'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

Hidenori Yamada, Taro Tamada, Megumi Kosaka, Kohei Miyata, Shinya Fujiki, Masaru Tano, Masayuki Moriya, Mamoru Yamanishi, Eijiro Honjo, Hiroko Tada, Takeshi Ino, Hiroshi Yamaguchi, Junichiro Futami, Masaharu Seno, Takashi Nomoto, Tomoko Hirata, Motonobu Yoshimura, Ryota Kuroki

Research output: Contribution to journalArticle

27 Citations (Scopus)

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. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish
Pages (from-to)1389-1397
Number of pages9
JournalProtein Science
Volume16
Issue number7
DOIs
Publication statusPublished - Jul 2007

Fingerprint

Crystal symmetry
Ribonucleases
Crystallization
Leucine
Crystal lattices
Engineers
Crystals
Proteins
Pancreatic Ribonuclease
Leucine Zippers
X ray crystallography
X Ray Crystallography
Ports and harbors
Crystal structure
Molecules

Keywords

  • Crystallization
  • Leucine zipper
  • RNase 1
  • Symmetry

ASJC Scopus subject areas

  • Biochemistry

Cite this

'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. / Yamada, Hidenori; Tamada, Taro; Kosaka, Megumi; Miyata, Kohei; Fujiki, Shinya; Tano, Masaru; Moriya, Masayuki; Yamanishi, Mamoru; Honjo, Eijiro; Tada, Hiroko; Ino, Takeshi; Yamaguchi, Hiroshi; Futami, Junichiro; Seno, Masaharu; Nomoto, Takashi; Hirata, Tomoko; Yoshimura, Motonobu; Kuroki, Ryota.

In: Protein Science, Vol. 16, No. 7, 07.2007, p. 1389-1397.

Research output: Contribution to journalArticle

Yamada, Hidenori ; Tamada, Taro ; Kosaka, Megumi ; Miyata, Kohei ; Fujiki, Shinya ; Tano, Masaru ; Moriya, Masayuki ; Yamanishi, Mamoru ; Honjo, Eijiro ; Tada, Hiroko ; Ino, Takeshi ; Yamaguchi, Hiroshi ; Futami, Junichiro ; Seno, Masaharu ; Nomoto, Takashi ; Hirata, Tomoko ; Yoshimura, Motonobu ; Kuroki, Ryota. / '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. In: Protein Science. 2007 ; Vol. 16, No. 7. pp. 1389-1397.
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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. Published by Cold Spring Harbor Laboratory Press.",
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AU - Kosaka, Megumi

AU - Miyata, Kohei

AU - Fujiki, Shinya

AU - Tano, Masaru

AU - Moriya, Masayuki

AU - Yamanishi, Mamoru

AU - Honjo, Eijiro

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AU - Ino, Takeshi

AU - Yamaguchi, Hiroshi

AU - Futami, Junichiro

AU - Seno, Masaharu

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