Ding Zhaofeng, Lee Gui-in, Liang Xiangyang, Gallazzi Fabio, Arunima A, Van Doren Steven R
Department of Biochemistry, 117 Schweitzer Hall, University of Missouri, Columbia, Missouri 65211, USA.
Biochemistry. 2005 Aug 2;44(30):10119-34. doi: 10.1021/bi050414a.
A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to (15)N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nanosecond-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nanosecond-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e., conserved residues and residues of the subsite for the key pT+3 peptide position. This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S(2)) of three recognition loops, a loop nearby, seven strands from the beta-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the beta-sandwich. Line broadening evidence of microsecond- to millisecond-scale fluctuations occurs across the six-stranded beta-sheet and nearby edges of the beta-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces microsecond- to millisecond-scale fluctuations to more residues of the long 8/9 recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding.
根据在11.7和14.1 T下的（15）N NMR弛豫结果，拟南芥受体激酶相关蛋白磷酸酶（KAPP）的激酶相互作用FHA结构域（KI-FHA）的主链刚性净增加伴随着来自其CLV1受体样激酶伙伴的磷酸苏氨酸肽的结合。游离KI-FHA的所有环都显示出纳秒级运动的证据。许多相同的残基具有偏离结构预测的剩余偶极耦合。CLV1 pT868肽的结合似乎减少了所有环的纳秒级波动，包括识别环一半的残基。发现对亲和力重要的残基是刚性的，即保守残基和关键pT + 3肽位置的亚位点残基。这种行为与SH2和PTB结构域对pTyr肽的识别相似。磷酸苏氨酸肽结合增加了三个识别环、一个附近的环、β-三明治的七条链和一个远端环的KI-FHA主链刚性（S(2)）。为了补偿刚性增加的趋势，结合增强了识别表面外围四个环和β-三明治另一侧两个环中几个位点的快速流动性。在六链β-折叠和β-三明治附近边缘出现了微秒到毫秒级波动的线宽证据；这形成了一个通过内部侧链堆积和氢键连接的网络。一小片缓慢波动的残基与人类Chfr的FHA结构域晶体中片段交换二聚化的位点重合。磷酸肽结合将微秒到毫秒级波动引入到KI-FHA长8/9识别环的更多残基中。这个FHA结构域的刚性似乎整体上与pThr肽结合相关联。
