The interaction of the human GGA1 GAT domain with rabaptin-5 is mediated by residues on its three-helix bundle.
GGA proteins regulate clathrin-coated vesicle trafficking by interacting with multiple proteins during vesicle assembly. As part of this process, the GAT domain of GGA is known to interact with both ARF and Rabaptin-5. Particularly, the GAT domains of GGA1 and -2, but not of GGA3, specifically bind with a coiled-coil region of Rabaptin-5. Rabaptin-5 interacts with Rab5 and is an essential component of the fusion machinery for targeting endocytic vesicles to early endosomes. The recently determined crystal structure of the GGA1 GAT domain has provided insights into its interactions with partner proteins. Here, we describe mutagenesis studies on the GAT-Rabaptin-5 interaction. The results demonstrate that a hydrophobic surface patch on the C-terminal three-helix bundle motif of the GAT domain is directly involved in Rabaptin-5 binding. A GGA3-like mutation, N284S, in this Rabaptin-5 binding patch of GGA1 led to a reduced level of Rabaptin-5 binding. Furthermore, a reversed mutation, S293N, in GGA3 partially establishes Rabaptin-5 binding ability in its GAT domain. These results provide a structural explanation for the binding affinity difference among GGA proteins. The current results also suggest that the binding of GAT to Rabaptin-5 is independent of its interaction with ARF.
Zhai
Peng
P
Crystallography Research Program, Oklahoma Medical Research Foundation, 825 NE 13(th) Street, Oklahoma City, Oklahoma 73104, USA.
He
Xiangyuan
X
Liu
Jian
J
Wakeham
Nancy
N
Zhu
Guangyu
G
Li
Guangpu
G
Tang
Jordan
J
Zhang
Xuejun C
XC
D020727:ADP-Ribosylation Factors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D004705:Endocytosis / Q000235:genetics; D006056:Golgi Apparatus / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation; D011108:Polymers; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D033921:Vesicular Transport Proteins / Q000737:chemistry*; D020691:rab GTP-Binding Proteins / Q000737:chemistry*
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C407306:GGA adaptor proteins; D010455:Peptides; D011108:Polymers; C097227:RABEP1 protein, human; D033921:Vesicular Transport Proteins; C020749:GAT; D020727:ADP-Ribosylation Factors; D020691:rab GTP-Binding Proteins