Structure determination of GGA-GAE and gamma1-ear in complex with peptides: crystallization of low-affinity complexes in membrane traffic.
Crystallization of protein-protein complexes is an important step in the studies of biological functions of proteins. However, weak and transient, even though specific, interactions often present difficulties in crystallization of protein complexes due to the heterogeneity of the sample mixture. For example, the gamma1-ear domain of the AP-1 complex and the GAE domain of GGA1, responsible for the interaction with accessory proteins involved in vesicular transport, are known to interact with target proteins with affinities of the order of 1-100 microM. Such low affinities have hampered crystallization trials of the complexes. To overcome this problem, the gamma1-ear and GAE domains were first co-crystallized with excess amounts of the peptides. Co-crystals of both domains were obtained and the complex structures were determined at 2.5-2.9 A resolution. Based on the crystal packing of gamma1-ear and the cognate peptide, gamma1-ear fused with a peptide tag at the N-terminus was prepared. The peptide-tagged gamma1-ear readily crystallized and the crystal diffracted far better, 1.9-2.2 A resolution, compared with the co-crystallized complex, giving significantly more details without affecting the overall complex structure.
Yamada
Yusuke
Y
Structural Biology Research Center, Photon Factory, High Energy Accelerator Research Organization, Japan.
Inoue
Michio
M
Shiba
Tomoo
T
Kawasaki
Masato
M
Kato
Ryuichi
R
Nakayama
Kazuhisa
K
Wakatsuki
Soichi
S
D020727:ADP-Ribosylation Factors / Q000737:chemistry*; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry*; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D018808:Transcription Factor AP-1 / Q000737:chemistry*
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D010455:Peptides; D018808:Transcription Factor AP-1; D020727:ADP-Ribosylation Factors