Solution NMR Provides New Insight into Lipid-Protein Interaction

Graphical abstract

Despite a growing interest in the biological functions of cellular membranes that can be attributed to direct associations between lipids and membrane proteins, available information about the molecular details of these interactions is still limited. Protein-lipid interactions are frequently visualized by X-ray crystallography in crystals of membrane proteins with co-crystallized lipids or analyzed by mass spectrometry of solubilized protein-lipid complexes¹. However, it is often not clear if lipids identified by these techniques as interacting with certain proteins are also specifically bound to these proteins in membrane environments or if they just happen to be co-crystallized to maintain the stability of the membrane protein or simply “hang on” when the membrane protein “flies” through vacuum in the mass spectrometer.

NMR has a long history of studying lipid-protein interactions by both solution and solid-state methods. Not only are both types of NMR spectroscopy adequate to determine structures of membrane proteins², but both have also been continuously refined to study lipid-protein interactions of increasing complexity. In a recent study, we set out to characterize in great molecular detail the specific interactions that could be observed by solution NMR between the Outer Membrane Protein H (OprH) from Pseudomonas aeruginosa and lipopolysaccharide (LPS)³.

The Gram-negative bacterium P. aeruginosa is clinically very important because it is a leading cause of nosocomial infections, as well as chronic lung infections in cystic fibrosis patients. LPS, which makes up the outer leaflet of the P. aeruginosa’s outer membrane (OM), contributes substantially to the unusually high intrinsic antibiotic resistance of this bacterium. OprH is up-regulated in the outer membrane in response to Mg²⁺-limited growth conditions and in early stages of bacterial infections. The protein is believed to crosslink the highly negatively charged polar carbohydrate moieties of LPS and thereby to act as a surrogate for Mg²⁺ and Ca²⁺ in the outer membrane. As such, it might tighten the OM and protect the bacterium from penetration by a range of antibiotics.

It has been shown previously that the interaction of LPS with the 8-stranded β-barrel protein OprH is mainly mediated through the outer loops 2 and 3 of the protein⁴. Therefore and to simplify NMR assignments, we used an OprH construct with loops 1 and 4 deleted (OprHΔL1ΔL4) in our recent work. OprHΔL1ΔL4 reconstituted in dihexanoyl-sn-glycero-3-phosphocholine (DHPC) lipid micelles yielded well-resolved NMR spectra with significantly less spectral overlap than wild-type OprH.

To determine which specific residues of OprHΔL1ΔL4 interact with LPS, ¹⁵N-¹H TROSY spectra with and without LPS were recorded. We found the most significant backbone chemical shift changes at the base of loop 1, in loop 2, at the base of loop 3, and in β-strand 8. To directly observe the side chains that interact with LPS we performed ¹H–¹³C HMQC and H(C)(CC)-TOCSY-(CO)-[¹⁵N,¹H]-TROSY experiments. Among several observed side chains, Arg113 of loop 3 exhibited the largest chemical shift changes upon the addition of LPS. Interestingly, Arg113 belongs to a set of highly conserved basic residues in the loops of all OprH family of proteins. This group of conserved residues also includes Lys70 and Arg72 in loop 2, Lys103 at the base of loop 3 and Arg145 at the base of loop 4 (see Figure 1).

Figure 1.

Structures of OprH from P. aeruginosa (PDB code: 2LHF) and interacting rough (Rd) LPS. Charged side chains that have been identified as contributors to LPS interaction sites are highlighted in red (from³ with permission).

To further analyze the ability of these conserved basic residues to bind LPS to OprH, we individually mutated Lys70, Arg72, Lys103, Arg113, and Arg145 to glutamines. We immobilized LPS and applied a solid-phase enzyme-linked immunosorbent assay (ELISA) to measure the binding of refolded wild-type and mutant OprH proteins in DHPC lipid micelles. All residues contributed to LPS binding and the contributions decreased in the order Arg72 > Lys103 ≈ Lys70 > Arg113 > Arg145. The triple mutation Lys70/Arg72/Lys103 to glutamines eliminated LPS binding almost completely. Interestingly the interactions with these side chains were so strong that the side-chain NMR signals disappeared and therefore, unlike with Arg113, no side-chain chemical shift changes could be observed with these residues. Significant contributions of the same basic side chains to the observed interactions between LPS and OprH have been confirmed by recent molecular dynamics simulations⁵.

Solution NMR experiments are also useful to estimate dissociation constants of specific lipid-protein interactions. To quantitatively measure the binding of LPS to OprH, we titrated LPS to OprH and some of its mutants and measured chemical shifts of the ¹⁵N-¹H TROSY cross-peaks from several loop 3 residues. These shifts followed Langmuir binding isotherms, from which dissociation constants K_D could be derived. The K_D‘s for binding of LPS to wild-type and ΔL1ΔL4 OprH were found to be approximately 0.2 mM and the K_D for binding of LPS to the Arg72 to glutamine mutant was approximately 0.6 mM. A dissociation constant of ~200 μM might appear weak, but this binding is actually highly efficient if one considers the very high density of LPS in the bacterial OM. Given that the local concentration of LPS in the membrane is close to 1 M, a K_D of a few hundred μM is sufficient to occupy each binding site on OprH with a molecule of LPS.

Lipid-protein interactions have been characterized by solution and solid-state NMR in several other systems to various degrees of detail, but it is beyond the scope of this short viewpoint to enumerate them here. However, in addition to the identification of several key residues that are responsible for the binding of a highly specialized lipid to an integral membrane protein, the work described here demonstrates the versatility of contemporary solution NMR methods to identify binding sites and dissociation constants of selectively binding membrane lipids to membrane proteins that cannot be easily achieved by other analytical methods.