Tiina Salminen

BODIL: a molecular modeling environment for structure-function analysis and drug design.

BODIL is a molecular modeling environment geared to help the user to quickly identify key features of proteins critical to molecular recognition, especially (1) in drug discovery applications, and (2) to understand the structural basis for function. The program incorporates state-of-the-art graphics, sequence and structural alignment methods, among other capabilities needed in modern structure–function–drug target research. BODIL has a flexible design that allows on-the-fly incorporation of new modules, has intelligent memory management, and fast multi-view graphics. A beta version of BODIL and an accompanying tutorial are available at http://www.abo.fi/fak/mnf/bkf/research/johnson/bodil.html

A Cell Surface Amine Oxidase Directly Controls Lymphocyte Migration

Lymphocytes leave the blood using a sequential adhesion cascade. Vascular adhesion molecule-1 (VAP-1) is a surface-expressed endothelial glycoprotein, which belongs to a distinct subgroup of monoamine oxidases. We show here that catalytic activity of VAP-1 on primary endothelial cells directly regulates lymphocyte rolling under defined laminar shear. VAP-1 seems to bind to a primary amino group presented on the lymphocyte surface and oxidatively deaminate it in a reaction, which results in the formation of a transient covalent bond between the two cell types. Instead, soluble reaction products (aldehydes and hydrogen peroxide) are not needed for the VAP-1-dependent rolling. Enzymatic regulation of lymphocyte adhesion to endothelium provides a previously unrecognized rapid way of controlling the extravasation process.

Three-dimensional Models of α2A-Adrenergic Receptor Complexes Provide a Structural Explanation for Ligand Binding

We have compared bacteriorhodopsin-based (α2A-ARBR) and rhodopsin-based (α2A-ARR) models of the human α2A-adrenengic receptor (α2A-AR) using both docking simulations and experimental receptor alkylation studies with chloroethylclonidine and 2-aminoethyl methanethiosulfonate hydrobromide. The results indicate that the α2A-ARR model provides a better explanation for ligand binding than does our α2A-ARBRmodel. Thus, we have made an extensive analysis of ligand binding to α2A-ARR and engineered mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The representative docked ligand conformation was chosen using extensive docking simulations coupled with the identification of favorable interaction sites for chemical groups in the receptor. These ligand-protein complex studies provide a rational explanation at the atomic level for the experimentally observed binding affinities of each of these ligands to the α2A-adrenergic receptor.

Carbohydrates located on the top of the cap contribute to the adhesive and enzymatic functions of vascular adhesion protein-1

Vascular adhesion protein 1 (VAP‐1) is an endothelial adhesion molecule with an enzymatic activity. It deaminates biogenic amines, resulting in the formation of aldehydes and hydrogen peroxide. During the enzymatic reaction a transient Schiff base is formed between endothelial VAP‐1 and its leukocytic ligand, and this interaction is important for lymphocyte adhesion. VAP‐1 monomer has six potential N‐linked, and three putative O‐linked glycosylation sites and an SSSS sequence potentially forming an attachment site for an adjacent O‐linked site. In this work we modeled the carbohydrate decorations on a structural model of VAP‐1, and studied which of those potential glycosylation sites are utilized, and whether those decorations accessible to a lymphocyte ligand are important in lymphocyte adhesion and enzymatic activity of VAP‐1. We show that, unlike the O‐linked attachment sites, all six N‐linked glycosylation sites are in use. Furthermore, mutation of the N‐linked attachment sites strategically located on the top of the molecule reduces lymphocyte adhesion in non‐static conditions, and enhances the catalytic activity of membrane‐bound human VAP‐1 in static conditions, suggesting that glycosylation regulates the functional properties of VAP‐1.

The unique substrate specificity of human AOC2, a semicarbazide-sensitive amine oxidase

Semicarbazide-sensitive amine oxidases (SSAOs) catalyze oxidative deamination of primary amines, but the true physiological function of these enzymes is still poorly understood. Here, we have studied the functional and structural characteristics of a human cell-surface SSAO, AOC2, which is homologous to the better characterized family member, AOC3. The preferred in vitro substrates of AOC2 were found to be 2-phenylethylamine, tryptamine and p-tyramine instead of methylamine and benzylamine, the favored substrates of AOC3. Molecular modeling suggested structural differences between AOC2 and AOC3, which provide AOC2 with the capability to use the larger monoamines as substrates. Even though AOC2 mRNA was expressed in many tissues, the only tissues with detectable AOC2-like enzyme activity were found in the eye. Characterization of AOC2 will help in evaluating the contribution of this enzyme to the pathological processes attributed to the SSAO activity and in designing specific inhibitors for the individual members of the SSAO family.

Crystallization and X-ray analysis of bovine glycolipid transfer protein.

Glycolipid-transfer protein (GLTP) is a 24 kDa basic cytosolic protein that facilitates the transfer of glycolipids between bilayer membranes in vitro, but its in vivo function is unknown. Human, bovine, porcine and murine GLTPs have recently been cloned and share high sequence identity to each other. The three-dimensional structure of GLTP has not yet been solved and no structures of any proteins related to GLTP are known. Therefore, the structure of GLTP might reveal a currently unknown fold. Here, the crystallization and preliminary X-ray analysis of bovine GLTP are reported for the first time. Protein prepared by recombinant techniques using an Escherichia coli expression system has been crystallized using the vapour-diffusion method. The crystals belong to space group P2(1), with unit-cell parameters a = 55.4, b = 34.9, c = 58.5 A, alpha = gamma = 90, beta = 116 degrees. The crystals diffract to 1.6 A resolution and a 97.1% complete data set with an R(merge) of 6.7% has been collected from a single crystal at 100 K using synchrotron radiation.

Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases

The Synechocystis sp. PCC 6803 genome harbours a deg gene family consisting of three members, degP (htrA, slr1204), degQ (hhoA, sll1679) and degS (hhoB, sll1427). We studied the environmental regulation of the Synechocystis sp. PCC 6803 deg genes at the level of transcription and protein structures of the gene products to evaluate their hypothetical role in D1 protein turnover. Northern blotting showed that transcription of the deg genes is differentially regulated, supporting a view of distinct roles of Degs in cellular processes. The oligomerization state as well as the three dimensional structures of the Synechocystis sp. PCC 6803 Deg proteases were predicted based on an amino acid sequence alignment and comparison of the Deg crystal structures from human, Escherichia coli and Thermotoga maritima. The structures of the Synechocystis sp. PCC 6803 Degs resemble more the Thermotoga maritima Deg enzyme structure than the Escherichia coli one. Moreover, the structures of the LA-loops hint towards a homotrimeric form of the Synechocystis sp. PCC 6803 Deg proteases.

Structural modeling and environmental regulation of arginine decarboxylase in Synechocystis sp. PCC 6803

Arginine decarboxylase (ADC) is the first enzyme in the alternative route to putrescine in the polyamine biosynthesis pathway in bacteria and plants. In this study, we have focused on the effects of various types of short-term stresses on the transcript amount and specific activity of Synechocystis sp. PCC 6803 ADC. Our results reveal that the steady-state transcript accumulation and enzyme activity are not connected in a simple manner, since only photoheterotrophy and synergistic salt and high-light stress affected both parameters similarly. Changes in the steady-state ADC mRNA accumulation under the other short-term stress conditions studied had only a small impact on enzyme activity, suggesting post-translational regulation. Based on structural modeling, Synechocystis ADCs have a putative extra domain, which might be involved in the post-translational regulation of ADC activity in Synechocystis. In addition, two symmetric inter-subunit disulfide bonds seem to stabilize the dimeric structure of ADCs. There are two genes coding for ADC and agmatinase, another polyamine pathway enzyme, in Synechocystis genome, while the genes coding for ornithine decarboxylase and for some other enzymes in the polyamine pathway were not identified with homology searches.

Transcriptional regulation and structural modelling of the Synechocystis sp. PCC 6803 carboxyl-terminal endoprotease family.

The Synechocystis sp. PCC 6803 ctp gene family members ctpA (slr0008), ctpB (slr0257) and ctpC (slr1751), encoding carboxyl-terminal endoproteases (Ctps), were studied at levels of gene transcription and protein structure. Northern blot analysis revealed differential activation and accumulation of the ctp transcripts upon induction of various environmental conditions, including light, temperature, salinity and growth mode, supporting the view of distinct roles of Ctps in Synechocystis sp. PCC 6803 cellular processes. Amino acid sequence comparison of 16 ctp gene products showed that they fall into three distinct groups: the eukaryotic CtpA-like proteins, the prokaryotic CtpA-like proteins and the prokaryotic CtpB/C-like proteins. Structural models of the Synechocystis sp. PCC 6803 Ctps, constructed based on the amino acid sequence alignment and the crystal structure of the Scenedesmus obliquus D1 processing protease, revealed that although the overall structure of the Synechocystis sp. PCC 6803 Ctps is very similar, differences exist in the putative membrane contact regions and in the active site environment.

Antiferritin VL homodimer binds human spleen ferritin with high specificity

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.