The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.     

Structural properties of recombinant nonrepetitive and repetitive parts of major ampullate spidroin 1 from Euprosthenops australis: implications for fiber formation

Spider dragline silk proteins, spidroins, have a tripartite composition; a nonrepetitive N-terminal domain, a central repetitive region built up from many iterated poly-Ala and Gly rich blocks, and a C-terminal nonrepetitive domain. It is generally believed that the repetitive region forms intermolecular contacts in the silk fibers, while precise functions of the terminal domains have not been established. Herein, thermal, pH, and salt effects on the structure and aggregation and/or polymerization of recombinant N- and C-terminal domains, a repetitive segment containing four poly-Ala and Gly rich coblocks, and combinations thereof were studied. The N- and C-terminal domains have mainly alpha-helical structure, and interestingly, both form homodimers. Dimerization of the end domains allows spidroin multimerization independent of the repetitive part. Reduction of an intersubunit disulfide in the C-terminal domain lowers the thermal stability but does not affect dimerization. The repetitive region shows helical secondary structure but appears to lack stable folded structure. A protein composed of this repetitive region linked to the C-terminal domain has a mainly alpha-helical folded structure but shows an abrupt transition to beta-sheet structures upon heating. At room temperature, this protein self-assembles into macroscopic fibers within minutes. The secondary structures of none of the domains are altered by pH or salt. However, high concentrations of phosphate affect the tertiary structure and accelerate the aggregation propensity of the repetitive region. Implications of these results for dragline spidroin behavior in solution and silk fiber formation are discussed.     

Comparative analysis of polyphenol oxidase from plant and fungal species

Polyphenol oxidase from plants and fungi is a metalloenzyme containing a type-3 copper center and is homologous to oxygen-carrying hemocyanin of molluscs. Molluscan hemocyanin consists of two domains, an N-terminal domain containing the copper center and a smaller C-terminal domain, connected by an alpha-helical linker. It is presumed that the same is true of polyphenol oxidase from plants and fungi although the structure of a polyphenol oxidase containing the C-terminal domain has not been determined. We show that a number of important structural features are conserved in the N-terminal domains of polyphenol oxidases from various plants and fungi, including a tyrosine motif which can be considered a landmark indicating the beginning of the linker region connecting the N- and C-terminal domains. Our sequence alignments and secondary structure predictions indicate that the C-terminal domains of polyphenol oxidases are likely to be similar in tertiary structure to that of hemocyanin. Detailed bioinformatics analyses of the linker regions predict that this section of the polypeptide chain is intrinsically disordered (lacking fixed tertiary structure) and contains a site of proteolytic processing as well as a potential phosphorylation site.     

Analysis and Expansion of the Role of the Escherichia coli Protein ProQ

The decrease in proline transport by the proline porter ProP in a ΔproQ strain has been well documented; however, the reason for this phenotype remains undefined. Previous studies have speculated that ProQ facilitates translation of proP mRNA. Here, we demonstrate that ProQ is enriched in the polysome fractions of sucrose gradient separations of E. coli lysates and the 30S fractions of lysates separated under conditions causing ribosomal subunit dissociation. Thus, ProQ is a bona fide ribosome associated protein. Analysis of proQ constructs lacking predicted structural domains implicates the N-terminal domain in ribosome association. Association with the ribosome appears to be mediated by an interaction with the mRNA being translated, as limited treatment of lysates with Micrococcal Nuclease maintains ribosome integrity but disrupts ProQ localization with polysomes. ProQ also fails to robustly bind to mRNA-free 70S ribosomes in vitro. Interestingly, deletion of proP does not disrupt the localization of ProQ with translating ribosomes, and deletion of proP in combination with the proU operon has no effect on ProQ localization. We also demonstrate that ProQ is necessary for robust biofilm formation, and this phenotype is independent of ProP. Binding studies were carried out using tryptophan fluorescence and in vitro transcribed proP mRNAs. proP is transcribed from two differentially regulated promoters, and ProQ interacts with proP mRNA transcribed from both promoters, as well as a control mRNA with similar affinities. In total, these data suggest that ProQ is positioned to function as a novel translational regulator, and its cellular role extends beyond its effects on proline uptake by ProP.     

Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings

I-TevI is a member of the GIY-YIG family of homing endonucleases. It is folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis andNMR spectroscopy to define the configuration of theN-terminal domain and its relationship to the flexible linker. The catalytic domain is an alpha/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structured domain corresponds precisely to the GIY-YIG module defined by sequence comparisons of 57 proteins including more than 30 newly reported members of the family. Although much of the unstructured linker is not essential for activity, residues 93-116 are required, raising the possibility that this region may adopt an alternate conformation upon DNA binding. Two invariant residues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, have properties of catalytic residues. Furthermore, the GIY-YIG sequence elements for which the module is named form part of a three-stranded antiparallel beta-sheet that is important for I-TevI structure and function.     

BBK32, a fibronectin binding MSCRAMM from Borrelia burgdorferi, contains a disordered region that undergoes a conformational change on ligand binding

BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBK32 is composed of two domains, an N-terminal segment lacking well defined secondary structure and a C-terminal segment composed largely of alpha-helices. Analysis of purified recombinant forms of the two domains by circular dichroism spectroscopy, gel permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal-extended, unstructured segment and a C-terminal globular domain in BBK32. Solid phase binding experiments suggest that the unstructured N-terminal domain binds fibronectin. Analysis of changes in circular dichroism spectra of the N-terminal segment of BBK32 upon binding of the N-terminal domain of fibronectin revealed an increase in beta-sheet content in the complex. Hence, BBK32, which belongs to a different family of proteins and shows no overall sequence similarity with the fibronectin binding MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) of Gram-positive bacteria, binds fibronectin by a mechanism that is reminiscent of the "tandem beta-zipper" previously demonstrated for the fibronectin binding of streptococcal adhesins.     

Impaired transport of intrinsically disordered proteins through the Sec61 and SecY translocon; implications for prion diseases

The prion protein (PrP) is composed of two major domains of similar size. The structured C-terminal domain contains three alpha-helical regions and a short two-stranded beta-sheet, while the N-terminal domain is intrinsically disordered. The analysis of PrP mutants with deletions in the C-terminal globular domain provided the first hint that intrinsically disordered domains are inefficiently transported into the endoplasmic reticulum through the Sec61 translocon. Interestingly, C-terminally truncated PrP mutants have been linked to inherited prion disease in humans and are characterized by inefficient ER import and the formation of neurotoxic PrP conformers. In a recent study we found that the Sec61 translocon in eukaryotic cells as well as the SecY translocon in bacteria is inherently deficient in translocating intrinsically disordered proteins. Moreover, our results suggest that translocon-associated components in eukaryotic cells enable the Sec61 complex to transport secretory proteins with extended unstructured domains such as PrP and shadoo.     

Folding and stability of the isolated Greek key domains of the long-lived human lens proteins gammaD-crystallin and gammaS-crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.     

Solution structure of the two-iron rubredoxin of Pseudomonas oleovorans determined by NMR spectroscopy and solution X-ray scattering and interactions with rubredoxin reductase

Here we provide insights into the molecular structure of the two-iron 19-kDa rubredoxin (AlkG) of Pseudomonas oleovorans using solution-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering studies. Sequence alignment and biochemical studies have suggested that AlkG comprises two rubredoxin folds connected by a linker region of approximately 70 amino acid residues. The C-terminal domain (C-Rb) of this unusual rubredoxin, together with approximately 35 amino acid residues of the predicted linker region, was expressed in Escherichia coli, purified in the one-iron form and the structure of the cadmium-substituted form determined at high-resolution by NMR spectroscopy. The structure shows that the C-Rb domain is similar in fold to the conventional one-iron rubredoxins from other organisms, whereas the linker region does not have any discernible structure. This tandem "flexible-folded" structure of the polypeptide chain derived for the C-Rb protein was confirmed using solution X-ray scattering methods. X-ray scattering studies of AlkG indicated that the 70-amino acid residue linker forms a structured, yet mobile, polypeptide segment connecting the globular N- and C-terminal domains. The X-ray scattering studies also showed that the N-terminal domain (N-Rb) has a molecular conformation similar to that of C-Rb. The restored molecular shape indicates that the folded N-Rb and C-Rb domains of AlkG are noticeably separated, suggesting some domain movement on complex formation with rubredoxin reductase to allow interdomain electron transfer between the metal centers in AlkG. This study demonstrates the advantage of combining X-ray scattering and NMR methods in structural studies of dynamic, multidomain proteins that are not suited to crystallographic analysis. The study forms a structural foundation for functional studies of the interaction and electron-transfer reactions of AlkG with rubredoxin reductase, also reported herein.     

Calorimetric dissection of thermal unfolding of OspA,a predominantly beta-sheet protein containing a single-layer beta-sheet

Outer surface protein A (OspA) from Borrelia burgdorferi is a predominantly beta-sheet protein comprised of beta-strands beta1-beta21 and a short C-terminal alpha-helix. It contains two globular domains (N and C-terminal domains) and a unique single-layer beta-sheet (central beta-sheet) that connects the two domains. OspA contains an unusually large number of charged amino acid residues. To understand the mechanism of stabilization of this unique beta-sheet protein, thorough thermodynamic investigations of OspA and its truncated mutant lacking a part of the C-terminal domain were conducted using calorimetry and circular dichroism. The stability of OspA was found to be sensitive to pH and salt concentration. The heat capacity curve clearly consisted of two components, and all the thermodynamic parameters were obtained for each step. The thermodynamic parameters associated with the two transitions are consistent with a previously proposed model, in which the first transition corresponds to the unfolding of the C-terminal domain and the last two beta-strands of the central beta-sheet, and the second transition corresponds to that of the N-terminal domain and the first beta-strand of the central beta-sheet in the second peak. The ratio of calorimetric and van't Hoff enthalpies indicates that the first peak includes another thermodynamic intermediate state. Large heat capacity changes were observed for both transitions, indicative of large changes in the exposure of hydrophobic surfaces associated with the transitions. This observation demonstrates that hydrophobic parts are buried efficiently in the native structure in spite of the low content of hydrophobic residues in OspA. By decomposing the enthalpy, entropy, and Gibbs free energy into contributions from different interactions, we found that the enthalpy changes for hydrogen bonding and polar interactions are exceptionally large, indicating that OspA maintains its stability by making full use of its unique beta-sheet and high content of polar residues. These thermodynamic analyses demonstrated that it is possible to maintain protein tertiary structure by making effective use of an unusual amino acid composition.     

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.     

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.     

The phycocyanin-associated rod linker proteins of the phycobilisome of Gloeobacter violaceus PCC 7421 contain unusually located rod-capping domains

Gloeobacter violaceus PCC 7421 is a unique cyanobacterium that has no thylakoids and whose genome has been sequenced [Y. Nakamura, T. Kaneko, S. Sato, M. Mimuro, H. Miyashita, T. Tsuchiya, S. Sasamoto, A. Watanabe, K. Kawashima, Y. Kishida, C. Kiyokawa, M. Kohara, M. Matsumoto, A. Matsuno, N. Nakazaki, S. Shimpo, C. Takeuchi, M. Yamada, S. Tabata, Complete Genome Structure of Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids. DNA Research 10 (2003) 137-145]. Phycobilisomes of G. violaceus were isolated and analyzed by SDS-PAGE followed by N-terminal sequencing. Three rod-linker subunits (CpeC, CpeD and CpeE) were identified as predicted from the genome sequence. The cpcC1 and cpcC2 genes at order locus named (OLN) glr0950 and gll 3219 encoding phycocyanin-associated linker proteins from G. violaceus are 56 and 55 amino acids longer at the N-terminus than the open reading frame proposed in the genome. The two amino acid extensions showed a 66% identity to one another. Also, the N-terminal extensions of these sequences were similar to domains in both the rod-capping-linker protein CpcD2 and to the C-terminus domain of the phycoerythrin-associated linker protein CpeC. These domains are not only unusual in their N-terminal location, but are unusual in that they are more closely related in sequence similarity to the C-terminus domain of the phycoerythrin-associated linker, CpeC of G. violaceus, than to the C-terminus domain of phycocyanin-associated linker CpcC in other cyanobacteria. These linker proteins with unique special domains are indicators of the unusual structure of the phycobilisomes of G. violaceus.