Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily.
The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a beta-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355-Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 x 10(2) M(-1). We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin-relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355-Glu401), as the formation of an extra alpha-helix was predicted. An NMR structural determination confirmed the formation of an alpha-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentration-dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal alpha-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain.
Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045, Japan.
Satoshi
S
Ohnishi
Naoya
N
Tochio
Tadashi
T
Tomizawa
Ryogo
R
Akasaka
Takushi
T
Harada
Eiko
E
Seki
Manami
M
Sato
Satoru
S
Watanabe
Yukiko
Y
Fujikura
Seizo
S
Koshiba
Takaho
T
Terada
Mikako
M
Shirouzu
Akiko
A
Tanaka
Takanori
T
Kigawa
Shigeyuki
S
Yokoyama
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry* / Q000235:genetics; D018797:Cell Cycle Proteins / Q000737:chemistry*; D019281:Dimerization; D014644:Genetic Variation; D051528:Guanylate Kinases; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010649:Phenylalanine / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D048868:Adaptor Proteins, Signal Transducing; D015815:Cell Adhesion Molecules; D015816:Cell Adhesion Molecules, Neuronal; D018797:Cell Cycle Proteins; C415864:SAV1 protein, human; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D014364:Tryptophan; D051528:Guanylate Kinases; C113338:MAGI1 protein, human