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Solution NMR Provides New Insight into Lipid−Protein Interaction Iga Kucharska* and Lukas K. Tamm Center for Membrane and Cell Physiology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22903, United States
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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 addition of LPS. Interestingly, Arg113 belongs to a set of highly conserved basic residues in the loops of all members of the 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). 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 following order: Arg72 > Lys103 ≈ Lys70 > Arg113 > Arg145. The triple mutation of Lys70, Arg72, and 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 simulations.5 Solution NMR experiments are also useful for estimating 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 (KD) could be derived. The KD values for binding of LPS to wild-type and ΔL1ΔL4 OprH were found to be approximately 0.2 mM, and the KD for binding of LPS to the Arg72 to glutamine mutant was approximately 0.6 mM. A dissociation constant of ∼200 μM might appear to be 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 KD of a few hundred micromolar 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
espite 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 cocrystallized lipids or analyzed by mass spectrometry of solubilized protein−lipid complexes.1 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 cocrystallized 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 use in studying lipid−protein interactions by both solution and solid-state methods. Both types of NMR spectroscopy are adequate for determining structures of membrane proteins,2 and 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 outer membrane (OM) of P. aeruginosa, contributes substantially to the unusually high intrinsic antibiotic resistance of this bacterium. OprH is upregulated in the outer membrane in response to Mg2+limited growth conditions and in early stages of bacterial infections. The protein is believed to cross-link 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 eight-stranded β-barrel protein OprH is mainly mediated through outer loops 2 and 3 of the protein.4 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 those of 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 © XXXX American Chemical Society
Received: April 13, 2017 Revised: May 13, 2017
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DOI: 10.1021/acs.biochem.7b00336 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
Figure 1. Structures of OprH from P. aeruginosa (Protein Data Bank entry 2LHF) and interacting rough (Rd) LPS. Charged side chains that have been identified as contributors to LPS interaction sites are colored red. Reproduced with permission from ref 3. Copyright 2016 American Chemical Society.
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.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Iga Kucharska: 0000-0001-6150-3419 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Saliba, A. E., Vonkova, I., and Gavin, A. C. (2015) The systematic analysis of protein-lipid interactions comes of age. Nat. Rev. Mol. Cell Biol. 16, 753−761. (2) Liang, B., and Tamm, L. K. (2016) NMR as a tool to investigate the structure, dynamics and function of membrane proteins. Nat. Struct. Mol. Biol. 23, 468−474. (3) Kucharska, I., Liang, B., Ursini, N., and Tamm, L. K. (2016) Molecular interactions of lipopolysaccharide with an outer membrane protein from Pseudomonas aeruginosa probed by solution NMR. Biochemistry 55, 5061−5072. (4) Edrington, T. C., Kintz, E., Goldberg, J. B., and Tamm, L. K. (2011) Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa. J. Biol. Chem. 286, 39211−39223. (5) Lee, J., Patel, D. S., Kucharska, I., Tamm, L. K., and Im, W. (2017) Refinement of OprH-LPS interactions by molecular simulations. Biophys. J. 112, 346−355.
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DOI: 10.1021/acs.biochem.7b00336 Biochemistry XXXX, XXX, XXX−XXX
