The envelope glycoproteins (Env) of human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate virus binding to the cell surface receptor CD4 on target cells to initiate infection 1 . Env is a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120), and forms trimers on the surface of the viral membrane. Using cryo-electron tomography combined with three-dimensional image classification and averaging, we report the threedimensional structures of trimeric Env displayed on native HIV-1 in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures 2,3 of the monomeric gp120 core in the b12-and CD4/17b-bound conformations into the density maps derived by electron tomography, we derive molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. We demonstrate that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This appears to be coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings elucidate the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells. It is estimated that over 33 million individuals are at present infected with HIV (http://www.unaids.org). The development of an effective vaccine is therefore a challenge of fundamental medical interest. It has been widely recognized that a better understanding of the structure of trimeric Env in its various conformational states is likely to be an important element in the overall strategy for vaccine development 4 . Although X-ray crystallographic methods have led to atomic models for HIV-1 gp120 monomers complexed to antibodies in the presence and absence of CD4 (refs 2 , 3, 5 ), determination of the structures of intact trimers on native viruses has nevertheless remained elusive. Theoretical models for the structure of the trimer that take into account constraints determined from biochemical and mutagenesis studies of monomeric gp120 (refs 6 , 7 ) have been advanced, but the advent of electron tomographic methods 8 provides a unique opportunity for direct experimental determination of the structure of the intact trimer on the virus in its native state. Here we report structural analysis of native HIV-1 Env using alignment and classification procedures that take into account the missing wedge that arises from the limited angular range used for data collection in electron tomography. Our approach takes advantage of complexes containing monomeric gp120 for
The high water permeability characteristic of mammalian red cell membranes is now known to be caused by the protein AQP1. This channel freely permits movement of water across the cell membrane, but it is not permeated by other small, uncharged molecules or charged solutes. AQP1 is a tetramer with each subunit containing an aqueous pore likened to an hourglass formed by obversely arranged tandem repeats. Cryoelectron microscopy of reconstituted AQP1 membrane crystals has revealed the three-dimensional structure at 3-6 A. AQP1 is distributed in apical and basolateral membranes of renal proximal tubules and descending thin limbs as well as capillary endothelia. Ten mammalian aquaporins have been identified in water-permeable tissues and fall into two groupings. Orthodox aquaporins are water-selective and include AQP2, a vasopressin-regulated water channel in renal collecting duct, in addition to AQP0, AQP4, and AQP5. Multifunctional aquaglyceroporins AQP3, AQP7, and AQP9 are permeated by water, glycerol, and some other solutes. Aquaporins are being defined in numerous other species including amphibia, insects, plants, and microbials. Members of the aquaporin family are implicated in numerous physiological processes as well as the pathophysiology of a wide range of clinical disorders.
he ongoing global pandemic of the novel SARS-CoV-2 coronavirus presents an urgent need for the development of effective preventative and treatment therapies. The viral spike (S)-protein is a prime target for such therapies because of its critical role in the virus life cycle. The transmembrane CoV S-protein spike trimer is composed of interwoven protomers that include an N-terminal receptor-binding S1 subunit and a C-terminal S2 subunit that contains the fusion elements (Fig. 1a,b) 1. The S1 subunit is subdivided into the N-terminal domain (NTD) followed by the receptor-binding domain (RBD) and two structurally conserved subdomains (SD1 and SD2). Together, these domains cap the S2 subunit, protecting the conserved fusion machinery. Binding to the host receptor via the RBD in S1 is followed by proteolytic cleavage of the spike by host proteases 2. Large conformational changes in the S-protein result in S1 shedding and exposure of the fusion machinery in S2. Class I fusion proteins, such as the CoV-2 S-protein, undergo large conformational changes during the fusion process and must, by necessity, be highly flexible and dynamic. Indeed, cryo-EM structures of the SARS-CoV-2 spike reveal considerable flexibility and dynamics in the S1 subunit 1,2 , especially around the RBD, which exhibits two discrete conformational states-a 'down' state that is shielded from receptor binding and an 'up' state that is receptor-accessible. The wealth of structural information for β-CoV S-proteins, including the recently determined cryo-EM structures of the SARS-CoV-2 spike 1-11 , has provided a rich source of detailed geometric information from which to begin precise examination of the macromolecular transitions underlying triggering of this fusion machine. Several structures of soluble ectodomain constructs that retain the complete S1 subunit and the surface-exposed S2 subunit have been determined. These include SARS-CoV-2 1,3 , SARS 4-8 , MERS 4,9 and other human 1,10 and murine 11 β-CoV S-proteins.
Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca2+)–permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo–electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP2, and Ca2+, as well as in complex with the menthol analog WS-12 and PIP2. Our structures reveal the binding sites for cooling agonists and PIP2in TRPM8. Notably, PIP2binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP2and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.
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