Solution NMR studies of periplasmic binding proteins and their interaction partners

Abstract

Periplasmic binding proteins (PBPs) are a crucial part of ATP-binding cassette import systems in Gram-negative bacteria. Central to their function is the ability to undergo a large-scale conformational rearrangement from open-unliganded to closed-liganded, which signals the presence of substrate and starts its translocation. Over the years, PBPs have been extensively studied not only owing to their essential role in nutrient uptake but also because they serve as excellent models for both practical applications (e.g., biosensor technology) and basic research (e.g., allosteric mechanisms). Although much of our knowledge at atomic level has been inferred from the detailed, static pictures afforded by crystallographic studies, nuclear magnetic resonance (NMR) has been able to fill certain gaps in such body of work, particularly with regard to dynamic processes. Here, we review NMR studies on PBPs, and their unique insights on conformation, dynamics, energetics, substrate binding, and interactions with related transport proteins. Based on the analysis of recent paramagnetic NMR results, as well as crystallographic and functional observations, we propose a mechanism that could explain the ability of certain PBPs to achieve a closed conformation in absence of ligand while others seem to remain open until ligand-mediated closure.

Figure 1

Crystallographic models of maltose-binding protein (MBP), a representative PBP. The N-domain is colored red, the C-domain blue, and the linker segments green; backbone traces are shown along with the translucent surface representation of all heavy atoms. The open-unliganded conformation (PDB ID 1OMP) is displayed in (A), and the closed, maltotriose-loaded (PDB ID 3MBP) in (B). Maltotriose atoms are displayed as magenta spheres. N-domain backbone atoms were used to align both structures prior to their lateral translation into (A) and (B). All graphical representations of atomic coordinates were generated with PyMOL (http://pymol.org).

Figure 2

Thermodynamic linkage relationships involving MBP. The ligand is represented by a magenta circle. The protein is colored yellow and appears either in an unfolded state (line) or in a folded one (other). Folded protein displays either an open conformation (top left corner) or a closed one (right corners); the hinge is denoted by a black circle. The balancing interface and the nomenclature for the free-energy changes between thermodynamic states are indicated.

Figure 3

Dependence on interdomain closure angle of the relative free energy of a wild-type MBP polypeptide that adopts either the wild-type conformation or that of hinge mutants I329C, I329W, I329F, I329C*, and I329W/A96W (the asterisk indicates derivatization with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole). The closed-liganded state (‘closed-bound’) is arbitrarily set as the energy origin. The energy of a hypothetical closed-unliganded conformation (‘closed-free’) is calculated by extrapolation of the linear correlation extracted from the free-energy change of unfolding ( $\mathrm{\Delta }{G}_{F\to U}^{\mathit{Open}}$) for the unliganded MBP set, which has the wild-type conformation (‘open-free’) as the most stable member. A dashed line indicates the point where unliganded, folded MBP is as stable as the unfolded state. Figure adapted from Ref. (40); Copyright (2003) National Academy of Sciences, USA.

Figure 4

Paramagnetic NMR strategy for structure determination of a minor closed-unliganded PBP conformation in equilibrium with a major open one. A red star represents a spin label, chemically connected to the ‘lip’ of one domain. The induced distance-dependent paramagnetic relaxation enhancement (PRE) on the protein is graphically indicated via a color gradient, red being the strongest and yellow the weakest. PRE_i, the measured PRE on site i, located in the domain that does not contain the label, considerably reflects that from the minor conformation ( ${\mathit{PRE}}_{\mathit{minor}}^i$) despite the low population of the latter (p_minor). Symbols labeled ‘major’ refer to the major conformation (with population p_major ≫ p_minor) and are analogous to those associated with the minor species. Although a balancing interface such as that in MBP is implied, it might not apply to all PBPs (14).

Figure 5

Conformational differences between crystallographic closed-liganded MBP (PDB ID 3MBP; magenta) and the solution, minor, semi-closed-unliganded species (PDB ID 2V93; green). Both structures are superimposed via the N-domain, shown only for the closed-liganded model (gray).

Figure 6

Hydrogen bond connectivity within hinge segments of open-unliganded crystal forms of GlnBP (PDB ID 1GGG), MBP (PDB ID 1OMP), GGBP (PDB ID 2FW0), and ChoX (PDB ID 3HCQ). Residue numbers at the end of each segment are indicated. Green, dashed lines denote hydrogen bonds. Side chains involved in hydrogen bonding are shown, as well as the complete polypeptide backbone (only heavy atoms included: C, gray; O, red; N, blue). Backbone covalent connections to the PBP domains are indicated by black, dotted lines. The domains are represented by red and blue rectangles (e.g., the N- and C-domains of MBP are denoted red and blue, respectively, as in Figure 1). Figure adapted from Ref. (30).

Figure 7

Crystal structure of the complete maltose importer (PDB ID 2R6G), an example of the ABC transporter superfamily. Protein backbone trace is shown, with the PBP (MBP) in yellow, and the two TMDs (MalF and MalG) and ABCs in blue and green, respectively, with different tones for each monomer. Atoms of a maltose substrate molecule are shown as magenta spheres occupying the transmembrane binding site. Atoms of two ATP molecules are displayed as red spheres at their corresponding ABC sites. The position of the membrane (horizontal lines) was chosen to correspond to the predicted buried residues of the TMDs.

Figure 8

Schematic representation of inward-facing (A) and outward-facing (B) conformations of ABC importers. The color codes of the different subunits are indicated in the caption to Figure 7; the latter corresponds to an outward-facing configuration (B).