Biomedical subjects

Tiina A Salminen

Publications and source records attributed to Tiina A Salminen.

11 recordsLinked to original sources

PubMed2006

Functional classification of amino acid decarboxylases from the alanine racemase structural family by phylogenetic studies.

Arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) are involved in the biosynthesis of putrescine, which is the precursor of other polyamines in animals, plants, and bacteria. These pyridoxal-5'-phosphate-dependent decarboxylases belong to the alanine racemase (AR) structural family together with diaminopimelate decarboxylase (DapDC), which catalyzes the final step of lysine biosynthesis in bacteria. We have constructed a multiple-sequence alignment of decarboxylases in the AR structural family and, based on the alignment, inferred phylogenetic trees. The phylogenetic tree consists of 3 distinct clades formed by ADC, DapDC, and ODC that diverged from an ancestral decarboxylase. The ancestral decarboxylase probably was able to recognize several substrates, and in archaea and bacteria, ODC may have retained the ability to bind other amino acids. Previously, a paralogue of ODC has been proposed to account for ADC activity detected in mammalian cells. According to our results, this appears unlikely, emphasizing the need for more caution in functional assignment made using sequence data and illustrating the continuing value of phylogenetic analysis in clarifying relationships and putative functions.

Alanine Racemase↗

PubMed2006

Controlling quaternary structure assembly: subunit interface engineering and crystal structure of dual chain avidin.

Dual chain avidin (dcAvd) is an engineered avidin form, in which two circularly permuted chicken avidin monomers are fused into one polypeptide chain. DcAvd can theoretically form two different pseudotetrameric quaternary assemblies because of symmetry at the monomer-monomer interfaces. Here, our aim was to control the assembly of the quaternary structure of dcAvd. We introduced the mutation I117C into one of the circularly permuted domains of dcAvd and scanned residues along the 1-3 subunit interface of the other domain. Interestingly, V115H resulted in a single, disulfide locked quaternary assembly of dcAvd, whereas I117H could not guide the oligomerisation process even though it stabilised the protein. The modified dcAvd forms were found to retain their characteristic pseudotetrameric state both at high and low pH, and were shown to bind D-biotin at levels comparable to that of wild-type chicken avidin. The crystal structure of dcAvd-biotin complex at 1.95 Angstroms resolution demonstrates the formation of the functional dcAvd pseudotetramer at the atomic level and reveals the molecular basis for its special properties. Altogether, our data facilitate further engineering of the biotechnologically valuable dcAvd scaffold and gives insights into how to guide the quaternary structure assembly of oligomeric proteins.

Animals↗

PubMed2006

Binding properties of HABA-type azo derivatives to avidin and avidin-related protein 4.

The chicken genome encodes several biotin-binding proteins, including avidin and avidin-related protein 4 (AVR4). In addition to D-biotin, avidin binds an azo dye compound, 4-hydroxyazobenzene-2-carboxylic acid (HABA), but the HABA-binding properties of AVR4 are not yet known. Differential scanning calorimetry, UV/visible spectroscopy, and molecular modeling were used to analyze the binding of 15 azo molecules to avidin and AVR4. Significant differences are seen in azo compound preferences for the two proteins, emphasizing the importance of the loop between strands beta3 and beta4 for azo ligand recognition; information on these loops is provided by the high-resolution (1.5 A) X-ray structure for avidin reported here. These results may be valuable in designing improved tools for avidin-based life science and nanobiotechnology applications.

Animals↗

PubMed2006

Characterization of SCP-2 from Euphorbia lagascae reveals that a single Leu/Met exchange enhances sterol transfer activity.

Sterol carrier protein-2 (SCP-2) is a small intracellular basic protein domain implicated in peroxisomal beta-oxidation. We extend our knowledge of plant SCP-2 by characterizing SCP-2 from Euphorbia lagascae. This protein consists of 122 amino acids including a PTS1 peroxisomal targeting signal. It has a molecular mass of 13.6 kDa and a pI of 9.5. It shares 67% identity and 84% similarity with SCP-2 from Arabidopsis thaliana. Proteomic analysis revealed that E. lagascae SCP-2 accumulates in the endosperm during seed germination. It showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC). The transfer of BODIPY-PC was almost completely inhibited after addition of phosphatidylinositol, palmitic acid, stearoyl-CoA and vernolic acid, whereas sterols only had a very marginal inhibitory effect. We used protein modelling and site-directed mutagenesis to investigate why the BODIPY-PC transfer mediated by E. lagascae SCP-2 is not sensitive to sterols, whereas the transfer mediated by A. thaliana SCP-2 shows sterol sensitivity. Protein modelling suggested that the ligand-binding cavity of A. thaliana SCP-2 has four methionines (Met12, 14, 15 and 100), which are replaced by leucines (Leu11, 13, 14 and 99) in E. lagascae SCP-2. Changing Leu99 to Met99 was sufficient to convert E. lagascae SCP-2 into a sterol-sensitive BODIPY-PC-transfer protein, and correspondingly, changing Met100 to Leu100 abolished the sterol sensitivity of A. thaliana SCP-2.

Amino Acid Sequence↗

PubMed2005

Structural evidence for adaptive ligand binding of glycolipid transfer protein.

Glycolipids participate in many important cellular processes and they are bound and transferred with high specificity by glycolipid transfer protein (GLTP). We have solved three different X-ray structures of bovine GLTP at 1.4 angstroms, 1.6 angstroms and 1.8 angstroms resolution, all with a bound fatty acid or glycolipid. The 1.4 angstroms structure resembles the recently characterized apo-form of the human GLTP but the other two structures represent an intermediate conformation of the apo-GLTPs and the human lactosylceramide-bound GLTP structure. These novel structures give insight into the mechanism of lipid binding and how GLTP may conformationally adapt to different lipids. Furthermore, based on the structural comparison of the GLTP structures and the three-dimensional models of the related Podospora anserina HET-C2 and Arabidopsis thaliana accelerated cell death protein, ACD11, we give structural explanations for their specific lipid binding properties.

Amino Acid Sequence↗

PubMed2005

Avidin related protein 2 shows unique structural and functional features among the avidin protein family.

BACKGROUND: The chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin. RESULTS: In the present study, we have employed multiple biochemical methods to better understand the structure-function relationship of AVR proteins focusing on AVR2. Firstly, we have solved the high-resolution crystal structure of AVR2 in complex with a bound ligand, D-biotin. The AVR2 structure reveals an overall fold similar to the previously determined structures of avidin and AVR4. Major differences are seen, especially at the 1-3 subunit interface, which is stabilized mainly by polar interactions in the case of AVR2 but by hydrophobic interactions in the case of AVR4 and avidin, and in the vicinity of the biotin binding pocket. Secondly, mutagenesis, competitive dissociation analysis and differential scanning calorimetry were used to compare and study the biotin-binding properties as well as the thermal stability of AVRs and avidin. These analyses pinpointed the importance of residue 109 for biotin binding and stability of AVRs. The I109K mutation increased the biotin-binding affinity of AVR2, whereas the K109I mutation decreased the biotin-binding affinity of AVR4. Furthermore, the thermal stability of AVR2(I109K) increased in comparison to the wild-type protein and the K109I mutation led to a decrease in the thermal stability of AVR4. CONCLUSION: Altogether, this study broadens our understanding of the structural features determining the ligand-binding affinities and stability as well as the molecular evolution within the protein family. This novel information can be applied to further develop and improve the tools already widely used in avidin-biotin technology.

Amino Acid Sequence↗

PubMed2005

Crystal structure of the human vascular adhesion protein-1: unique structural features with functional implications.

The expression of human vascular adhesion protein-1 (hVAP-1) is induced at sites of inflammation where extravasation of lymphocytes from blood to the peripheral tissue occurs. We have solved the X-ray structure of hVAP-1, a human copper amine oxidase (CAO), which is distinguished from other CAOs in being membrane-bound. The dimer structure reveals some intriguing features that may have fundamental roles in the adhesive and enzymatic functions of hVAP-1, especially regarding the role of hVAP-1 in inflammation, lymphocyte attachment, and signaling. Firstly, Leu469 at the substrate channel may play a key role in controlling the substrate entry; depending on its conformation, it either blocks or gives access to the active site. Secondly, sugar units are clearly observed at two of the six predicted N-glycosylation sites. Moreover, mutagenesis analysis showed that all of the predicted sites were glycosylated in the protein used for crystallization. Thirdly, the existence of a solvent-exposed RGD motif at the entrance to each active site in hVAP-1 suggests that it may have a functional role.

Amine Oxidase (Copper-Containing)↗

PubMed2004

Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli.

Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.

Amino Acid Sequence↗

PubMed2004

Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2.

This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.

Agar↗

PubMed2003

Jararhagin-derived RKKH peptides induce structural changes in alpha1I domain of human integrin alpha1beta1.

Integrin alpha(1)beta(1) is one of four collagen-binding integrins in humans. Collagens bind to the alphaI domain and in the case of alpha(2)I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In alpha(2)I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligand-bound "open" conformation of alpha(2)I differs significantly from the "closed" conformation seen in the structure of apo-alpha(2)I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to alpha(1)I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of alpha(1)I with Mg(2+) and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-alpha(1)I ("closed" conformation) and a model structure of the alpha(1)I ("open" conformation) based on the closely related structure of alpha(2)I reveals that the binding site is partially blocked to ligands by Glu(255) and Tyr(285) in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of alpha(2)I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of alpha(1)I and shown experimentally for alpha(1)I using a fluorescent hydrophobic probe.

Amino Acid Sequence↗

PubMed2003

Crystallization and preliminary X-ray analysis of the human vascular adhesion protein-1.

Human vascular adhesion protein-1 (VAP-1) is a membrane-bound multifunctional glycoprotein with both adhesive and enzymatic properties. The protein belongs to the copper-containing amine oxidase (CAO) family, which use 2,4,5-trihydroxyphenylalanine quinone as a cofactor. Here, the crystallization and preliminary X-ray analysis of a mammalian CAO, human VAP-1, is reported. The protein was expressed in Chinese hamster ovary cells as a full-length form with an N-terminal transmembrane region and multiple glycosylation sites. Hexagonal crystals with unit-cell parameters a = b = 225.9, c = 218.7 A, alpha = beta = 90, gamma = 120 degrees were obtained using the vapour-diffusion method. Data from three different crystals were collected at 100 K using synchrotron radiation and were processed to 3.2 A resolution with 95.9% completeness and an R(merge) of 19.6%.

Amine Oxidase (Copper-Containing)↗