosidase signal, a measure of homotypic affinity, that is elicited by the QSOX2 TMD is close to that of the glycophorin A TMD,12 a structurally well-characterized reference known to dimerize with high affinity.16 To test which of the residues that comprise the highly conserved face of the TMD helix [664CvxLYxxSSxxLxxMY (Figure S1)] support helix−helix interaction, we conducted scanning mutagenesis (Figure 1). Most residues were mutated to Ala; A670 was converted to Leu, and C664 was exchanged for Ser to test whether a potentially existing weak Cys−Cys H-bond could be exchanged
ABSTRACT: Little is known about how a membrane can regulate interactions between transmembrane helices. Here, we show that strong self-interaction of the transmembrane helix of human quiescin sulfhydryl oxidase 2 rests on a motif of conserved amino acids comprising one face of the helix. Atomistic molecular dynamics simulations suggest that water molecules enter the helix− helix interface and connect serine residues of both partner helices. In addition, an interfacial tyrosine can interact with noninterfacial water or lipid. Dimerization of this transmembrane helix might therefore be controlled by membrane properties controlling water permeation and/ or by the lipid composition of the membrane.
F
olding and oligomerization of integral membrane proteins frequently depend on sequence-specific interactions between their helical transmembrane domains (TMDs) (reviewed in refs 1−3). These interactions can rest on different types of physical forces apart from the ubiquitous van der Waals contacts. For example, small residues, often arranged in motifs such as the GxxxG motif or variants thereof, facilitate Cαhydrogen-bond (H-bond) formation.4 High-affinity TMD− TMD interactions can also be driven by strong interhelical Hbonds between the polar side chains of Asn, Gln,5,6 Ser, Thr,7 and His residues.8 There are rare instances in which water has also been implicated in stabilizing TMD−TMD interactions. In one example, the interface of the BNIP TMD dimer can be accessed by water that H-bonds to a Ser residue being part of a His-Ser node.8 Similarly, water is found within the EphA2 TMD−TMD interface at a site next to a glutamate.9 Water also appears to be retained by basic residues being membraneembedded as part of TMD helices.10,11 Here, we describe interhelical H-bonding via water molecules located at an evolutionarily conserved sequence position within the TMD helix−helix interface of human quiescin sulfhydryl oxidase 2 (QSOX2). Originally, high-affinity self-interaction of the QSOX2 TMD helix was predicted by its pronounced onesided amino acid conservation.12 Enzymes of the QSOX family catalyze the oxidation of sulfhydryl groups in peptide and protein thiols to disulfides. Alternative splicing leads to isoforms that form either Nout membrane proteins with a single C-terminal TMD or secretory variants lacking a TMD (reviewed in ref 13). The possibility that the TMD could be involved in QSOX dimerization has previously been suggested © XXXX American Chemical Society
Figure 1. Experimental assessment of QSOX2 TMD−TMD interaction. (A) TMD sequence used to construct the ToxR/TMD/ MalE hybrid proteins (lowercase letters signify invariant residues of the host protein). (B) Relative homotypic affinities as analyzed by the ToxR system in the E. coli inner membrane. The β-galactosidase activity of the human QSOX2 TMD (=100%, upper dotted vertical line) is compared to that of the glycophorin A TMD (GpA1315). Point mutations introduced into the QSOX2 TMD reduce the magnitude of the signal up to the level of the nondimerizing GpA13 mutant G83A (lower dotted vertical line) (means ± SE; n = 12 data points). deltaTM denotes a construct without the TMD. Received: November 17, 2015 Revised: January 28, 2016
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DOI: 10.1021/acs.biochem.5b01239 Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
Rapid Report
Figure 2. Computational model of the QSOX2 TMD dimer and its solvation by water and lipids. (A) Dimer model derived by averaging data from 10 molecular dynamics simulations in a membrane (interfacial residues as well as the highly conserved C664 are shown as licorice, and interhelical Hbonds are shown as dashed black lines). Two water molecules (van der Waals spheres) bridge both monomers by binding to S672 and S671. Horizontal dashed lines denote the position of lipid phosphorus atoms. (B) Helix−helix contacts based on side chain−side chain heavy atom distances (
