Author(s): Thomas R. Ayres
Affiliation(s): Center for Astrophysics and Space Astronomy, 389 UCB, University of Colorado, Boulder, CO 80309, [...]
|
||||||||||||||||||
|
Author(s): J. F. Gallimore, A. Yzaguirre, J. Jakoboski, M. J. Stevenosky, D. J. Axon, S. A. Baum, C. L. Buchanan, M. Elitzur, M. Elvis, C. P. O’Dea, and A. Robinson
Mammalian mitochondrial DNA replication intermediates are essentially duplex, but contain extensive tracts of RNA/DNA hybrid. J Mol Biol. 2010 Feb 22; Authors: Pohjoismäki JL, Holmes JB, Wood SR, Yang MY, Yasukawa T, Reyes A, Bailey LJ, Cluett TJ, Goffart S, Willcox S, Rigby RE, Jackson AP, Spelbrink JN, Griffith JD, Crouch RJ, Jacobs HT, Holt IJ We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mtDNA replication intermediates (mtRIs) are essentially duplex throughout their length, but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mtDNA replication involving RNA incorporation throughout the lagging strand (RITOLS). PMID: 20184890 [PubMed - as supplied by publisher] [...]
Crystal structure of P58(IPK) TPR fragment reveals the mechanism for its molecular chaperone activity in UPR. J Mol Biol. 2010 Feb 22; Authors: Tao J, Petrova K, Ron D, Sha B P58(IPK) may function as an ER molecular chaperone to maintain protein folding homeostasis during Unfolded Protein Responses (UPR). P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism how P58(IPK) functions to promote protein folding within ER, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5A resolution by SAD method. The crystal structure of P58(IPK) revealed three domains (I, II and III) with similar folds and each domain contains three TPR motifs. An In vitro ELISA assay indicates that P58(IPK) acts as a molecular chaperone by interacting with the misfolded proteins such as Luciferase and Rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that may be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK). PMID: 20184891 [PubMed - as supplied by publisher] [...]
The Membrane Complex between Transducin and Dark-state Rhodopsin exhibits large-amplitude Interface Dynamics on the sub-musec timescale: Insights from all-atom MD simulations. J Mol Biol. 2010 Feb 22; Authors: Sgourakis NG, Garcia AE Rhodopsin, the prototype class A G-protein coupled receptor, is a very important model system for all 7-transmembrane domain proteins. Characterization of the interactions between rhodopsin and transducin, its intracellular G-protein counterpart, as well as fluctuations in these interactions due to thermal motions are required for an understanding of early events in the mechanism of signal transduction. Here, we use all-atom Molecular Dynamics simulations of a transmembrane protein complex between rhodopsin and the heterotrimeric transducin (Galphabetagamma) in an all-atom DOPC membrane/water environment. Based on the analysis of a musec-timescale simulation trajectory we characterize the dynamics of the system and their effects in the structural features of the protein subunits. Our simulations describe a highly dynamic interaction interface, where the system is alternating between distinct domain orientations at the 10-100ns timescale that can be further classified into interaction modes involving contacts between distinct structural features on the protein subunits. We relate our results with experimental measurements from a variety of studies and high-resolution models of activated rhodopsin. Monitoring key structural features that are involved in the activation process along our simulation trajectory indicates the presence of extensive dynamics in the dark-adapted state, including a motion of Y223 from Helix 3 towards the ionic lock interactions of the conserved ERY motif. The dynamic picture shown here is consistent with a framework in which the dark-state fluctuations sample conformations consistent with the activated state. These results provide an atomic-level description of the dynamics of the full complex and further suggest novel mutagenesis experiments that can be used to investigate the stability and dynamics of this model membrane protein receptor system. PMID: 20184892 [PubMed - as supplied by publisher] [...]
Disulfide-Reduced ALS Variants of Cu, Zn Superoxide Dismutase Exhibit Increased Populations of Unfolded Species. J Mol Biol. 2010 Feb 22; Authors: Kayatekin C, Zitzewitz JA, Matthews CR Cu, Zn superoxide dismutase (SOD1) is a dimeric metal binding enzyme responsible for the dismutation of toxic superoxide to hydrogen peroxide and oxygen in cells. Mutations at dozens of sites in SOD1 induce amyotrophic lateral sclerosis (ALS), a fatal gain-of-function neurodegenerative disease whose molecular basis is unknown. To obtain insights into effects of the mutations on the folded and unfolded populations of immature monomeric forms whose aggregation or self-association may be responsible for ALS, the thermodynamic and kinetic folding properties of a set of disulfide-reduced and disulfide-oxidized Zn-free and Zn-bound stable monomeric SOD1 variants were compared to the wild-type (WT) protein. The most striking effect of the mutations on the monomer stability was observed for the disulfide-reduced metal-free variants. Whereas the WT and S134N monomers are >95% folded at neutral pH and 37 degrees C, A4V, L38V, G93A, and L106V ranged from 50% to ~90% unfolded. The reduction of the disulfide-bond was also found to reduce the apparent Zn affinity of the WT monomer by 750-fold, into the nanomolar range where it may be unable to compete for free Zn in the cell. With the exception of the S134N metal-binding variant, the Zn affinity of disulfide-oxidized SOD1 monomers showed little sensitivity to amino acid replacements. These results suggest a model for SOD1 aggregation where the constant synthesis of ALS-variants of SOD1 on ribosomes provides a pool of species in which the increased population of the unfolded state may favor aggregation over productive folding to the stable native dimeric state. PMID: 20184893 [PubMed - as supplied by publisher] [...]
Comprehensive analysis of HAMP domains: Implications for transmembrane signal transduction. J Mol Biol. 2010 Feb 22; Authors: Dunin-Horkawicz S, Lupas AN Homodimeric receptors with one or two transmembrane segments per monomer are universal to life and represent the largest and most diverse group of cellular transmembrane receptors. They frequently share domain types across phyla and in some cases they have been recombined experimentally into functional chimeras, for example the bacterial aspartate chemoreceptor with the human insulin receptor, suggesting that they have a common mechanism. The nature of this mechanism, however, is still debated. We have proposed a new model for the transduction mechanism by axial helix rotation, based on the structure of a wide-spread domain, HAMP, that frequently occurs in direct continuation of the last transmembrane segment, primarily in histidine kinases and chemoreceptors. Here we show by statistical analysis that HAMP domain sequences have biophysical properties compatible with the two conformations proposed by the model. The analysis also identifies three networks of co-evolving residues, which allow to subdivide the mechanism into individual steps. The most extended of these networks is specific to membrane-bound HAMP domains and most likely accepts the signal from the transmembrane helices. In a classification based on sequence clustering, these HAMPs form a central supercluster, surrounded by smaller clusters of divergent HAMPs, which typically combine into arrays of up to 31 consecutive copies and accept conformational input from other HAMP domains. Unexpectedly, the classifcation shows a division betwen domains of histidine kinases and those of chemoreceptors; thus, except for a few versatile lineages, HAMP domains are largely specific for one particular output domain. Within proteins using a given output domain, HAMP domains also show extensive co-evolution with histidine kinases, but not with chemoreceptors. We attribute the greater capability for recombination among chemoreceptors to their acquisition of a reversible modification system, which acts as a capacitor for the initially deleterious effects of combining domains optimized in different contexts. PMID: 20184894 [PubMed - as supplied by publisher] [...]
The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis bound to Ornithine, a Competitive Inhibitor. J Mol Biol. 2010 Feb 22; Authors: Sankaranarayanan R, Cherney MM, Garen C, Garen G, Niu C, Yuan M, James MN Ornithine acetyltransferase in Mycobacterium tuberculosis (Mtb OAT; E.C.2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to L-glutamate (GLU). Mtb OAT is a member of the N-terminal nucleophile (Ntn) fold family of enzymes. The crystal structures of Mtb OAT in the native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 A resolution, respectively. ORN is a competitive inhibitor of this enzyme against GLU as the substrate. Although, the acyl-enzyme complex of OAT from S.clavuligerus (Sclav OAT) has been determined, ours is the first crystal structure to be reported of an OAT in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, the stabilization of the C-terminal residues by ORN reduces the size of the active site pocket volume in the structure of ORN complex. The interactions involving ORN with the protein residues of Mtb OAT unambiguously delineate the active site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal the important interactions between the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly 128 and with the side-chain oxygen of Thr 127. These interactions likely help in the stabilization of the oxyanion formation during the enzymatic reaction and also would polarize the carbonyl-carbon to oxygen bond thereby enabling the side-chain atom O(gamma1) of Thr 200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN. PMID: 20184895 [PubMed - as supplied by publisher] [...]
Conversion of a regulatory into a degradative protease. J Mol Biol. 2010 Feb 22; Authors: Hasenbein S, Meltzer M, Hauske P, Kaiser M, Huber R, Clausen T, Ehrmann M The PDZ protease DegS senses mislocalized outer membrane proteins and initiates the sigma E pathway in the bacterial periplasm. This unfolded protein response pathway is activated by processing of the anti sigma factor RseA by DegS and other proteases acting downstream of DegS. Since DegS mediates the rate limiting step of sigma E induction, its activity must be highly specific and tightly regulated. While DegS is structurally and biochemically well studied, the determinants of its pronounced substrate specificity are unknown. We therefore performed swapping experiments by introducing elements of the homologous but unspecific PDZ protease DegP. Introduction of loop L2 of DegP into DegS converted the enzyme into a non specific protease, while swapping of PDZ domains did not. Therefore, loop L2 of the protease domain is a key determinant of substrate specificity. Interestingly, swapping of loop L2 did not affect the tight regulation of DegS. In addition, the combined introduction of loop L2 and PDZ domain 1 of DegP into DegS converted DegS even further into a DegP like protease. These and other data suggest that homologous enzymes with distinct activities and regulatory features can be converted by simple genetic modifications. PMID: 20184896 [PubMed - as supplied by publisher] [...]
Crystal structure of prostate secretory protein PSP94 shows an edge-to-edge association of two monomers to form a homodimer. J Mol Biol. 2010 Feb 22; Authors: Kumar A, Jagtap DD, Mahale SD, Kumar M Several recent genome-wide association studies have linked the human MSMB gene, encoding prostate secretory protein of 94 residues (PSP94), with prostate cancer susceptibility. PSP94 is one of the most abundant proteins form prostatic secretions and a primary constituent of human semen. PSP94 suppresses tumor growth and metastasis and its expression gradually decreases during progression of the prostate cancer. It is a rapidly evolving protein with homologues present in several species with ten conserved cysteine residues. PSP94 homologues show high affinity binding with different proteins from cysteine rich secretory protein (CRISP) family, some of which have been shown to be ion channel blockers. Here, we report the crystal structure of human PSP94 at 2.3 A resolution. The structure shows that the amino and the carboxyl ends of the polypeptide chain are held in close proximity facing each other. A strong hydrogen bond between these ends, which are located respectively on the first and the last beta-strands, leads to formation of an almost straight edge in PSP94 structure. Crystal structure shows that these edges from two PSP94 monomers associate in antiparallel fashion leading to formation of a dimer. Our studies further show that dimers dissociate into monomers at acidic pH, possibly through distortion of the straight edge. Further, based on several observations, we propose that PSP94 binds to CRISPs and immunoglobulin G through the same edge, which is involved in the formation of PSP94 dimeric interface. PMID: 20184897 [PubMed - as supplied by publisher] [...] |
||||||||||||||||||
|
|
||||||||||||||||||
