Molecular Biophysics
Structural and functional interactions between the Ca2+-, ATP-, and caffeine-binding sites of skeletal muscle ryanodine receptor (RyR1)
Ryanodine receptor type 1 (RyR1) releases Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle cells to initiate muscle contraction. Multiple endogenous and exogenous effectors regulate RyR1, such as ATP, Ca2+, caffeine (Caf), and ryanodine. Cryo-EM identified binding sites for the three coactivators Ca2+, ATP, and Caf. However, the mechanism of coregulation and synergy between these activators remains to be determined. Here, we used [3H]ryanodine ligand-binding assays and molecular dynamics simulations to test the hypothesis that both the ATP- and Caf-binding sites communicate with the Ca2+-binding site to sensitize RyR1 to Ca2+.ATP utilization by a DEAD-box protein during refolding of a misfolded group I intron ribozyme
DEAD-box helicase proteins perform ATP-dependent rearrangements of structured RNAs throughout RNA biology. Short RNA helices are unwound in a single ATPase cycle, but the ATP requirement for more complex RNA structural rearrangements is unknown. Here we measure the amount of ATP used for native refolding of a misfolded group I intron ribozyme by CYT-19, a Neurospora crassa DEAD-box protein that functions as a general chaperone for mitochondrial group I introns. By comparing the rates of ATP hydrolysis and ribozyme refolding, we find that several hundred ATP molecules are hydrolyzed during refolding of each ribozyme molecule.ATP-dependent interactions of a cargo protein with the transmembrane domain of a polypeptide processing and secretion ABC transporter
Powered by the energy of ATP binding and hydrolysis, protease-containing ABC transporters (PCATs) export amphipathic and hydrophilic bacteriocin and quorum-sensing proteins across the membrane hydrophobic barrier. The cargo proteins have N-terminal leader peptides that are cleaved off by the cysteine protease domain, referred to as the C39 domain, or referred to as the peptidase (PEP) domain. The sequence and structural determinants of the interaction between PCATs and cargo proteins are poorly understood, yet this interaction is a central aspect of the transport mechanism.A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2′-OH of GTP
Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3′-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion.Autophosphorylation activates c-Src kinase through global structural rearrangements
The prototypical kinase c-Src plays an important role in numerous signal transduction pathways, where its activity is tightly regulated by two phosphorylation events. Phosphorylation at a specific tyrosine by C-terminal Src kinase inactivates c-Src, whereas autophosphorylation is essential for the c-Src activation process. However, the structural consequences of the autophosphorylation process still remain elusive. Here we investigate how the structural landscape of c-Src is shaped by nucleotide binding and phosphorylation of Tyr416 using biochemical experiments, hydrogen/deuterium exchange MS, and atomistic molecular simulations.Permeant-specific gating of connexin 30 hemichannels
Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca2+-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in Xenopus laevis oocytes.Conserved functional consequences of disease-associated mutations in the slide helix of Kir6.1 and Kir6.2 subunits of the ATP-sensitive potassium channel
Cantu syndrome (CS) is a condition characterized by a range of anatomical defects, including cardiomegaly, hyperflexibility of the joints, hypertrichosis, and craniofacial dysmorphology. CS is associated with multiple missense mutations in the genes encoding the regulatory sulfonylurea receptor 2 (SUR2) subunits of the ATP-sensitive K+ (KATP) channel as well as two mutations (V65M and C176S) in the Kir6.1 (KCNJ8) subunit. Previous analysis of leucine and alanine substitutions at the Val-65-equivalent site (Val-64) in Kir6.2 indicated no major effects on channel function.ATP hydrolysis Promotes Duplex DNA Release by the RecA Presynaptic Complex
Homologous recombination is an important DNA repair pathway that plays key roles in maintaining genome stability. Escherichia coli RecA is an ATP-dependent DNA-binding protein that catalyzes the DNA strand exchange reactions in homologous recombination. RecA assembles into long helical filaments on single-stranded DNA, and these presynaptic complexes are responsible for locating and pairing with a homologous duplex DNA. Recent single molecule studies have provided new insights into RecA behavior, but the potential influence of ATP in the reactions remains poorly understood.A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors
Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding.
