Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130 -132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme.Protein-tyrosine phosphatases (PTPs) 1 are key regulators of signal transduction processes (1, 2). The family of classical PTPs can be divided into two broad categories as intracellular and receptor-like PTPs covering a total of 17 subtypes (3). Receptor-like PTPs contain an extracellular domain, a single transmembrane domain, and one or two cytoplasmic PTP domains. Intracellular PTPs generally contain one PTP domain and an N-or C-terminal domain that targets the enzymes to specific subcellular localizations, as exemplified by the targeting of PTP1B to the endoplasmic reticulum (4).PTP1B and TC-PTP are two closely related intracellular enzymes. PTP1B was the first protein-tyrosine phosphatase to be identified and characterized (5, 6). Shortly after this landmark event, PTP1B was cloned from a placenta cDNA library (7), and TC-PTP was cloned from a peripheral human T cell cDNA library (8). Despite its name, TC-PTP is ubiquitously expressed (9). Alternative splicing gives rise to two forms of TC-PTP that differ in the C termini, a 45-kDa form that is targeted to the nucleus and a 48-kDa form that localizes to the endoplasmic reticulum via a hydrophobic C-terminal region (10). TC-PTP is tightly regulated during the cell cycle and seems to play an important role in mitogenesis (9). In a recent study, it was shown that cellular stress causes reversible cytoplasmic accumulation of the 45-kDa form of TC-PTP (i.e. the nuclear form) (11).Although they have a sequence identity of about 74% in the catalytic domains (see Fig. 1), TC-PTP and PTP1B clearly fulfill different biological functions, as has been demonstrated in knock-out mice. Thus, although PTP1B knock-out mice show increased insulin sensitivity and resistance to diet-induced obesity and are viable with a normal life span (12, 13), TC-PTP knock-out mice die at 3-5 weeks of age (14).In accordance with these in vivo observations, substrate trapping experiment...

The serotonin transporter (SERT) is one of the neurotransmitter transporters that plays a critical role in the regulation of endogenous amine concentrations and therefore is an important target for therapeutic agents affecting the central nervous system. The recently published, high resolution X-ray structure of the closely related amino acid transporter, Aquifex aeolicus leucine transporter (LeuT), provides an opportunity to develop a three-dimensional model of the structure of SERT. We present herein a homology model of SERT using LeuT as the template and containing escitalopram as a bound ligand. Our model explains selectivities known from mutational studies and varying ligand data, which are discussed and illustrated in the paper.

IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.

We have performed molecular dynamics simulations of a homology model of the human serotonin transporter (hSERT) in a membrane environment and in complex with either the natural substrate 5-HT or the selective serotonin reuptake inhibitor escitalopram. We have also included a transporter homologue, the Aquifex aeolicus leucine transporter (LeuT), in our study to evaluate the applicability of a simple and computationally attractive membrane system. Fluctuations in LeuT extracted from simulations are in good agreement with crystallographic B factors. Furthermore, key interactions identified in the X-ray structure of LeuT are maintained throughout the simulations indicating that our simple membrane system is suitable for studying the transmembrane protein hSERT in complex with 5-HT or escitalopram. For these transporter complexes, only relatively small fluctuations are observed in the ligand-binding cleft. Specific interactions responsible for ligand recognition, are identified in the hSERT-5HT and hSERT-escitalopram complexes. Our findings are in good agreement with predictions from mutagenesis studies.

Liposomale Wirkstofftransportsysteme, in denen Vorstufen durch krankheitsspezifische Enzyme aktiviert werden, haben ein großes Potenzial für die Therapie von Erkrankungen wie Krebs. Eine neuartige Wirkstoffvorstufe auf Phospholipidbasis kann stabile kleine unilamellare Vesikel aufbauen (siehe Bild). Die Aktivierung dieser Vesikel durch das Enzym sPLA2 löst eine Cyclisierungsreaktion aus, die zur Freisetzung des Wirkstoffs führt.

Molecular structures of exopolysaccharides are required to understand their functions and the relationships between the structure and physical and rheological properties. Small-angle X-ray scattering and dynamic light scattering were used in conjunction with molecular modeling to characterize solution structures of three lactic acid bacterial heteroexopolysaccharides (HePS-1, HePS-2, and HePS-3). Values of radius of gyration R, cross-sectional radius of gyration R, approximate length L, and hydrodynamic diameter were not directly proportional to the molar mass and indicated the HePSs adopted a compact coil-like rather than an extended conformation. Constrained molecular modeling of 15000 randomized HePS-1 conformers resulted in five best-fit structures with R factor of 3.9-4.6% revealing random coil-like structure. Φ and Ψ angle analysis of glycosidic linkages in HePS-1 structures suggests Galf residues significantly influence the conformation. Ab initio scattering modeling of HePS-2 and HePS-3 gave excellent curve fittings with χ of 0.43 and 0.34 for best-fit models, respectively, compatible with coil-like conformation. The findings disclose solution behavior of HePS relevant for their interactions with biomacromolecules, for example, milk proteins.

Constant-pressure molecular-dynamics simulations of phospholipid membranes in the fluid Lα phase reveal strong correlations between equilibrium fluctuations of volume and energy on the nanosecond time-scale. The existence of strong volume-energy correlations was previously deduced indirectly by Heimburg from experiments focusing on the phase transition between the Lα and the L β phases. The correlations, which are reported here for three different membranes (DMPC, DMPS-Na, and DMPSH), have volume-energy correlation coefficients ranging from 0.81 to 0.89. The DMPC membrane was studied at two temperatures showing that the correlation coefficient increases as the phase transition is approached.Biological membranes are essential parts of living cells. They not only act as passive barriers between outside and inside, but also play an active role in various biological mechanisms. The major constituent of biological membranes are phospholipids. Pure phospholipid membranes often serve as a models for the more complex biological membranes. Close to physiological temperatures membranes undergo a transition from the high-temperature fluid L α phase (often referred to as the "biologically relevant phase") to a low-temperature ordered gel phase L β . In the melting regime response functions such as heat capacity, volume-expansion coefficient, and area-expansion coefficient increase dramatically. Also, the characteristic time for the collective degrees of freedom increases and becomes longer than milliseconds. Some time ago Heimburg found that the slow, dominating parts of heat capacity and volume-expansion coefficient of DMPC as a function of temperature can be superimposed close to the melting temperature T m [1] (see also Refs. 2 and 3). Thus the response functions are related in such a way that a single function describes the temperature dependence of both.The fluctuation-dissipation (FD) theorem connects (linear) response functions to equilibrium fluctuations. The isobaric heat capacity c p can be calculated from enthalpy fluctuations as follows:2 ), where . . . is an average in the constant temperature and pressure ensemble and ∆ is deviation from the average value. Similarly, volume fluctuations are connected to the isothermal volume compressibility by the expression κ T = (∆V ) 2 /(V k B T ). If the response functions were described by a single parameter, fluctuations are also described by a single parameter [1,4] and the microstates were connected via the relation ∆H i = γ vol ∆V i . At constant pressure this relation applies if and only if ∆E i = γ vol ∆V i (where E is energy), which is the relation investigated below. This situation is referred to as a that of a single-parameter description [4]. A single-parameter description applies to a good approximation for several models of van der Waals bonded liquids as well as for experimental super-critical argon [5].Unfortunately, molecular-dynamics simulations are not possible for investigating "single parameter"-ness of membranes close to T m , because the relaxation ti...

To gain insight into the mobility of the occupied ligand-binding pocket of the Rhizomucor miehei lipase we have conducted a rigorous molecular dynamics analysis. The covalently attached inhibitor, ethylhexylphosphonate, was employed as a mimic of the putative tetrahedral intermediate in the esterolytic reaction. Our results show that in this lipase, ligand recognition is influenced by the flexibility of the binding pocket, a feature that is common to many other enzymes. Several regions around the active site were found to move significantly to adapt to the inhibitor. These motions are correlated to the flexibility of the inhibitor. In particular, the hexyl chain of the inhibitor shows considerable mobility, and adjacent residues in the binding cleft accommodate to this flexibility. Pronounced fluctuations in the binding pocket induced by the flexibility of the inhibitor are observed in the hinge region F79-S82, the active site loop region W88-V95 and the protein regions P209-F215/H257-Y260. The flexibility in the regions F79-S82 and H257-Y260, where the shorter ethyl chain is located, indicates that additional space in this binding cleft region is available for accommodating a larger moiety. Fluctuations in the region W88-V95 and P209-F215 are due to the relatively short flexible hexyl carbon chain. This part of the binding pocket could be stiffened by the presence of a longer carbon chain. Though the inhibitor is covalently attached through the phosphonate moiety, interaction of the remainder of the molecule and the enzyme are determined by hydrophobic interactions, where the Van der Waals energies are approximately 25% lower than the electrostatic contributions.