Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.

Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 A resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs.