ABSTRACT
Fibrillin-rich microfibrils are important structural elements widespread throughout connective tissues. Genetic defects identified in the Ca(2+) binding sites of fibrillin have severe effects and in addition Ca(2+) has a marked effect on the microfibrillar structure. We have studied the role of Ca(2+) on the mechanical behavior of fibrillin-rich microfibrils using the micro-needle technique. We find that Ca(2+)-depletion results in a 50% decrease in rest length and reduces the stiffness of fibrillin-rich microfibrils. At high strain, irreversible damage occurs. This behavior is consistent with Ca(2+) stabilization of interactions between consecutive EGF-like domains and breakdown in the quaternary structure upon over-extension.
Subject(s)
Calcium/metabolism , Microfilament Proteins/chemistry , Microfilament Proteins/metabolism , Animals , Cattle , Elasticity , Fibrillins , Microfibrils/chemistry , Microfibrils/metabolism , Microinjections/methods , Models, Molecular , Protease Inhibitors/metabolismABSTRACT
Here we report the first three-dimensional structure of a phosphoribosylpyrophosphate (PRPP) synthetase. PRPP is an essential intermediate in several biosynthetic pathways. Structures of the Bacillus subtilis PRPP synthetase in complex with analogs of the activator phosphate and the allosteric inhibitor ADP show that the functional form of the enzyme is a hexamer. The individual subunits fold into two domains, both of which resemble the type I phosphoribosyltransfereases. The active site is located between the two domains and includes residues from two subunits. Phosphate and ADP bind to the same regulatory site consisting of residues from three subunits of the hexamer. In addition to identifying residues important for binding substrates and effectors, the structures suggest a novel mode of allosteric regulation.
Subject(s)
Bacillus subtilis/enzymology , Ribose-Phosphate Pyrophosphokinase/chemistry , Ribose-Phosphate Pyrophosphokinase/metabolism , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Allosteric Site , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallization , Crystallography, X-Ray , Enzyme Activation , Models, Molecular , Molecular Sequence Data , Protein Conformation , Ribose-Phosphate Pyrophosphokinase/antagonists & inhibitors , Sequence Alignment , Structure-Activity Relationship , Sulfates/metabolismABSTRACT
cDNAs specifying four active phosphoribosyl diphosphate synthase isozymes were isolated from an Arabidopsis thaliana cDNA library. In contrast to other phosphoribosyl diphosphate synthases the activity of two of the A. thaliana isozymes are independent of Pi. Amino acid sequence comparison and phylogenetic analysis indicate that these two isozymes belong to a novel class of phosphoribosyl diphosphate synthases.
