Structural and biophysical studies of the human IL-7/IL-7Ralpha complex.

IL-7 and IL-7Ralpha bind the gamma(c) receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Ralpha ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the gamma(c) family. IL-7 binds glycosylated IL-7Ralpha 300-fold more tightly than unglycosylated IL-7Ralpha, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Ralpha reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Ralpha locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Ralpha. The IL-7/IL-7Ralpha structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases.

Influence of ions, hydration, and the transcriptional inhibitor P4N on the conformations of the Sp1 binding site.

Three crystal structures containing the entire Sp1 consensus sequence d(GGGGCGGGG) with two or three additional base-pairs on either the 5' or 3' ends and overhangs have been determined. Despite the different lengths of DNA in the pseudo-dodecamers and pseudo-tridecamer, all three structures form A-DNA duplexes that share a common set of crystal contacts, including a T*(G.C) base triplet and a 5'-overhang that flips out and away from the helical axes to form a Hoogsteen base-pair with the 3'-overhang of a symmetry mate. The global conformations of the three structures differ, however, in the widths of their respective major grooves, the lengths of the molecules, and the extent of crystal packing. The structures were determined from crystals grown in an unusual precipitant for A-DNA, polyethylene glycol (PEG) 400, in combination with polyamines or ions; cobalt hexamine for the pseudo-tridecamer, and spermidine for the pseudo-dodecamers. As the Sp1 binding site is a target for antiviral and anticancer drugs, pseudo-dodecamer crystals were soaked with one such antiviral and anticancer compound, P4N. Although P4N was not visualized unambiguously in the electron density maps, the effect of the drug is evident from significant differences in the lattice constants, crystal packing, and overall conformation of the structure.

Crystallization and preliminary X-ray characterization of the relaxase domain of F factor TraI.

Conjugative plasmids are capable of transferring a copy of themselves in single-stranded form from donor to recipient bacteria. Prior to transfer, one plasmid strand must be cleaved in a sequence-specific manner by a relaxase or mobilization protein. TraI is the relaxase for the conjugative plasmid F factor. A 36 kDa N-terminal fragment of TraI possesses the single-stranded DNA-binding and cleavage activity of the protein. Crystals of the 36 kDa TraI fragment in native and selenomethionine-labeled forms were grown by sitting-drop vapor-diffusion methods using PEG 1000 as the precipitant. Crystallization in the presence of chloride salts of magnesium and strontium was required to obtain crystals yielding high-resolution diffraction. To maintain high-resolution diffraction upon freezing, crystals had to be soaked in crystallization buffer with stepwise increases of ethylene glycol. The resulting crystals were trigonal and diffracted to a resolution of 3.1 A or better using synchrotron radiation.

Subdomain organization and catalytic residues of the F factor TraI relaxase domain.

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.