Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Structure-functional analyses of CRHSP-24 plasticity and dynamics in oxidative stress response

The cold shock domain (CSD) is an evolutionarily conserved nucleic acid binding domain that exhibits binding activity to RNA, ssDNA, and dsDNA. Mammalian CRHSP-24 contains CSD, but its structure-functional relationship has remained elusive. Here we report the crystal structure of human CRHSP-24 and characterization of the molecular trafficking of CRHSP-24 between stress granules and processing bodies in response to oxidative stress. The structure of CRHSP-24 determined by single-wavelength anomalous dispersion exhibits an α-helix and a compact β-barrel formed by five curved anti-parallel β strands. Ligand binding activity of the CSD is orchestrated by residues Ser(41) to Leu(43). Interestingly, a phosphomimetic S41D mutant abolishes the ssDNA binding in vitro and causes CRHSP-24 liberated from stress granules in vivo without apparent alternation of its localization to the processing bodies. This new class of phosphorylation-regulated interaction between the CSD and nucleic acids is unique in stress granule plasticity. Importantly, the association of CRHSP-24 with stress granules is blocked by PP4/PP2A inhibitor calyculin A as PP2A catalyzes the dephosphorylation of Ser(41) of CRHSP-24. Therefore, we speculate that CRHSP-24 participates in oxidative stress response via a dynamic and temporal association between stress granules and processing bodies.     

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.     

Chloroplast β-Barrel Proteins Are Assembled into the Mitochondrial Outer Membrane in a Process That Depends on the TOM and TOB Complexes

Membrane-embedded β-barrel proteins are found in the outer membranes (OM) of Gram-negative bacteria, mitochondria and chloroplasts. In eukaryotic cells, precursors of these proteins are synthesized in the cytosol and have to be sorted to their corresponding organelle. Currently, the signal that ensures their specific targeting to either mitochondria or chloroplasts is ill-defined. To address this issue, we studied targeting of the chloroplast β-barrel proteins Oep37 and Oep24. We found that both proteins can be integrated in vitro into isolated plant mitochondria. Furthermore, upon their expression in yeast cells Oep37 and Oep24 were exclusively located in the mitochondrial OM. Oep37 partially complemented the growth phenotype of yeast cells lacking Porin, the general metabolite transporter of this membrane. Similarly to mitochondrial β-barrel proteins, Oep37 and Oep24 expressed in yeast cells were assembled into the mitochondrial OM in a pathway dependent on the TOM and TOB complexes. Taken together, this study demonstrates that the central mitochondrial components that mediate the import of yeast β-barrel proteins can deal with precursors of chloroplast β-barrel proteins. This implies that the mitochondrial import machinery does not recognize signals that are unique to mitochondrial β-barrel proteins. Our results further suggest that dedicated targeting factors had to evolve in plant cells to prevent mis-sorting of chloroplast β-barrel proteins to mitochondria.     

The three domains of the mitochondrial outer membrane protein Mim1 have discrete functions in assembly of the TOM complex

The assembly of mitochondrial outer membrane proteins is an essential process, mediated by the SAM complex and a set of additional protein modules. We show that one of these, Mim1, is anchored in the outer membrane with its N-terminus exposed to the cytosol and its C-terminus in the mitochondrial intermembrane space. Using an in vitro assay to measure the multi-step pathway for assembly of Tom40 into the TOM complex, we find that an “early reaction” mediated by the SAM complex is regulated by the N-terminal domain of Mim1. In addition, a “late reaction” catalysed by the Sam37 subunit of the SAM complex is also influenced by Mim1. Thus, Mim1 participates at multiple stages in the assembly of the TOM complex.     

Ion channel formation by N-terminal domain: a common feature of OprFs of Pseudomonas and OmpA of Escherichia coli

The proteolytic fragments of OprFs of Pseudomonas aeruginosa and Pseudomonas fluorescens were identified, respectively, as the first 175 and 177 amino acids from the N-terminal domain. They induced ion channels after reincorporation into planar lipid bilayers (85 and 75 pS, respectively, in 1 M NaCl). A similar conductance value (72 pS) was found for the eight beta-strand OmpA N-terminal domain (OmpA171) of Escherichia coli. We conclude that the N-terminal domain of OprFs is sufficient to induce ion channels and the comparison with OmpA171, provides strong evidence of the existence of an eight-stranded beta-barrel in the N-terminal domain of OprFs.     

Novel fold and carbohydrate specificity of the potent anti-HIV cyanobacterial lectin from Oscillatoria agardhii

Oscillatoria agardhii agglutinin (OAA) is a recently discovered cyanobacterial lectin that exhibits potent anti-HIV activity. Up to now, only its primary structure and carbohydrate binding data have been available. To elucidate the structural basis for the antiviral mechanism of OAA, we determined the structure of this lectin by x-ray crystallography at 1.2 Å resolution and mapped the specific carbohydrate recognition sites of OAA by NMR spectroscopy. The overall architecture of OAA comprises 10 β-strands that fold into a single, compact, β-barrel-like domain, creating a unique topology compared with all known protein structures in the Protein Data Bank. OAA sugar binding was tested against Man-9 and various disaccharide components of Man-9. Two symmetric carbohydrate-binding sites were located on the protein, and a preference for Manα(1–6)Man-linked sugars was found. Altogether, our structural results explain the antiviral activity OAA and add to the growing body of knowledge about antiviral lectins.     

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a β-barrel containing 19 membrane spanning β-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential β-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 β-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.     

Acidification Asymmetrically Affects Voltage-dependent Anion Channel Implicating the Involvement of Salt Bridges

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to “gate” between an open and several “closed” states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pK_a values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.