Architecture of a serine recombinase-DNA regulatory complex.

An essential feature of many site-specific recombination systems is their ability to regulate the direction and topology of recombination. Resolvases from the serine recombinase family assemble an interwound synaptic complex that harnesses negative supercoiling to drive the forward reaction and promote recombination between properly oriented sites. To better understand the interplay of catalytic and regulatory functions within these synaptic complexes, we have solved the structure of the regulatory site synapse in the Sin resolvase system. It reveals an unexpected synaptic interface between helix-turn-helix DNA-binding domains that is also highlighted in a screen for synapsis mutants. The tetramer defined by this interface provides the foundation for a robust model of the synaptic complex, assembled entirely from available crystal structures, that gives insight into how the catalytic activity of Sin and other serine recombinases may be regulated.

Metal ion-mediated reduction in surface entropy improves diffraction quality of crystals of the IRAK-4 death domain.

Interleukin-1 receptor-associated kinase-4 (IRAK-4) is an essential component of innate immunity in mice and humans. IRAK-4 is a bipartite protein composed of a death domain (DD) that mediates molecular recognition, and a catalytic kinase domain. Structure determination of the proteolytically stable, soluble IRAK-4 DD was hampered by poor diffraction quality. Addition of manganese (II) chloride to the crystallization solution produced significant improvements in diffraction, and the structure has been determined to 1.7-Angstrom resolution. Examination of the IRAK-4 DD crystal structure reveals a single manganese ion coordinated to surface residues lysine-21 and aspartate-24. Coordination of the manganese ion resulted in a reduction in the surface entropy at this region of the molecule, by generating a contact-forming and conformationally homogenous surface patch. Prior studies have shown that surface entropy reduction by mutation of surface residues with large flexible side chains (i.e., Lys and Glu) to smaller side chains results in the production of diffraction-quality crystals. The intrinsic high surface entropy of Lys residues can also be decreased by reductive methylation. Our results suggest that screening of manganese ions as a crystallization additive may also facilitate ordered crystallization by reduction of surface entropy. Given the quick and inexpensive nature of screening, this technique is likely to be amenable to high-throughput methods such as those employed by Protein Structure Initiatives.

Cutting edge: molecular structure of the IL-1R-associated kinase-4 death domain and its implications for TLR signaling.

IL-1R-associated kinase (IRAK) 4 is an essential component of innate immunity. IRAK-4 deficiency in mice and humans results in severe impairment of IL-1 and TLR signaling. We have solved the crystal structure for the death domain of Mus musculus IRAK-4 to 1.7 A resolution. This is the first glimpse of the structural details of a mammalian IRAK family member. The crystal structure reveals a six-helical bundle with a prominent loop, which among IRAKs and Pelle, a Drosophila homologue, is unique to IRAK-4. This highly structured loop contained between helices two and three, comprises an 11-aa stretch. Although innate immune domain recognition is thought to be very similar between Drosophila and mammals, this structural component points to a drastic difference. This structure can be used as a framework for future mutation and deletion studies and potential drug design.