Structure alignment of membrane proteins: Accuracy of available tools and a consensus strategy

Abstract

Protein structure alignment methods are used for the detection of evolutionary and functionally related positions in proteins. A wide array of different methods are available, but the choice of the best method is often not apparent to the user. Several studies have assessed the alignment accuracy and consistency of structure alignment methods, but none of these explicitly considered membrane proteins, which are important targets for drug development and have distinct structural features. Here, we compared 13 widely used pairwise structural alignment methods on a test set of homologous membrane protein structures (called HOMEP3). Each pair of structures was aligned and the corresponding sequence alignment was used to construct homology models. The model accuracy compared to the known structures was assessed using scoring functions not incorporated in the tested structural alignment methods. The analysis shows that fragment-based approaches such as FR-TM-align are the most useful for aligning structures of membrane proteins. Moreover, fragment-based approaches are more suitable for comparison of protein structures that have undergone large conformational changes. Nevertheless, no method was clearly superior to all other methods. Additionally, all methods lack a measure to rate the reliability of a position within a structure alignment. To solve both of these problems, we propose a consensus-type approach, combining alignments from four different methods, namely FR-TM-align, DaliLite, MATT, and FATCAT. Agreement between the methods is used to assign confidence values to each position of the alignment. Overall, we conclude that there remains scope for the improvement of structural alignment methods for membrane proteins.

Keywords: beta barrel; conformational change; flexible alignment; homology modeling; integral membrane protein; protein structure; structure comparison.

Figure 1. Composition of the HOMEP3 data set of homologous membrane protein structures

A. Distribution of family size for a given number of membrane-spanning segments for α-helical (blue) and β-barrel proteins (green). B. Distribution of families with different numbers of proteins. Most families of α-helical proteins (blue) contain 2-4 known protein structures, whereas the β-barrel families (green) contain more proteins per family.

Figure 2. Alternate conformations in the family of major facilitator superfamily transporters

Two structures reflect inward-facing conformations (GlpT and LacY; PDB codes: 1PW4 and 2CFQ) and two reflect outward-facing conformations (FucP and XylE; PDB codes: 3O7Q and 4GC0). The proteins are shown as cartoon helices, viewed from along the plane of the membrane with the outside of the cell toward the top, and colored according to a rainbow, from blue (N-terminal) to red (C-terminal).

Figure 3. A consensus-type structure-based alignment fragment with confidence values

Two protein structures were aligned with four different structural alignment methods, FR-TM-align, FATCAT, MATT and DaliLite. The resulting alignments were then fused using the sequence of one of the protein structures as a reference. Depending on the agreement between the four methods, confidence values were assigned as very strong (i.e., all methods concur, confidence value of 9, dark green), strong (three methods agree, confidence value of 6, pale green), moderate (two methods agree, confidence value of 3, orange), and weak (only one method found this solution, confidence value of 1, red).

Figure 4. Correlation of residue accuracy with confidence values based on the consensus between FR-TM-align, FATCAT, MATT and DaliLite alignments

From all consensus alignments of the α-helical subset of HOMEP3, the position-specific confidence level was extracted for positions in which amino acids were aligned, i.e., excluding gapped positions. For each considered position, the distance (in Å) of the corresponding Cα-atom in the homology model to that in the native X-ray structure was calculated. This value then was averaged over the models built based on each of the alignments from the four structural alignment methods. The plot contains the normalized distribution of averaged Cα distances for each of the confidence levels (see Fig. 3).