Structural insights on TRPV5 gating by endogenous modulators.

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery

The C-H Peripheral Stalk Base: A Novel Component in V1-ATPase Assembly

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V1Vo-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V 1-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE 3G 3H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembl

Structural and Functional Insights into the Evolution and Stress Adaptation of Type II Chaperonins

Chaperonins are essential biological complexes assisting protein folding in all kingdoms of life. Whereas homooligomeric bacterial GroEL binds hydrophobic substrates non-specifically, the heterooligomeric eukaryotic CCT binds specifically to distinct classes of substrates. Sulfolobales, which survive in a wide range of temperatures, have evolved three different chaperonin subunits (α, β, γ) that form three distinct complexes tailored for different substrate classes at cold, normal, and elevated temperatures. The larger octadecameric β complexes cater for substrates under heat stress, whereas smaller hexadecameric αβ complexes prevail under normal conditions. The cold-shock complex contains all three subunits, consistent with greater substrate specificity. Structural analysis using crystallography and electron microscopy reveals the geometry of these complexes and shows a novel arrangement of the α and β subunits in the hexadecamer enabling incorporation of the γ subunit.Accepted versio

Single-Ring Intermediates Are Essential for Some Chaperonins

Chaperonins are macromolecular complexes found throughout all kingdoms of life that assist unfolded proteins reach a biologically active state. Historically, chaperonins have been classified into two groups based on sequence, subunit structure, and the requirement for a co-chaperonin. Here, we present a brief review of chaperonins that can form double- and single-ring conformational intermediates in their protein-folding catalytic pathway. To date, the bacteriophage encoded chaperonins ϕ-EL and OBP, human mitochondrial chaperonin and most recently, the bacterial groEL/ES systems, have been reported to form single-ring intermediates as part of their normal protein-folding activity. These double-ring chaperonins separate into single-ring intermediates that have the ability to independently fold a protein. We discuss the structural and functional features along with the biological relevance of single-ring intermediates in cellular protein folding. Of special interest are the ϕ-EL and OBP chaperonins which demonstrate features of both group I and II chaperonins in addition to their ability to function via single-ring intermediates

Biochemical Characterization and X-ray Crystallography of a Lysozyme Encoded by Pseudomonas aeruginosa Bacteriophage SN

Bacteriophage SN is a virulent phage that selectively infects the bacterium Pseudomonas aeruginosa. It was isolated from Lake Chernoe in Russia and it is related to the PB1-like species of the Myoviridae family. The DNA genome is composed of 66,391 base pairs, has 89 predicted open reading frames, and encodes more than 20 structural proteins. One of the open reading frames of this newly discovered bacteriophage has high sequence identity to other lysozyme and chitinase genes. It is therefore assumed that this protein encoded by bacteriophage SN is utilized for digestion of the host cell wall prior to injection of the nucleic acid into the host. Determining the high-resolution structure of the protein will aid in later determination of location within the intact phage. The gene of interest has been cloned into E. coli by PCR amplification and ligation into the PET30a vector. This protein expressed to high levels and the product has been purified to homogeneity. The protein demonstrated specific enzymatic activity by lysis of Micrococcus luteus cells in suspension as purity increased. Crystals of the protein currently diffract to 3.5 Å, the data gathered from optimized crystals will be used to determine the X-ray structure of the lysozyme

Translocation of the papillomavirus L2/vDNA complex across the limiting membrane requires the onset of mitosis

<div><p>The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the <i>trans</i>-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions. We find that L2 translocation requires transport to the TGN and is strictly dependent on entry into mitosis, coinciding with mitotic entry in synchronized cells. Cell cycle arrest causes retention of L2/vDNA at the TGN; only release and progression past G2/M enables translocation across the limiting membrane and subsequent infection. Microscopy of EdU-labeled vDNA reveals a rapid and dramatic shift in vDNA localization during early mitosis. At late G2/early prophase vDNA egresses from the TGN to a pericentriolar location, accumulating there through prometaphase where it begins to associate with condensed chromosomes. By metaphase and throughout anaphase the vDNA is seen bound to the mitotic chromosomes, ensuring distribution into both daughter nuclei. Mutations in a newly defined chromatin binding region of L2 potently blocked translocation, suggesting that translocation is dependent on chromatin binding during prometaphase. This represents the first time a virus has been shown to functionally couple the penetration of limiting membranes to cellular mitosis, explaining in part the tropism of HPV for mitotic basal keratinocytes.</p></div

Structural insights on TRPV5 gating by endogenous modulators.

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery