On the accuracy of homology modeling and sequence alignment methods applied to membrane proteins

Abstract

In this study, we investigate the extent to which techniques for homology modeling that were developed for water-soluble proteins are appropriate for membrane proteins as well. To this end we present an assessment of current strategies for homology modeling of membrane proteins and introduce a benchmark data set of homologous membrane protein structures, called HOMEP. First, we use HOMEP to reveal the relationship between sequence identity and structural similarity in membrane proteins. This analysis indicates that homology modeling is at least as applicable to membrane proteins as it is to water-soluble proteins and that acceptable models (with C alpha-RMSD values to the native of 2 A or less in the transmembrane regions) may be obtained for template sequence identities of 30% or higher if an accurate alignment of the sequences is used. Second, we show that secondary-structure prediction algorithms that were developed for water-soluble proteins perform approximately as well for membrane proteins. Third, we provide a comparison of a set of commonly used sequence alignment algorithms as applied to membrane proteins. We find that high-accuracy alignments of membrane protein sequences can be obtained using state-of-the-art profile-to-profile methods that were developed for water-soluble proteins. Improvements are observed when weights derived from the secondary structure of the query and the template are used in the scoring of the alignment, a result which relies on the accuracy of the secondary-structure prediction of the query sequence. The most accurate alignments were obtained using template profiles constructed with the aid of structural alignments. In contrast, a simple sequence-to-sequence alignment algorithm, using a membrane protein-specific substitution matrix, shows no improvement in alignment accuracy. We suggest that profile-to-profile alignment methods should be adopted to maximize the accuracy of homology models of membrane proteins.

FIGURE 1

Structural relationship between membrane protein models and their templates. The sequence identity of the structure-based (correct) alignment is plotted against (A) the Cα-RMSD and (B) the GDT_TS scores of the corresponding model compared to the native structure. Data are shown for the whole protein (•) and for the transmembrane regions (□). Six models had RMSD values of between 10 and 40 Å; for clarity these points are plotted at RMSD = 10 Å.

FIGURE 2

Relationship between model quality and model-building difficulty. (Top) Alignment accuracy measured by AL0 for the whole protein (•) and transmembrane regions (□). (Bottom) Structural accuracy measured by GDT_TS for the whole protein (•) and transmembrane regions (□).

FIGURE 3

Accuracy of membrane protein sequence alignments/homology models obtained from different sequence alignment methods as a function of sequence identity. Results are given for (A) the whole protein and (B) the transmembrane regions. The average AL0 score is given over all alignments/models within a window of 10% sequence identity, and error bars indicate the standard deviation over that window. Numbers correspond to the number of alignments in each window and apply to both plots. Abbreviations: seq-seq, sequence-to-sequence alignment; seq-profile, sequence-to-profile alignment; and MSA, multiple sequence alignment. The two HMAP labels indicate profile-to-profile alignments.

FIGURE 4

Accuracy of bipartite sequence-to-sequence alignments of membrane proteins obtained with different substitution matrices. See legend to Fig. 3 for more details.