Home About us Contact
|
Home About us Contact | ||
|
|
||
Major Conformational Changes (major + conformational_change)
Selected AbstractsTransmembrane signal transduction of the ,IIb,3 integrinPROTEIN SCIENCE, Issue 7 2002Kay E. Gottschalk Abstract Integrins are composed of noncovalently bound dimers of an ,- and a ,-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix,helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the ,IIb,3 integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of ,IIb,3 is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism. [source] The structure of dihydrodipicolinate reductase (DapB) from Mycobacterium tuberculosis in three crystal formsACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2010Robert Janowski Dihydrodipicolinate reductase (DHDPR, DapB) is an enzyme that belongs to the l -lysine biosynthetic pathway. DHDPR reduces the ,,,-unsaturated cyclic imine 2,3-dihydrodipicolinic acid to yield the compound 2,3,4,5-tetrahydrodipicolinic acid in a pyridine nucleotide-dependent reaction. The substrate of this reaction is the unstable product of the preceding enzyme dihydrodipicolinate synthase (DHDPS, DapA). Here, the structure of apo-DHDPR from Mycobacterium tuberculosis is reported in two orthorhombic crystal forms, as well as the structure of DHDPR from M. tuberculosis in complex with NADH in a monoclinic crystal form. A comparison of the results with previously solved structures of this enzyme shows that DHDPR undergoes a major conformational change upon binding of its cofactor. This conformational change can be interpreted as one of the low-frequency normal modes of the structure. [source] A new crystal form of human tear lipocalin reveals high flexibility in the loop region and induced fit in the ligand cavityACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2009Daniel A. Breustedt Tear lipocalin (TLC) with the bound artificial ligand 1,4-butanediol has been crystallized in space group P21 with four protein molecules in the asymmetric unit and its X-ray structure has been solved at 2.6,Å resolution. TLC is a member of the lipocalin family that binds ligands with diverse chemical structures, such as fatty acids, phospholipids and cholesterol as well as microbial siderophores and the antibiotic rifampin. Previous X-ray structural analysis of apo TLC crystallized in space group C2 revealed a rather large bifurcated ligand pocket and a partially disordered loop region at the entrace to the cavity. Analysis of the P21 crystal form uncovered major conformational changes (i) in ,-strands B, C and D, (ii) in loops 1, 2 and 4 at the open end of the ,-barrel and (iii) in the extended C-terminal segment, which is attached to the ,-barrel via a disulfide bridge. The structural comparison indicates high conformational plasticity of the loop region as well as of deeper parts of the ligand pocket, thus allowing adaptation to ligands that differ vastly in size and shape. This illustrates a mechanism for promiscuity in ligand recognition which may also be relevant for some other physiologically important members of the lipocalin protein family. [source] Native and transformed ,2 -macroglobulin in plasma from patients with multiple sclerosisACTA NEUROLOGICA SCANDINAVICA, Issue 1 2003M. Gunnarsson Multiple sclerosis (MS) is an inflammatory demyelinating disease with unknown etiology. Various proteinases have been observed in increased levels in the central nervous system of patients with MS, which may contribute to the release of immunogenic myelin components. ,2 -Macroglobulin (,2M) inhibits a broad spectrum of proteinases sterically, undergoing major conformational changes induced by the proteinases themselves. Moreover, ,2M acts as a carrier of several cytokines in the systemic circulation. By use of radial immunodiffusion, we determined the total ,2M levels in plasma from 28 MS patients and 15 control subjects [14 patients with other neurologic diseases (OND) and one healthy individual]. No significant differences in total ,2M concentration were observed between the MS patients and the control subjects. A comparison of the degree of ,2M transformation in MS patients with different disease courses and controls was performed, using monoclonal antibodies (mAbs) specific for binding to native and transformed ,2M, respectively. The fractions of transformed ,2M were significantly increased in patients with secondary or primary progressive disease course compared with the controls. No significant differences were obtained using a native-specific mAb. At least a major proportion of ,2M from the MS patients was able to change conformation from its native to its transformed state, as demonstrated by a shift in mAb reactivity, following methylamine treatment of the plasma samples. In conclusion, the results indicate that plasma ,2M may be inactivated at a higher degree in patients with chronic progressive MS compared with patients with OND. This may influence the levels of proteinases and cytokines in the systemic circulation and may furthermore have diagnostic implications. [source] Alteration of the Diastereoselectivity of 3-Methylaspartate Ammonia Lyase by Using Structure-Based MutagenesisCHEMBIOCHEM, Issue 13 2009Hans Raj Abstract 3-Methylaspartate ammonia-lyase (MAL) catalyzes the reversible amination of mesaconate to give both (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products. The deamination mechanism of MAL is likely to involve general base catalysis, in which a catalytic base abstracts the C3 proton of the respective stereoisomer to generate an enolate anion intermediate that is stabilized by coordination to the essential active-site MgII ion. The crystal structure of MAL in complex with (2S,3S)-3-methylaspartic acid suggests that Lys331 is the only candidate in the vicinity that can function as a general base catalyst. The structure of the complex further suggests that two other residues, His194 and Gln329, are responsible for binding the C4 carboxylate group of (2S,3S)-3-methylaspartic acid, and hence are likely candidates to assist the MgII ion in stabilizing the enolate anion intermediate. In this study, the importance of Lys331, His194, and Gln329 for the activity and stereoselectivity of MAL was investigated by site-directed mutagenesis. His194 and Gln329 were replaced with either an alanine or arginine, whereas Lys331 was mutated to a glycine, alanine, glutamine, arginine, or histidine. The properties of the mutant proteins were investigated by circular dichroism (CD) spectroscopy, kinetic analysis, and 1H NMR spectroscopy. The CD spectra of all mutants were comparable to that of wild-type MAL, and this indicates that these mutations did not result in any major conformational changes. Kinetic studies demonstrated that the mutations have a profound effect on the values of kcat and kcat/KM; this implicates Lys331, His194 and Gln329 as mechanistically important. The 1H NMR spectra of the amination and deamination reactions catalyzed by the mutant enzymes K331A, H194A, and Q329A showed that these mutants have strongly enhanced diastereoselectivities. In the amination direction, they catalyze the conversion of mesaconate to yield only (2S,3S)-3-methylaspartic acid, with no detectable formation of (2S,3R)-3-methylaspartic acid. The results are discussed in terms of a mechanism in which Lys331, His194, and Gln329 are involved in positioning the substrate and in formation and stabilization of the enolate anion intermediate. [source] | ||||||||||