Prediction of membrane protein structures with complex topologies using limited constraints

Abstract

Reliable structure-prediction methods for membrane proteins are important because the experimental determination of high-resolution membrane protein structures remains very difficult, especially for eukaryotic proteins. However, membrane proteins are typically longer than 200 aa and represent a formidable challenge for structure prediction. We have developed a method for predicting the structures of large membrane proteins by constraining helix-helix packing arrangements at particular positions predicted from sequence or identified by experiments. We tested the method on 12 membrane proteins of diverse topologies and functions with lengths ranging between 190 and 300 residues. Enforcing a single constraint during the folding simulations enriched the population of near-native models for 9 proteins. In 4 of the cases in which the constraint was predicted from the sequence, 1 of the 5 lowest energy models was superimposable within 4 A on the native structure. Near-native structures could also be selected for heme-binding and pore-forming domains from simulations in which pairs of conserved histidine-chelating hemes and one experimentally determined salt bridge were constrained, respectively. These results suggest that models within 4 A of the native structure can be achieved for complex membrane proteins if even limited information on residue-residue interactions can be obtained from protein structure databases or experiments.

Fig. 1.

Ab initio folding protocol with long-range interactions. Interactions can be predicted from sequence information using a database of TMH pairs of known structure (Fig. S1) or can be inferred from experiments (see Materials and Methods). Once an interaction is selected, the two helices connected through space by that interaction are inserted and folded in the membrane. Adjacent individual TMHs are then randomly selected and folded in the membrane by Monte-Carlo fragment insertion sampling. After all TMHs are assembled in the membrane, the initial chain break is closed.

Fig. 2.

Prediction of membrane protein structures. Superposition between the most accurate (highest maxsub) models of the 5 lowest all-atom energy models (magenta) and X-ray structure of: chain A of cytochrome c (A), Bacteriorhodopsin (B), chain H of fumarate reductase (E), and chain D of cytochrome bc1 (F). Because individual subunits of the Lactose permease virtually expose pore-lining polar residues to the lipids, near-native structures cannot be selected by energy alone. The cluster size was used as an initial filter for the selection of the models. Superposition between the most accurate (highest maxsub) of the lowest all-atom energy model in the 2 largest clusters (magenta) and X-ray structure of: N-terminal subunit of lactose permease (C, view from the channel), C-terminal subunit of lactose permease (D, view from the channel).