title: 'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines.
abstract: A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.
authors.affiliation: Department of Medical and Bioengineering Science, Graduate School of Natural Science and Technology, Okayama University, Japan. ymadah@cc.okayama-u.ac.jp
authors.forename: Hidenori
authors.initials: H
authors.lastname: Yamada
authors.forename: Taro
authors.initials: T
authors.lastname: Tamada
authors.forename: Megumi
authors.initials: M
authors.lastname: Kosaka
authors.forename: Kohei
authors.initials: K
authors.lastname: Miyata
authors.forename: Shinya
authors.initials: S
authors.lastname: Fujiki
authors.forename: Masaru
authors.initials: M
authors.lastname: Tano
authors.forename: Masayuki
authors.initials: M
authors.lastname: Moriya
authors.forename: Mamoru
authors.initials: M
authors.lastname: Yamanishi
authors.forename: Eijiro
authors.initials: E
authors.lastname: Honjo
authors.forename: Hiroko
authors.initials: H
authors.lastname: Tada
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ino
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Junichiro
authors.initials: J
authors.lastname: Futami
authors.forename: Masaharu
authors.initials: M
authors.lastname: Seno
authors.forename: Takashi
authors.initials: T
authors.lastname: Nomoto
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hirata
authors.forename: Motonobu
authors.initials: M
authors.lastname: Yoshimura
authors.forename: Ryota
authors.initials: R
authors.lastname: Kuroki
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D012259:Ribonuclease, Pancreatic