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. 2017 Nov 8;139(44):15774-15783.
doi: 10.1021/jacs.7b07505. Epub 2017 Oct 27.

Combination of Cα-H Hydrogen Bonds and van der Waals Packing Modulates the Stability of GxxxG-Mediated Dimers in Membranes

Samantha M Anderson  1 , Benjamin K Mueller  1 , Evan J Lange  1 , Alessandro Senes  1
Affiliations

Combination of Cα-H Hydrogen Bonds and van der Waals Packing Modulates the Stability of GxxxG-Mediated Dimers in Membranes

Samantha M Anderson et al. J Am Chem Soc. .

Abstract

The GxxxG motif is frequently found at the dimerization interface of a transmembrane structural motif called GASright, which is characterized by a short interhelical distance and a right-handed crossing angle between the helices. In GASright dimers, such as glycophorin A (GpA), BNIP3, and members of the ErbB family, the backbones of the helices are in contact, and they invariably display networks of 4 to 8 weak hydrogen bonds between Cα-H carbon donors and carbonyl acceptors on opposing helices (Cα-H···O═C hydrogen bonds). These networks of weak hydrogen bonds at the helix-helix interface are presumably stabilizing, but their energetic contribution to dimerization has yet to be determined experimentally. Here, we present a computational and experimental structure-based analysis of GASright dimers of different predicted stabilities, which show that a combination of van der Waals packing and Cα-H hydrogen bonding predicts the experimental trend of dimerization propensities. This finding provides experimental support for the hypothesis that the networks of Cα-H hydrogen bonds are major contributors to the free energy of association of GxxxG-mediated dimers. The structural comparison between groups of GASright dimers of different stabilities reveals distinct sequence as well as conformational preferences. Stability correlates with shorter interhelical distances, narrower crossing angles, better packing, and the formation of larger networks of Cα-H hydrogen bonds. The identification of these structural rules provides insight on how nature could modulate stability in GASright and finely tune dimerization to support biological function.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
The GASright dimerization motif. (a) The GASright motif is a right-handed helical dimer with a short interhelical distance (6.3–7.5 Å) and a right-handed crossing angle of approximately −40°. The GxxxG sequence pattern at the crossing point (red) allows the backbones to come into contact. (b) The contact enables formation of networks of weak interhelical H bonds between Cα–H donors and carbonyl oxygen acceptors (shown in detail in c).
Figure 2
Figure 2
Energy distribution of CATM predicted GASright dimers in human single-pass sequences. (a) Histogram of calculated energies of human GASright dimers. CATM identified 1141 sequences that produced a model with a negative (favorable) energy of association. (b) Extensive complementary packing, as well as (c) the characteristic networks of Cα–H hydrogen bonds displayed by the lowest energy structures, chondroitin sulfate glucuronyltransferase (Uniprot accession Q9P2E5).
Figure 3
Figure 3
Experimental design. (a) TOXCAT is an in vivo assay based on a construct in which the TM domain under investigation is fused to the ToxR transcriptional activator. TM association results in the expression of a reporter gene in E. coli cells, which can be quantified. (b) To reduce variability in TOXCAT, the eight interfacial amino acids identified by CATM in the wild-type sequences (top) were “stitched” into a standardized poly-Leu sequence (bottom). Standardization of the predicted constructs retains the geometry of the interface while controlling the length of the TM helix, the position of the crossing point, and the hydrophobicity of the TM segment.
Figure 4
Figure 4
Comparison of CATM energies with apparent TOXCAT dimerization. (a) Comparison of CATM energy score of 26 sequences and their TOXCAT signal (measured as the enzymatic activity of the reporter gene CAT). The points are color-coded according to the grouping in (b). The error bars represent the standard deviation among replicates. The dashed line represents the linear regression fit of the data, with the exclusion of the outlier point highlighted in gray (R2 = 0.647, p < 0.000005). (b) Same data as in (a), grouped and averaged in five bins based on CAT activity from weak (>25%, magenta) to very strong (>125%, blue), in 25% intervals. The error bars represent the standard error of the average. The dashed line is the linear regression of the data (R2 = 0.931, p < 0.01). The groups are the base of the analysis reported in Figure 5.
Figure 5
Figure 5
Sequence and structural bias occur in groups with different stabilities. (a) Sequences of the 26 constructs ranked by TOXCAT signal showing the groups, as defined in Figure 4. GxxxG motifs are underlined with a solid line, GxxxG-like motifs with a dotted line. Color coding as in Figure 4b. (b) Nomenclature of the interfacial positions, as defined previously. The sequence and structural biases of the groups of experimental constructs (orange symbols) are illustrated for (c) the number of Cα–H hydrogen bonds, which increases with stability, (d) the interhelical distance, and (e) crossing-angle, which decrease with stability, and the fraction of sequences containing (f) GxxxG and (g) Gly at the N1 position, which also increase. Data also reported in Table 1. The same trends are observed in groups of different stabilities computed from the entire data set of 670 structures predicted from the human proteome (blue symbols).
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