October 1, 2015 (Vol. 35, No. 17)

Kathy Liszewski

Research Is Seeing the Emergence of Many Promising Techniques, Including Novel Sample Delivery Systems

Membrane proteins, such as receptors, ion channels, and transporters, comprise nearly 30% of all proteins in eukaryotic cells. They also constitute more than 50% of all drug targets.

Yet, membrane proteins continue to present considerable challenges to the field of structural biology. Their surface is relatively hydrophobic, usually requiring potentially harmful detergent solubilization. Conformational flexibility and instability also may create roadblocks for the expression and purification required for structural analysis.

The recent Argonne National Laboratory Conference on Membrane Protein Structures highlighted advances in the field such as use of smaller and more intense beams for X-ray micro-crystallography, novel protein engineering of fusion proteins for structure determination, nanodiscs that mimic native cell environments, visualization strategies employing single particle electron microscopy, and bacterial nanopore studies that may help surmount antibiotic resistance.

X-Ray Micro-Crystallography

X-ray crystallography has been a workhorse technology for structural biologists for many years. Scientists generate a minute crystal by carefully optimizing conditions, shoot a high-powered X-ray beam at it, measure the angle and intensity of the diffracted beams, and derive a complete or partial structure by analyzing the results with sophisticated analytical programs.

“Membrane proteins are notoriously difficult to crystallize, and often yield very small, weakly diffracting, radiation-sensitive crystals that are intractable to large-beam crystallography. However, high-resolution structures can be obtained by using a micro-beam,” noted Robert F. Fischetti, Ph.D., associate division director and group leader, X-ray Science Division, Argonne National Laboratory.

Dr. Fischetti said the Advanced Photon Source (APS), a DOE user facility at Argonne, leads the field in deriving membrane protein structures.

“G-Protein Coupled Receptors (GPCRs) are one very important class of membrane proteins. There are more than 800 GPCRs, and over 40% of all drugs target them. Of the 30 known protein structures, 21 were solved at the APS.”

According to Dr. Fischetti, a number of key improvements and innovative approaches are needed.

“Stability of the beam intensity and the relative alignment of the beam and crystal are paramount in micro-crystallography. One problem is that X-ray beams cause both primary and secondary (diffusional) structural damage to the crystal. To overcome that issue smaller, hotter beams and more rapid detectors are being used in the race against radiation damage.”

Dr. Fischetti said the field is also seeing the emergence of breakthrough techniques, including novel sample delivery systems such as the acoustic drop and microfluidic technologies. Further, throughput is advancing.

“We’re approaching the ability to perform data collection on many thousands of microcrystals complexed to a variety of compounds. This is enabling pharmaceutical applications.”

One of the most exciting changes at APS and throughout the scientific community is the development of a new storage ring magnet lattice design, the multibend achromat (MBA). The technology promises a revolutionary increase in brightness that could reach two to three orders of magnitude beyond the current capability.

According to Dr. Fischetti, “This fourth generation storage ring will be nearly diffraction-limited and provide key improvements such as focusing X-rays down to the nanometer level with much higher intensity than is currently available. We expect the proposed MBA to be available in the 2020s. With this and other advances, it is clear that we are entering a new frontier in X-ray science.”

Schematic view of the planned upgrade of the GM/CA beamline 23-ID-D at the Advanced Photon Source (APS) at Argonne National Laboratory. Top panel: cartoon of the X-ray optics to focus the beam. Bottom panel: elevation view of the endstation focusing optics, sample goniometry, and detector. The beam line upgrade will reduce the minimum beam size from 5 µm to 1 µm in the near future. The proposed APS-MBA upgrade will allow the beam to be focused to <500 nm with a 100-fold increase in intensity. The small, high intensity X-ray beam will enable structure determination for some of the most challenging problems in structural biology.

Disease-Related Receptors

Many physiological processes are controlled and regulated by conformational changes in GPCRs and other integral membrane proteins. “We are studying at the atomic level how allosteric changes in such proteins regulate cell signaling,” explained Daniel M. Rosenbaum, Ph.D., assistant professor, biophysics, biochemistry, University of Texas Southwestern Medical Center.

In particular, Dr. Rosenbaum and his laboratory use protein engineering, X-ray crystallography, and NMR spectroscopy to study the structure and dynamics of molecules involved in hormone signaling and lipid homeostasis.

“GPCRs and other membrane proteins are not easily amenable to structural studies,” he said. “This limitation can often be overcome by protein engineering methods such as creating fusion proteins or thermostable mutants and using lipid-mediated crystallization methods.”

For example, Dr. Rosenbaum and colleagues studied the human β2 adrenergic receptor (β2AR) that binds epinephrine and is involved in the fight or flight response. Using the inactive structure of β2AR as guide, the team designed a β2AR agonist that could be covalently attached to a specific site on the receptor. “With this approach, we were able to crystallize a covalent agonist-bound β2AR fusion protein in lipid bilayers and determine its structure at 3.5 angstroms resolution.”

Another example of using fusion proteins to overcome membrane protein crystallization limitations is that of the human orexin receptor, OX2R. The orexin system modulates behaviors in mammals such as sleep, arousal, and feeding. Dysfunctions can lead to narcolepsy and cataplexy. The FDA recently approved the first-in-class drug, suvorexant, which became available in early 2015.

Dr. Rosenbaum and colleagues used lipid-mediated crystallization and protein engineering with a novel fusion chimera to solve the structure of the OX2R, bound to suvorexant at 2.5 angstom resolution.

“Elucidation of the molecular architecture of the human OX2R enhances our knowledge of how it recognizes ligands. Such studies provide powerful tools for designing improved therapeutics that can activate or inactivate orexin signaling.”

These studies have an overarching significance as well. “Looking at the bigger picture, these methods may lead to the design of new classes of small molecules that modulate key signaling pathways by controlling protein conformational changes within cellular membranes,” Dr. Rosenbaum concluded.

Nanodisc Technology

Although membrane proteins can be purified following cell lysis and detergent solubilization or after expression in heterologous systems, their true structure and function can be significantly compromised or lost entirely in the process. Ideally one would like membrane proteins to remain in a solubilized state for easier purification, functional assays, and structure determination. However, the native membrane environment is often necessary for full functionality. Detergents pose many technical obstacles including being hazardous for protein stability and interfering in many assay techniques.

Enter Nanodisc technology, a new approach for providing accessibility to the world of membrane proteins.

“We’ve always had a dream of engineering a process that would not only incorporate any membrane protein into a soluble bilayer structure, but also one that would employ a self-assembly process that would be applicable to all individual membrane proteins regardless of their structure and topology,” explained Stephen G. Sligar, Ph.D., director of the School of Molecular and Cellular Biology, University of Illinois, Urbana Champaign.

“Recently, that dream became realized by the creation of Nanodisc technology. Nanodiscs are self-assembling nanoscale phospholipid bilayers that are stabilized using engineered membrane scaffold proteins. The Nanodiscs allow membrane proteins to remain soluble and thus closely mimics native environment.”

There are many uses for the new technology according to Dr. Sligar. “Technological applications can take advantage of Nanodisc properties such as its small size, reduced light scattering, faster diffusion, enhanced stability, access to both sides of the bilayer and for surface attachment (e.g., surface plasmon resonance studies).”

Dr. Sligar and colleagues even demonstrated how to utilize the new technology for high throughput screening (HTS) assays.

“We wanted to identify antagonists that would interfere with the binding of membrane proteins to Alzheimer’s-associated amyloid β oligomers (AβOs), which are the neurotoxic ligands that instigate Alzheimer’s dementia. In collaboration with Professor William Klein and co-workers at Northwestern University, we created a solubilized membrane protein library (SMPL). This consisted of a complete set of membrane proteomes derived from biological tissue containing a heterogeneous mixture of individual proteins.

“Screening an extensive library of drug-like compounds and natural products identified yielded several ‘hits’, thus providing proof of concept for using SMPLs in HTS applications. An initial publication appeared recently in PLOS ONE.”

The results need to be confirmed in animal studies, Dr. Sligar noted. Overall, he is enthusiastic about the Nanodisc platform for uses that range from determination of structure/function to HTS applications.

“The unique properties of Nanodiscs make them ideal candidates to address important functional and structural questions involving membrane proteins in a more native environment.”

EM for Structural Analysis

Electron microscopy (EM) not only provides a straightforward approach to scrutinize the ultrastructure of cells and tissues, but it is also gaining momentum as a means to derive structural information on membrane proteins.

According to Bridget Carragher, Ph.D., co-director, Simons Electron Microscopy Center, New York Structural Biology Center, “EM is a widely applied technique to study the structure of proteins and membranes, but it is still less common than X-ray diffraction of prepared crystals. However, crystallization of membrane proteins has been particularly challenging. Since EM does not require obtaining crystals, it is becoming an increasingly used tool for performing structural analysis of membrane proteins and their complexes.”

As an example, Dr. Carragher described the use of single particle EM to directly visualize the conformational spectra of two homologous ATP-binding cassette (ABC) exporters. Single particle EM determines structure from multiple images of individual particles and uses methods like multivariate statistical analysis to separate heterogeneous particles into homogeneous classes.

“ABC transporters constitute a large family of membrane proteins that use the energy of ATP hydrolysis to translocate (either export or import) substances such as nutrients, lipids, and ions across the lipid bilayers,” said Dr. Carragher. “They are medically important because they also transport drugs and contribute to antibiotic or antifungal resistance.

“In a collaborative study, we utilized an unbiased approach employing newly developed amphiphiles in complex with lipids to create a membrane-mimicking environment for stabilizing membrane proteins. Visualization of the complexes using single particle EM analysis revealed striking conformational differences between the two transporters with respect to the effect of binding nucleotides and substrates. Overall, these studies provided a comprehensive view of the conformational flexibility of these two ABC exporters.”

As improvements continue to be made in the technology, resolution is nearing the 3 to 5 angstrom range, at least for some proteins and protein complexes.

“EM is becoming competitive with X-ray diffraction for solving some protein structures. It is not likely to replace other techniques, but rather will be complementary to them,” she added.

Bacterial Membrane Dynamics

The outer membranes of gram negative bacteria, such as Pseudomonas and E. coli, consist of multiple proteins and densely packed lipopolysaccharides (LPS or endotoxin). This structure provides a formidable barrier to antibiotics, most of which are targeted to intracellular processes.

“Understanding outer membrane structure and how molecules are recognized and transported across the bacterial membrane are critical to creating more effective antibiotics,” noted Lukas K. Tamm, Ph.D., professor molecular physiology and biological physics, University of Virginia.

The Tamm laboratory studies the dynamics of membrane proteins especially via solution NMR spectroscopy. His laboratory provided the first structure of the outer membrane ion channel of E. coli, OmpA. The group also studies OmpG, an outer membrane protein whose single polypeptide chain forms a membrane nanopore.

“Engineered protein nanopores have attracted interest to detect rare metal ions and neurotransmitters in solution, to sequence DNA and RNA, and to measure folding and unfolding kinetics of single proteins,” he explained. “We developed a new approach to loop immobilization that revealed cross-talk patterns between different loops of the OmpG nanopore. This will be useful to engineer new functions into OmpG and for analyzing other membrane nanopores.”

Dr. Tamm also studies the outer membrane protein H (OprH) from Pseudomonas aeruginosa, a multidrug resistant pathogen that is the most common cause of pneumonia and mortality in cystic fibrosis patients. It is the major cause of hospital-acquired infections.

“The impermeability of this pathogen’s outer membrane contributes substantially to its notorious antibiotic resistance. We utilized in vivo and in vitro assays that demonstrated the importance of the interaction of OprH with LPS in the outer membrane. Additionally, beyond determining the structure of OprH, our studies revealed that solution NMR can be a powerful tool for investigating interaction of integral membrane proteins with specific lipids. This cannot be easily done by crystallography.”

Dr. Tamm explained that there are many challenges remaining before antibiotic resistance can be overcome.

“The substrate is unknown for many of the outer membrane proteins. To develop better targeted antibiotics, it will be important to define specific substrates. Also, determining the structure of outer membrane proteins will likely also provide new insights for understanding how protein-lipid interactions contribute to antibiotic resistance. We aren’t there yet, but we are close to getting better answers.”

Structural model of a re-engineered nanopore [Lukas Tamm, Ph.D., University of Virginia]

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