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. 2016 Jul 20:6:29809.
doi: 10.1038/srep29809.

Structural and thermodynamic basis of proline-induced transmembrane complex stabilization

Thomas Schmidt  1 , Alan J Situ  1 , Tobias S Ulmer  1
Affiliations

Structural and thermodynamic basis of proline-induced transmembrane complex stabilization

Thomas Schmidt et al. Sci Rep. .

Abstract

In membrane proteins, proline-mediated helix kinks are indispensable for the tight packing of transmembrane (TM) helices. However, kinks invariably affect numerous interhelical interactions, questioning the acceptance of proline substitutions and evolutionary origin of kinks. Here, we present the structural and thermodynamic basis of proline-induced integrin αIIbβ3 TM complex stabilization to understand the introduction of proline kinks in membrane proteins. In phospholipid bicelles, the A711P substitution in the center of the β3 TM helix changes the direction of adjacent helix segments to form a 35 ± 2° angle and predominantly repacks the segment in the inner membrane leaflet due to a swivel movement. This swivel repacks hydrophobic and electrostatic interhelical contacts within intracellular lipids, resulting in an overall TM complex stabilization of -0.82 ± 0.01 kcal/mol. Thus, proline substitutions can directly stabilize membrane proteins and such substitutions are proposed to follow the structural template of integrin αIIbβ3(A711P).

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Figures

Figure 1
Figure 1. Transmembrane helix-helix interfaces in the neurotensin receptor 1.
(a) Proline kink-mediated helix-helix packing. (b,c) Wedging of either non-helical residues or an additional helix into a helix-helix interface. PDB entry 4bwb was used.
Figure 2
Figure 2. NMR spectra of the integrin αIIbβ3(A711P) TM complex.
(a) TROSY-type H-N correlation spectrum of disulfide-linked 2H/13C/15N-αIIb(A963C)–2H/13C/15N-β3(G690C/A711P). (b) 3D NOESY-TROSY strips of disulfide-linked 2H/15N-αIIb(A963C)–β3(G690C/A711P) and αIIb(A963C)–2H/15N-β3(G690C/A711P) illustrate interhelical NOEs. NOEs to protonated lipids are indicated by green lines. All spectra were recorded at 40 °C and 700 MHz.
Figure 3
Figure 3. Structure of the integrin αIIbβ3(A711P) TM complex.
(a) Comparison of integrin αIIbβ3(A711P) and αIIbβ3 TM complex structures. The structures were superimposed on the backbone heavy atoms of αIIb(W967-L979). (b) Chemical shift differences between αIIb backbone 15N nuclei of non-covalently linked αIIbβ3(A711P) and αIIbβ3 TM complexes. (c) Comparison of β3 sidechain orientations in the αIIbβ3(A711P) and αIIbβ3 TM complex structures. TM complex coordinates were superimposed as shown in panel a. (d) Comparison of αIIb(G972), β3(L712), β3(W715) and αIIb(R995) sidechain interactions between αIIbβ3(A711P) and αIIbβ3 TM complex structures. ΔΔG°,′ associated with the αIIb(G972A), β3(L712A), β3(W715Y) and αIIb(R995A) substitutions (Table 1) are indicated. (e) Comparison of β3 sidechain orientations when superimposing β3 backbone coordinates near the TM termini. PDB entries 2k9j (αIIbβ3) and 2n9y (αIIbβ3(A711P)) were used.
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