Imaging the spatial orientation of subunits within membrane receptors by atomic force microscopy.

Our experimental approach is based on the atomic force microscope (AFM) imaging of epitope-tagged subunits within membrane protein complexes purified in small amounts and decorated by anti-tag antibodies. Furthermore, we can produce simultaneous decoration of protein complexes using Fab fragments and IgG antibodies, which, combined with chemical modification of the substrate, allows us to determine the protein orientation across the cell membrane. Here, we describe a detailed protocol for membrane protein purification, AFM data collection, analysis, and interpretation of results. The protocol also covers basic AFM instrument settings and best practices for both observation of membrane protein complexes by AFM and automatic detection of the structures by an in-house algorithm. Once a sufficient number of membrane protein complexes have been visualized by AFM, data acquisition and processing can be completed in approximately 10 min using a scanning surface of 1 µm(2).

Structure and binding mechanism of vascular endothelial cadherin: a divergent classical cadherin.

Vascular endothelial cadherin (VE-cadherin), a divergent member of the type II classical cadherin family of cell adhesion proteins, mediates homophilic adhesion in the vascular endothelium. Previous investigations with a bacterially produced protein suggested that VE-cadherin forms cell surface trimers that bind between apposed cells to form hexamers. Here we report studies of mammalian-produced VE-cadherin ectodomains suggesting that, like other classical cadherins, VE-cadherin forms adhesive trans dimers between monomers located on opposing cell surfaces. Trimerization of the bacterially produced protein appears to be an artifact that arises from a lack of glycosylation. We also present the 2.1-Å-resolution crystal structure of the VE-cadherin EC1-2 adhesive region, which reveals homodimerization via the strand-swap mechanism common to classical cadherins. In common with type II cadherins, strand-swap binding involves two tryptophan anchor residues, but the adhesive interface resembles type I cadherins in that VE-cadherin does not form a large nonswapped hydrophobic surface. Thus, VE-cadherin is an outlier among classical cadherins, with characteristics of both type I and type II subfamilies.

Acid-sensing ion channel (ASIC) 1a undergoes a height transition in response to acidification.

The acid-sensing ion channel (ASIC) 1a is known to assemble as a homotrimer. Here, we used atomic force microscopy to image ASIC1a, integrated into lipid bilayers, at pH 7.0 and pH 6.0. The triangular appearance of the channel was clearly visible. A height distribution for the channels at pH 7.0 had two peaks, at 2 and 4 nm, likely representing the intracellular and extracellular domains, respectively. At pH 6.0 the 2-nm peak remained, but the higher peak shifted to 6 nm. Hence, the extracellular domain of the channel becomes 'taller' after acidification.

Demonstration of a direct interaction between sigma-1 receptors and acid-sensing ion channels.

The sigma-1 receptor is a widely expressed protein that interacts with a variety of ion channels, including the acid-sensing ion channel (ASIC) 1a. Here we used atomic force microscopy to determine the architecture of the ASIC1a/sigma-1 receptor complex. When isolated His(8)-tagged ASIC1a was imaged in complex with anti-His(6) antibodies, the angle between pairs of bound antibodies was 135 degrees , consistent with the known trimeric structure of the channel. When ASIC1a was coexpressed with FLAG/His(6)-tagged sigma-1 receptor, ASIC1a became decorated with small particles, and pairs of these particles bound at an angle of 131 degrees . When these complexes were incubated with anti-FLAG antibodies, pairs of antibodies bound at an angle of 134 degrees , confirming that the small particles were sigma-1 receptors. Of interest, we found that the sigma-1 receptor ligand haloperidol caused an approximately 50% reduction in ASIC1a/sigma-receptor binding, suggesting a way in which sigma-1 ligands might modulate channel properties. For the first time, to our knowledge, we have resolved the structure of a complex between the sigma-1 receptor and a target ion channel, and demonstrated that the stoichiometry of the interaction is 1 sigma-1 receptor/1 ASIC1a subunit.

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping.

Atomic force microscopy (AFM) allows the study of single protein-DNA interactions such as those observed with the Type I Restriction-Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATPgammaS, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein-DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.

Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging.

There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs.