Gábor Tusnády

PDB_TM: selection and membrane localization of transmembrane proteins in the protein data bank

PDB_TM is a database for transmembrane proteins with known structures. It aims to collect all transmembrane proteins that are deposited in the protein structure database (PDB) and to determine their membrane-spanning regions. These assignments are based on the TMDET algorithm, which uses only structural information to locate the most likely position of the lipid bilayer and to distinguish between transmembrane and globular proteins. This algorithm was applied to all PDB entries and the results were collected in the PDB_TM database. By using TMDET algorithm, the PDB_TM database can be automatically updated every week, keeping it synchronized with the latest PDB updates. The PDB_TM database is available at http://www.enzim.hu/PDB_TM.

Functional Multidrug Resistance Protein (MRP1) Lacking the N-terminal Transmembrane Domain

The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.

Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters

A group of ATP‐binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance‐associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP‐related proteins are also characterized by a special, common membrane topology pattern. The most studied ABC transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance (MDR) proteins, were shown to contain a tandem repeat of six transmembrane helices, each set followed by an ATP‐binding domain. According to the present study, in contrast to various membrane topology predictions proposed for the different MRP‐related proteins, they all seem to have a CFTR/MDR‐like core structure, and an additional, large, N‐terminal hydrophobic region. This latter domain is predicted to contain 4–6 (most probably 5) transmembrane helices, and is occasionally glycosylated on the cell surface. Since all the MRP‐related transporters were shown to interact with anionic compounds, the N‐terminal membrane‐bound domain may have a key role in these interactions.

Membrane Topology and Glycosylation of the Human Multidrug Resistance-associated Protein

The membrane topology of the human multidrug resistance-associated protein (MRP) was examined by flow cytometry phenotyping, immunoblotting, and limited proteolysis in drug-resistant human and baculovirus-infected insect cells, expressing either the glycosylated or the underglycosylated forms of this protein. Inhibition of N-linked glycosylation in human cells by tunicamycin did not inhibit the transport function or the antibody recognition of MRP, although its apparent molecular mass was reduced from 180 kDa to 150 kDa. Extracellular addition of trypsin or chymotrypsin had no effect either on the function or on the molecular mass of MRP, while in isolated membranes limited proteolysis produced three large membrane-bound fragments. These experiments and the alignment of the MRP sequence with the human cystic fibrosis transmembrane conductance regulator (CFTR) suggest that human MRP, similarly to CFTR, contains a tandem repeat of six transmembrane helices, each followed by a nucleotide binding domain, and that the C-terminal membrane-bound region is glycosylated. However, the N-terminal region of MRP contains an additional membrane-bound, glycosylated area with four or five transmembrane helices, which seems to be a characteristic feature of MRP-like ATP-binding cassette transporters.

On optimal matchings

Givenn random red points on the unit square, the transportation cost between them is tipically √n logn.

PDBTM: Protein Data Bank of transmembrane proteins after 8 years

The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the last 8 years not only the size of the PDBTM database has been steadily growing from ∼400 to 1700 entries but also new structural elements have been identified, in addition to the well-known α-helical bundle and β-barrel structures. Numerous ‘exotic’ transmembrane protein structures have been solved since the first release, which has made it necessary to define these new structural elements, such as membrane loops or interfacial helices in the database. This article reports the new features of the PDBTM database that have been added since its first release, and our current efforts to keep the database up-to-date and easy to use so that it may continue to serve as a fundamental resource for the scientific community.

BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced. During sequencing, the initially unmethylated cytosines are detected as thymines. The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web. It has the unique property of analyzing the primer pairs for mispriming sites on the bisulfite-treated genome and determines potential non-specific amplification products with a new search algorithm. The options of primer-design and analysis for mispriming sites can be used sequentially or separately, both on bisulfite-treated and untreated sequences. In silico and in vitro tests of the software suggest that new PCR strategies may increase the efficiency of the amplification.

SRide: a server for identifying stabilizing residues in proteins

Residues expected to play key roles in the stabilization of proteins [stabilizing residues (SRs)] are selected by combining several methods based mainly on the interactions of a given residue with its spatial, rather than its sequential neighborhood and by considering the evolutionary conservation of the residues. A residue is selected as a stabilizing residue if it has high surrounding hydrophobicity, high long-range order, high conservation score and if it belongs to a stabilization center. The definition of all these parameters and the thresholds used to identify the SRs are discussed in detail. The algorithm for identifying SRs was originally developed for TIM-barrel proteins [M. M. Gromiha, G. Pujadas, C. Magyar, S. Selvaraj, and I. Simon (2004), Proteins, 55, 316–329] and is now generalized for all proteins of known 3D structure. SRs could be applied in protein engineering and homology modeling and could also help to explain certain folds with significant stability. The SRide server is located at .

CCTOP: a Consensus Constrained TOPology prediction web server

The Consensus Constrained TOPology prediction (CCTOP; http://cctop.enzim.ttk.mta.hu) server is a web-based application providing transmembrane topology prediction. In addition to utilizing 10 different state-of-the-art topology prediction methods, the CCTOP server incorporates topology information from existing experimental and computational sources available in the PDBTM, TOPDB and TOPDOM databases using the probabilistic framework of hidden Markov model. The server provides the option to precede the topology prediction with signal peptide prediction and transmembrane-globular protein discrimination. The initial result can be recalculated by (de)selecting any of the prediction methods or mapped experiments or by adding user specified constraints. CCTOP showed superior performance to existing approaches. The reliability of each prediction is also calculated, which correlates with the accuracy of the per protein topology prediction. The prediction results and the collected experimental information are visualized on the CCTOP home page and can be downloaded in XML format. Programmable access of the CCTOP server is also available, and an example of client-side script is provided.

Membrane topology of human ABC proteins

In this review, we summarize the currently available information on the membrane topology of some key members of the human ABC protein subfamilies, and present the predicted domain arrangements. In the lack of high‐resolution structures for eukaryotic ABC transporters this topology is based only on prediction algorithms and biochemical data for the location of various segments of the polypeptide chain, relative to the membrane. We suggest that topology models generated by the available prediction methods should only be used as guidelines to provide a basis of experimental strategies for the elucidation of the membrane topology.