ABSTRACT
BACKGROUND: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds divalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'active' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflected the stabilization of an 'inactive' I-domain conformation. We set out to independently determine the structure of the CD11 b I domain and to evaluate the structural effects of divalent ion binding to this protein. RESULTS: We have determined the X-ray structure of a new crystal form of the CD11 b I domain in the absence of added metal ions by multiple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straight-forward assessment of the structural effects of divalent cation binding at the metal ion dependent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein conformation and metal-ion coordination of the I domain is the same as that observed for all previously reported CD11 a I-domain structures and a CD11 b I-domain complex with Mn2+. These structures define a majority conformation. CONCLUSIONS: Addition of the cations Mg2+, Mn2+ and Cd2+ to the metal-free I domain does not induce conformational changes in the crystalline environment. Moreover, we find that Ca2+ binds poorly to the I domain which serves to explain its failure to support adhesion. We show that the active conformation proposed by Lee et al, is likely to be a construct artifact and we propose that the currently available data do not support a dramatic structural transition for the I domain during counter-receptor binding.
Subject(s)
Macrophage-1 Antigen/chemistry , Macrophage-1 Antigen/metabolism , Metals/metabolism , Amino Acid Sequence , Binding Sites , Cadmium/chemistry , Cadmium/metabolism , Cations , Crystallography, X-Ray , Magnesium/chemistry , Magnesium/metabolism , Manganese/chemistry , Manganese/metabolism , Metals/chemistry , Models, Molecular , Protein ConformationABSTRACT
This report describes the results of a biosynthesis study of marcfortine A (MA). We report here that MA is derived from methionine, tryptophan, lysine and two isoprene units, the latter two being derived from acetic acid. From the 13C enrichment pattern of the pipecolic acid moeity we further conclude that this unit is derived from lysine via alpha-ketoglutarate. Therefore, we have accounted for the biogenesis of every carbon atom of MA and established the biosynthetic pathway for the pipecolic acid moiety of MA.
Subject(s)
Bridged Bicyclo Compounds/metabolism , Indolizines , Penicillium/metabolism , Spiro Compounds/metabolism , Acetates/metabolism , Bridged Bicyclo Compounds/chemistry , Carbon Isotopes , Lysine/metabolism , Magnetic Resonance Spectroscopy , Methionine/metabolism , Molecular Structure , Spiro Compounds/chemistryABSTRACT
A high-volume screen for anthelmintic microbial metabolites with an avermectinlike mode of action was developed. The primary screen used the free-living nematode Caenorhabditis elegans in a whole-organism assay. The specificity for avermectinlike compounds resides in the secondary screen, which takes advantage of the chloride channel-opening properties of the avermectins. By using standard microelectrode techniques, membrane conductance changes following exposure to extracts of microbial cultures were measured in the walking leg stretcher muscle fibers of the lined shore crab Pachygrapsus crassipes. The avermectins and related milbemycins give a characteristic response of rapid loss of membrane resistance coupled with a slight hyperpolarization of the membrane. This is partially (near 50%) reversible with the chloride channel blocker picrotoxinin. Four morphologically similar cultures that produced avermectinlike activities were identified by this screen. Isolation of the active components from one of these cultures (strain UC 8984) followed by nuclear magnetic resonance spectroscopy resulted in the identification of milbemycins alpha 1 and alpha 3. These metabolites are members of a large family of milbemycins produced by Streptomyces hygroscopicus subsp. aureolacrimosus NRRL 5739. Systematic studies revealed that strain UC 8984 is also a S. hygroscopicus strain, but which is taxonomically distinct from NRRL 5739.
Subject(s)
Caenorhabditis/metabolism , Ivermectin/analogs & derivatives , Streptomyces/metabolism , Animals , Anthelmintics/pharmacology , Anti-Bacterial Agents/pharmacology , Brachyura , Caenorhabditis/drug effects , Cells, Cultured , Chlorides/metabolism , Ion Channels/drug effects , Ivermectin/metabolism , Ivermectin/pharmacology , Macrolides , Picrotoxin/pharmacology , Streptomyces/drug effectsSubject(s)
Naphthacenes , Nogalamycin , Chemical Phenomena , Chemistry , Magnetic Resonance Spectroscopy , StereoisomerismABSTRACT
Reduction of the nitroso group in streptozotocin (1a) has led to cyclized products rather than a semicarbazide (1c). Some analogs (1e, 9a, 9b, 9c and 9d) of streptozotocin in which the nitroso group was replaced by other groups have been prepared.
