Kim JungMin, Lee Christina D, Rath Arianna, Davidson Alan R
Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
J Mol Biol. 2008 Mar 28;377(3):889-901. doi: 10.1016/j.jmb.2008.01.063. Epub 2008 Jan 31.
The yeast Fus1p SH3 domain binds to peptides containing the consensus motif, R(S/T)(S/T)SL, which is a sharp contrast to most SH3 domains, which bind to PXXP-containing peptides. Here, we have demonstrated that this domain binds to R(S/T)(S/T)SL-containing peptides derived from two putative in vivo binding partners from yeast proteins, Bnr1p and Ste5p, with K(d) values in the low micromolar range. The R(S/T)(S/T)SL consensus motif is necessary, but not sufficient for binding to the Fus1p SH3 domain, as residues lying N-terminal to the consensus motif also play a critical role in the binding reaction. Through mutagenesis studies and comparisons to other SH3 domains, we have discovered that the Fus1p SH3 domain utilizes a portion of the same binding surface as typical SH3 domains. However, the PXXP-binding surface, which plays the predominant role in binding for most SH3 domains, is debilitated in the WT domain by the substitution of unusual residues at three key conserved positions. By replacing these residues, we created a version of the Fus1p SH3 domain that binds to a PXXP-containing peptide with extremely high affinity (K(d)= 40 nM). Based on our data and analysis, we have clearly delineated two distinct surfaces comprising the typical SH3-domain-binding interface and show that one of these surfaces is the primary mediator of almost every "non-canonical" SH3-domain-mediated interaction described in the literature. Within this framework, dramatic alterations in SH3 domain specificity can be simply explained as a modulation of the binding strengths of these two surfaces.
酵母Fus1p的SH3结构域与包含共有基序R(S/T)(S/T)SL的肽段结合，这与大多数与含PXXP肽段结合的SH3结构域形成鲜明对比。在此，我们已证明该结构域与源自酵母蛋白Bnr1p和Ste5p的两个假定体内结合伙伴的含R(S/T)(S/T)SL肽段结合，解离常数（K(d)）值处于低微摩尔范围。R(S/T)(S/T)SL共有基序对于与Fus1p的SH3结构域结合是必要的，但并不充分，因为共有基序N端的残基在结合反应中也起着关键作用。通过诱变研究以及与其他SH3结构域的比较，我们发现Fus1p的SH3结构域利用了与典型SH3结构域相同结合表面的一部分。然而，在大多数SH3结构域结合中起主要作用的PXXP结合表面，在野生型结构域中因三个关键保守位置上异常残基的取代而减弱。通过替换这些残基，我们构建了一个Fus1p的SH3结构域变体，它以极高的亲和力（K(d)= 40 nM）与含PXXP的肽段结合。基于我们的数据和分析，我们清晰地描绘了构成典型SH3结构域结合界面的两个不同表面，并表明其中一个表面是文献中描述的几乎每一种“非典型”SH3结构域介导相互作用的主要介导者。在此框架内，SH3结构域特异性的显著改变可以简单地解释为这两个表面结合强度的调节。
