Identification of the WW domain-interaction sites in the unstructured N-terminal domain of EBV LMP 2A.
Epstein-Barr virus latency is maintained by the latent membrane protein (LMP) 2A, which mimics the B-cell receptor (BCR) and perturbs BCR signaling. The cytoplasmic N-terminal domain of LMP2A is composed of 119 amino acids. The N-terminal domain of LMP2A (LMP2A NTD) contains two PY motifs (PPPPY) that interact with the WW domains of Nedd4 family ubiquitin-protein ligases. Based on our analysis of NMR data, we found that the LMP2A NTD adopts an overall random-coil structure in its native state. However, the region between residues 60 and 90 was relatively ordered, and seemed to form the hydrophobic core of the LMP2A NTD. This region resides between two PY motifs and is important for WW domain binding. Mapping of the residues involved in the interaction between the LMP2A NTD and WW domains was achieved by chemical shift perturbation, by the addition of WW2 and WW3 peptides. Interestingly, the binding of the WW domains mainly occurred in the hydrophobic core of the LMP2A NTD. In addition, we detected a difference in the binding modes of the two PY motifs against the two WW peptides. The binding of the WW3 peptide caused the resonances of five residues (Tyr(60), Glu(61), Asp(62), Trp(65), and Gly(66)) just behind the N-terminal PY motif of the LMP2A NTD to disappear. A similar result was obtained with WW2 binding. However, near the C-terminal PY motif, the chemical shift perturbation caused by WW2 binding was different from that due to WW3 binding, indicating that the residues near the PY motifs are involved in selective binding of WW domains. The present work represents the first structural study of the LMP2A NTD and provides fundamental structural information about its interaction with ubiquitin-protein ligase.
Seo
Min-Duk
MD
National Research Laboratory, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul, Republic of Korea.
Park
Sung Jean
SJ
Kim
Hyun-Jung
HJ
Lee
Bong Jin
BJ
D020816:Amino Acid Motifs / Q000502:physiology; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D018716:Molecular Mimicry / Q000502:physiology; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism*; D017735:Virus Latency / Q000502:physiology*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D010455:Peptides; D011947:Receptors, Antigen, B-Cell; D014763:Viral Matrix Proteins; C465283:WWP2 protein, human; D044767:Ubiquitin-Protein Ligases