Protein-protein interactions of KChIP proteins and Kv4.2

To prove heteromeric assembly of KChIP proteins, the present study is carried out. The results of chemical crosslinking and pull down assay revealed that KChIP1, KChIP2.1, and KChIP2.2 could form homo- as well as hetero-oligomer, and this oligomerization exhibited a Ca(2+)-dependent manner. Moreover, homomeric and heteromeric assembly of KChIPs did not perturb their interaction with Kv4.2 K(+) channel, indicating that the region associated with oligomerization of KChIPs was distinct from that for binding with Kv4.2. Together with previous findings that the net effects of KChIP proteins on the molecular properties and trafficking of Kv channel were different, these observations open a fascinating possibility that the electrophysiological properties of Kv channel may be differently regulated by homomeric and heteromeric assembly of KChIPs.     

F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis

The Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro-synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.     

Deregulation of cyclin E in human cells interferes with prereplication complex assembly

Deregulation of cyclin E expression has been associated with a broad spectrum of human malignancies. Analysis of DNA replication in cells constitutively expressing cyclin E at levels similar to those observed in a subset of tumor-derived cell lines indicates that initiation of replication and possibly fork movement are severely impaired. Such cells show a specific defect in loading of initiator proteins Mcm4, Mcm7, and to a lesser degree, Mcm2 onto chromatin during telophase and early G1 when Mcm2-7 are normally recruited to license origins of replication. Because minichromosome maintenance complex proteins are thought to function as a heterohexamer, loading of Mcm2-, Mcm4-, and Mcm7-depleted complexes is likely to underlie the S phase defects observed in cyclin E-deregulated cells, consistent with a role for minichromosome maintenance complex proteins in initiation of replication and fork movement. Cyclin E-mediated impairment of DNA replication provides a potential mechanism for chromosome instability observed as a consequence of cyclin E deregulation.     

Design and crystal structure of bacteriophage T4 mini-fibritin NCCF

Fibritin is a fibrous protein that forms "whiskers" attached to the neck of bacteriophage T4. Whiskers interact with the long tail fibers regulating the assembly and infectivity of the virus. The fibritin trimer includes the N-terminal domain responsible for attachment to the phage particle and for the collar formation, the central domain forming a 500 A long segmented coiled-coil structure, and the C-terminal "foldon" domain. We have designed a "mini" fibritin with most of the coiled-coil domain deleted, and solved its crystal structure. The non-helical N-terminal part represents a new protein fold that tightly interacts with the coiled-coil segment forming a single domain, as revealed by calorimetry. The analysis of the crystal structure and earlier electron microscopy data on the collar-whisker complex suggests the necessity of other proteins to participate in the collar formation. Crystal structure determination of the N-terminal domain of fibritin is the first step towards elucidating the detailed structure and assembly mechanism of the collar-whisker complex.     

Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily

The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids). In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.08A resolution, respectively. Inspection of the cofactor-binding cavity led to the identification of a new interaction between side-chains of residues His222 and Lys270, which cover the central phosphate chain of the cofactor, reminiscent of the "safety-belt" found in other aldo-keto reductases. Testosterone is stabilized by a phenol/benzene tunnel composed of side-chains of numerous residues, among which Phe54, which forces the steroid to take up an orientation markedly contrasting with that found in HSD ternary complexes reported. Combining structural, site-directed mutagenesis, kinetic and fluorescence titration studies, we found that the selectivity of rb20alpha-HSD is mediated by (i) the relaxation of loop B (residues 223-230), partly controlled by the nature of residue 230, (ii) the nature of the residue found at position 54, and (iii) the residues found in the C-terminal tail of the protein especially the side-chain of the amino acid 306.     

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.     

A single subunit of a heterotrimeric CCAAT-binding complex carries a nuclear localization signal: piggy back transport of the pre-assembled complex to the nucleus

An unresolved question concerns the nuclear localization of the heterotrimeric CCAAT-binding complex, which is evolutionarily conserved in eukaryotic organisms including fungi, plants and mammals. All three subunits are necessary for DNA binding. In the filamentous fungus Aspergillus nidulans the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the HapB, HapC and HapE subunits. Here, by using various green fluorescent protein constructs, a nuclear localization signal sequence (NLS) of the HapB protein was identified, outside of the evolutionarily conserved domain. HapB-EGFP was transported into the nucleus in both DeltahapC and DeltahapE strains, indicating that its NLS interacts with the import machinery independently of the other Hap subunits. In contrast, HapC-EGFP did not enter the nucleus in the absence of HapE or HapB. A similar finding was made for HapE-EGFP, which did not localize to the nucleus in the absence of HapC or HapB. Addition of the HapB-NLS to either HapC or HapE led to nuclear localization of the respective protein fusions, indicating that both HapC and HapE lack a functional NLS. Furthermore, these data strongly suggest that HapC and HapE have first to form a heterodimer and can be transported only as a heterodimer via the HapB protein into the nucleus. Therefore, the HapB subunit is the primary cargo for the import machinery, while HapC and HapE are transported to the nucleus only as a heterodimer and in complex with HapB via a piggy back mechanism. This enables the cell to provide equimolar concentrations of all subunits to the nucleus.     

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and _L-crystallins was studied during thermal denaturation of _L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and _L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure _L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of _L-crystallin it binds with modified -crystallin or, alternatively, _L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -_L-crystallin complex size and relative contents of - and -_L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation.