Conformational Motions and Functionally Key Residues for Vitamin B12 Transporter BtuCD-BtuF Revealed by Elastic Network Model with a Function-Related Internal Coordinate

Abstract

BtuCD-BtuF from Escherichia coli is a binding protein-dependent adenosine triphosphate (ATP)-binding cassette (ABC) transporter system that uses the energy of ATP hydrolysis to transmit vitamin B12 across cellular membranes. Experimental studies have showed that during the transport cycle, the transporter undergoes conformational transitions between the "inward-facing" and "outward-facing" states, which results in the open-closed motions of the cytoplasmic gate of the transport channel. The opening-closing of the channel gate play critical roles for the function of the transporter, which enables the substrate vitamin B12 to be translocated into the cell. In the present work, the extent of opening of the cytoplasmic gate was chosen as a function-related internal coordinate. Then the mean-square fluctuation of the internal coordinate, as well as the cross-correlation between the displacement of the internal coordinate and the movement of each residue in the protein, were calculated based on the normal mode analysis of the elastic network model to analyze the function-related motions encoded in the structure of the system. In addition, the key residues important for the functional motions of the transporter were predicted by using a perturbation method. In order to facilitate the calculations, the internal coordinate was introduced as one of the axes of the coordinate space and the conventional Cartesian coordinate space was transformed into the internal/Cartesian space with linear approximation. All the calculations were carried out in this internal/Cartesian space. Our method can successfully identify the functional motions and key residues for the transporter BtuCD-BtuF, which are well consistent with the experimental observations.

Keywords: ABC transporter; BtuCD–BtuF; elastic network model; functional motions; internal coordinate; key residues; normal mode analysis; perturbation method.

Figure 1

The tertiary structure of BtuCD–BtuF complex at the nucleotide-bound intermediate state (protein data bank (PDB) code: 4FI3) (a) and the apo complex state (PDB code: 4DBL) (b). BtuCD consists of two transmembrane domains (TMDs, i.e., BtuC subunits) and two cytoplasmic nucleotide-binding domains (NBDs, i.e., BtuD subunits). BtuF binds to the periplasmic side of BtuCD. The TMDs of BtuCD form the substrate translocation channel, whose cytoplasmic gate is marked by red circle in panel (a). In this study, the extent of opening of the cytoplasmic gate is chosen as the internal coordinate that related to the channel-gating function of the protein. The coupling helices that connect TMDs and NBDs are also marked by red circles in the figure; The top view of NBDs is displayed in panel (c), in which the helical and RecA-like domains are highlighted by dotted circles; The residue displacements between the two states of BtuCD–BtuF are displayed (d); In order to intuitively display the calculation results, the residue displacements are also mapped onto the protein structure (e), where the yellow-red color indicates larger residue displacements and the blue color denotes lower displacements.

Figure 2

The calculated mean-square fluctuation of the internal coordinate (MSFIC) values for the first 30 slowest ANM modes. From this figure, it is found that the MSFIC values for modes 10, 13, 25 and 26 are relatively large.

Figure 3

The motions of modes 10 (a), 13 (b), 25 (c) and 26 (d), which are relevant to the channel-gating function of the transporter. The close-up views of the NBDs for all the modes are also displayed. In this figure, the structure of the transporter is displayed in gray tube, and the amplitude and direction of the motions are denoted by the length and direction of the blue arrows. The red arrows and dotted lines respectively represent the motion direction and axis of the subdomains of the protein.

Figure 4

The functional motion that contributes to the channel-gating of BtuCD–BtuF. The functional motion was obtained by adding up the first 30 slowest normal modes of the system according to their contributions to the channel-gating of the transporter. In this figure, the structure of the protein is displayed in gray tube, and the amplitude and direction of the functional motion are denoted by the length and direction of the blue arrows. The red arrows and dotted lines respectively represent the motion direction and axis of the subdomains of the system.

Figure 5

The functionally important residue interactions predicted by the perturbation method. The structure of BtuCD–BtuF is displayed in gray tube and the predicted key residue interactions are denoted by blue lines. According to their locations on protein structure, these key residue interactions are grouped into four regions that are denoted by ellipses and the numbers 1–4 in the figure.