Entangled Motifs in Membrane Protein Structures

Abstract

Entangled motifs are found in one-third of protein domain structures, a reference set that contains mostly globular proteins. Their properties suggest a connection with co-translational folding. Here, we wish to investigate the presence and properties of entangled motifs in membrane protein structures. From existing databases, we build a non-redundant data set of membrane protein domains, annotated with the monotopic/transmembrane and peripheral/integral labels. We evaluate the presence of entangled motifs using the Gaussian entanglement indicator. We find that entangled motifs appear in one-fifth of transmembrane and one-fourth of monotopic proteins. Surprisingly, the main features of the distribution of the values of the entanglement indicator are similar to the reference case of general proteins. The distribution is conserved across different organisms. Differences with respect to the reference set emerge when considering the chirality of entangled motifs. Although the same chirality bias is found for single-winding motifs in both membrane and reference proteins, the bias is reversed, strikingly, for double-winding motifs only in the reference set. We speculate that these observations can be rationalized in terms of the constraints exerted on the nascent chain by the co-translational bio-genesis machinery, which is different for membrane and globular proteins.

Keywords: chirality; co-translational folding; entanglement; membrane proteins.

Figure 1

Distribution of the values of the overall entanglement indicator $G_{\max }^{\prime }$ for different sets of protein domain structures: (a) $G_{\max }^{\prime }$ probability distributions for 1378 membrane protein domains (light blue filled bars) and for the reference set of 16,709 protein domain structures (dark blue solid line). (b) $G_{\max }^{\prime }$ probability distributions for the subsets of the 1378 membrane protein domains corresponding to some of the most represented organisms.

Figure 2

Comparison between monotopic and transmembrane sets of protein domain structures: (a) $G_{\max }^{\prime }$ probability distributions for 871 transmembrane protein domains (light blue) and for 494 monotopic protein domains (orange). (b) Survival function showing the fraction of structures in the data set with an entanglement indicator greater than $\left|G_{\max }^{\prime }\right|$. Black: Reference set. Light blue: transmembrane protein domains. Orange: Monotopic protein domains. Error bars refer to the 5% and 95% percentiles after 10,000 bootstrap samplings. (c) Distributions of the protein domain length for the different data sets. Black: Reference set. Light blue: transmembrane protein domains. Orange: Monotopic protein domains.

Figure 3

Scatter plot of $G_N^{\prime }$ and $G_C^{\prime }$ values. Each point in the plot refers to a single domain in the data sets. Light blue: 871 transmembrane protein domains. Orange: 494 monotopic protein domains. Grey: 13 not classified membrane protein domains. Dash lines refer to the the diagonal ($G_C^{\prime }=G_N^{\prime }$) and the anti-diagonal ($G_C^{\prime }=-G_N^{\prime }$) lines in the ($G_N^{\prime }$,$G_C^{\prime }$) plane.

Figure 4

Grouping fractions for different kinds of entangled motifs. The data report the fraction of the survived structures (i.e., with entanglement indicator greater than $\left|G_{\max }^{\prime }\right|$, see Figure 2b) that belong to a given group. In all groups, we consider only structures for which the most entangled N-thread and the most entangled C-thread share the same chirality. Solid black: N-threads with positive chirality ($G_N^{\prime }>G_C^{\prime }>0$). Dashed black: C-threads with positive chirality ($G_C^{\prime }>G_N^{\prime }>0$). Solid red: N-threads with negative chirality ($G_N^{\prime }<G_C^{\prime }<0$). Dashed red: C-threads with negative chirality ($G_C^{\prime }<G_N^{\prime }<0$). Error bars refer to the 5% and 95% percentiles after 10,000 bootstrap samplings: (a) Reference set. (b) transmembrane protein domains. (c) Monotopic protein domains.

Figure 5

Examples of transmembrane proteins with entangled motifs. Top: Cartoon structures. Bottom: Temptative planar structure projection to better appreciate the winding of the thread around the entangled loop and the corresponding chirality; secondary structure elements are shown as a guide. Blue: thread. Red: entangled loop. Grey: other portions of the protein chain. Yellow: residues at the loop ends that close the non-covalent lasso; all heavy atoms are shown in the cartoon structure for the yellow residues. (a) A subunit from the bacterial cellulose synthase (Bcs) 4P02 complex with a positive chirality N-thread entangled motif ($G_N^{\prime }=1.10>G_C^{\prime }=0.79$); membrane boundaries are shown as dashed black lines. (b) APC transporter 3GIA with a positive chirality C-thread double winding entangled motif ($G_N^{\prime }=2.32<G_C^{\prime }=2.39$); membrane boundaries are shown as dashed black lines.

Figure 6

Example of a transmembrane protein with coexisting entangled motifs with opposite chiralities. Left: Cartoon structure of the A chain from the CmeC outer membrane channel homo-trimer 4MT4 from Campylobacter jejuni; membrane boundaries are shown as dashed black lines. Right: Temptative planar structure projection to better appreciate the winding of the thread around the entangled loop and the corresponding chirality; secondary structure elements are shown as a guide. Blue: thread. Red: entangled loop. Grey: other portions of the protein chain. Yellow: residues at the loop ends that close the non-covalent lasso; all heavy atoms are shown in the cartoon structure for the yellow residues, whereas the non-covalent contact is shown as a yellow dotted line in the planar projection. (a) Positive chirality N-thread ($G_N^{\prime }=1.20$). (b) Negative chirality C-thread ($G_C^{\prime }=-1.19$).