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Calmodulin (CaM) regulates tetrameric N-methyl-D-aspartate receptors (NMDARs) by binding tightly to the C0 and C1 regions of its NR1 subunit. A crystal structure (2HQW; 1.96 Å) of calcium-saturated CaM bound to NR1C1 (peptide spanning 875–898) showed that NR1 S890, whose phosphorylation regulates membrane localization, was solvent protected, whereas the endoplasmic reticulum retention motif was solvent...
In the bacterial genetic-code system, the codon AUA is decoded as isoleucine by tRNA Ile2 with the lysidine residue at the wobble position. Lysidine is derived from cytidine, with ATP and L-lysine, by tRNA Ile lysidine synthetase (TilS), which is an N-type ATP pyrophosphatase. In this study, we determined the crystal structure of Aquifex aeolicus TilS, complexed with ATP, Mg ...
Transcriptional repressor FL11 from the hyperthermophilic archaeon, Pyrococcus OT3, was crystallized in its dimer form in complex with a DNA duplex, TGAAAWWWTTTCA. Chemical contacting of FL11 to the terminal 5 bps, and DNA bending by propeller twisting at WWW confirmed specificity of the interaction. Dimer-binding sites were identified in promoters of ∼200 transcription units coding, for example,...
Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic...
In designing mutagenesis experiments, it is often crucial to know how certain mutations will affect the structure and thermodynamic stability of the protein. Here, we present a methodology, Eris, to efficiently and accurately compute the stability changes of proteins upon mutations using our protein-modeling suite, Medusa. We evaluate the stability changes upon mutations for 595 mutants from five...
Potassium channels fluctuate between closed and open states. The detailed mechanism of the conformational changes opening the intracellular pore in the K + channel from Streptomyces lividans (KcsA) is unknown. Applying Monte Carlo normal mode following, we find that gating involves rotation and unwinding of the TM2 bundle, lateral movement of the TM2 helices away from the channel axis, and...
Radiation damage can be a problem when utilizing ionizing X-radiation in macromolecular crystallography. The dose dependence of radiation damage to eight lysozyme crystals at room temperature (292 K) was investigated in order to provide an accurate comparison with cryotemperature (100 K) results and to allow researchers to calculate expected maximum room-temperature-crystal lifetimes prior to data...
Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved βαβ-element, suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins. The basal fold within this metafold, the RIFT barrel, is also found in a wide range of enzymes, whose homologous relationship with the nucleic acid-binding group is unclear. We have characterized a protein...
Structural studies of large proteins and protein assemblies are a difficult and pressing challenge in molecular biology. Experiments often yield only low-resolution or sparse data that are not sufficient to fully determine atomistic structures. We have developed a general geometry-based algorithm that efficiently samples conformational space under constraints imposed by low-resolution density maps...
The human cytosolic thymidine kinase (TK) and structurally related TKs in prokaryotes play a crucial role in the synthesis and regulation of the cellular thymidine triphosphate pool. We report the crystal structures of the TK homotetramer from Thermotoga maritima in four different states: its apo-form, a binary complex with thymidine, as well as the ternary structures with the two substrates (thymidine/AppNHp)...
In Rhodobacter (Rba.) sphaeroides, the subunit PufX is involved in the dimeric organization of the core complex. Here, we report the 3D reconstruction at 12 Å by cryoelectron microscopy of the core complex of Rba. veldkampii, a complex of ∼300 kDa without symmetry. The core complex is monomeric and constituted by a light-harvesting complex 1 (LH1) ring surrounding a uniquely oriented reaction center...
Phosphorylation of endogenous inhibitor proteins for type-1 Ser/Thr phosphatase (PP1) provides a mechanism for reciprocal coordination of kinase and phosphatase activities. A myosin phosphatase inhibitor protein CPI-17 is phosphorylated at Thr38 through G-protein-mediated signals, resulting in a >1000-fold increase in inhibitory potency. We show here the solution NMR structure of phospho-T38-CPI-17...
Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of...
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