title: Metal ion-mediated reduction in surface entropy improves diffraction quality of crystals of the IRAK-4 death domain.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA. lasker@uiuc.edu
authors.forename: Michael V
authors.initials: MV
authors.lastname: Lasker
authors.forename: Santosh M
authors.initials: SM
authors.lastname: Kuruvilla
authors.forename: Mark M
authors.initials: MM
authors.lastname: Gajjar
authors.forename: Anubhav
authors.initials: A
authors.lastname: Kapoor
authors.forename: Satish K
authors.initials: SK
authors.lastname: Nair
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D020134:Catalytic Domain; D002712:Chlorides / Q000494:pharmacology; D018360:Crystallography, X-Ray / Q000379:methods*; D019277:Entropy; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases; D007477:Ions / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D017895:Manganese Compounds / Q000494:pharmacology; D051379:Mice; D016296:Mutagenesis; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry*; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction
chemical_list: D002712:Chlorides; D007477:Ions; D017895:Manganese Compounds; D001224:Aspartic Acid; D008345:Manganese; D017853:Phosphotransferases (Alcohol Group Acceptor); D053592:Interleukin-1 Receptor-Associated Kinases; C507774:Irak4 protein, mouse; D008239:Lysine; C025340:manganese chloride