Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively. These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.

Cation channels of the TRP family serve important physiological roles by opening in response to diverse intra-and extra-cellular stimuli which regulate their lower or upper gates. Despite extensive studies, the mechanism coupling these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as “pore turrets,” which are proximal to the upper gates. We establish the importance of the pore turret through activity assays and by solving structures of rat TRPV2 both with and without an intact turret at resolutions of 4.0 Å and 3.6 Å respectively. These structures resolve the full-length pore turret and reveal fully open and partially open states of TRPV2, both with unoccupied vanilloid pockets. Our results suggest a mechanism by which physiological signals, such as lipid binding, can regulate the lower gate and couple to the upper gate through a pore turret-facilitated mechanism.

The GTPase-accelerating protein (GAP) complex RGS9-1⅐G␤ 5 plays an important role in the kinetics of light responses by accelerating the GTP hydrolysis of G␣ t in vertebrate photoreceptors. Much, but not all, of this complex is tethered to disk membranes by the transmembrane protein R9AP. To determine the effect of the R9AP membrane complex on GAP activity, we purified recombinant R9AP and reconstituted it into lipid vesicles along with the photon receptor rhodopsin. Full-length RGS9-1⅐G␤ 5 bound to R9AP-containing vesicles with high affinity (K d < 10 nM), but constructs lacking the DEP (dishevelled/EGL-10/pleckstrin) domain bound with much lower affinity, and binding of those lacking the entire N-terminal domain (i.e. the dishevelled/EGL-10/pleckstrin domain plus intervening domain) was not detectable. Formation of the membranebound complex with R9AP increased RGS9-1 GAP activity by a factor of 4. Vesicle titrations revealed that on the time scale of phototransduction, the entire reaction sequence from GTP uptake to GAP-catalyzed hydrolysis is a membrane-delimited process, and exchange of G␣ t between membrane surfaces is much slower than hydrolysis. Because in rod cells different pools exist of RGS9-1⅐G␤ 5 that are either associated with R9AP or not, regulation of the association between R9AP and RGS9-1⅐G␤ 5 represents a potential mechanism for the regulation of recovery kinetics.Timely deactivation of G protein ␣ subunits is a key element of responses to the stimulation of G protein-coupled receptors. It plays an especially important role in fast cellular responses such as those of vertebrate photoreceptors. In the rod and cone cell outer segments, the recovery phase of light responses depends on the presence of a GTPase-accelerating protein (GAP) 1 complex RGS9-1⅐G␤ 5 (1-4). Whether and how the GAP activity of this complex is regulated is unknown.RGS9-1 contains multiple functional domains, including an RGS domain that is responsible for its GAP activity (3), a G protein ␥ subunit-like domain for G␤ 5L binding (4,5), an Nterminal domain that includes a DEP (dishevelled/EGL-10/ pleckstrin) domain (6) and an intermediate domain (7), and a C-terminal domain that is unique to RGS9-1 among all of the RGS proteins (8). All of these domains have been found to participate in the regulation of GAP activity and substrate specificity (9 -11). The inhibitory PDE␥ subunit of the effector regulated by G␣ t , cGMP phosphodiesterase (PDE6), interacts with both G␣ t and the catalytic core of RGS9-1 and enhances RGS9-1 GAP activity (12-14) in vitro, but it is not clear how this enhancement is accomplished in a physiological setting in which tight PDE␥ binding to PDE6 catalytic subunits blocks GAP enhancement (15-18).Additional possible mechanisms for regulation include a light-and calcium-regulated phosphorylation (19, 20) of RGS9-1 and interactions with the recently discovered membrane anchor protein, R9AP (7, 21). R9AP is a 25-kDa protein that is selectively expressed in photoreceptor outer segments. Homologues are apparen...

Photoreceptor-specific ciliopathies often affect a structure that is considered functionally homologous to the ciliary transition zone (TZ) called the connecting cilium (CC). However, it is unclear how mutations in certain ciliary genes disrupt the photoreceptor CC without impacting the primary cilia systemically. By applying stochastic optical reconstruction microscopy technology in different genetic models, we show that the CC can be partitioned into two regions: the proximal CC (PCC), which is homologous to the TZ of primary cilia, and the distal CC (DCC), a photoreceptor-specific extension of the ciliary TZ. This specialized distal zone of the CC in photoreceptors is maintained by SPATA7, which interacts with other photoreceptor-specific ciliary proteins such as RPGR and RPGRIP1. The absence of results in the mislocalization of DCC proteins without affecting the PCC protein complexes. This collapse results in destabilization of the axonemal microtubules, which consequently results in photoreceptor degeneration. These data provide a novel mechanism to explain how genetic disruption of ubiquitously present ciliary proteins exerts tissue-specific ciliopathy phenotypes.

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