Substrate Peptide Interactions with the E. Coli ClpA Hex

ClpA是一种依赖于ATP的伴侣，它形成一个六聚体环，在体外单独具有去折叠酶活性。其中两个环位于ClpP十四聚体的侧翼，形成了ClpAP蛋白酶。在这个复合体中，ClpA通过结合、展开底物和将底物转移到蛋白水解室来帮助蛋白水解室。在ClpA识别和降解的蛋白质中，有一些是通过添加由SsrA RNA编码的多肽序列AANDENYALAA(SsrA标签)进行修饰的。从缺少终止密码子的mRNAs合成的蛋白质和从含有稀有密码子的mRNAs合成的蛋白质都可以被SsrA系统标记。用11-氨基酸底物AANDENYALAA(SsrA)等温滴定量热法(ITC)测定了在pH 7.5、1 mM ATPGammaS存在下ClpA与多肽相互作用的热力学参数。光散射研究证实，含有1 mM ATPGammaS的ClpA在6~50℃的温度范围内保持六聚体形式，ITC测得的缔合常数logK=6.70+/-0.6(/M)比以前通过抑制酪蛋白降解测定的缔合常数高一个数量级。在不同温度下的滴定结果表明，在4~28℃温度范围内，结合热随温度升高而降低，热容变化为-1.2kcal/(Kmol)。热容的负变化为疏水相互作用作为底物与ClpA结合的驱动力提供了强有力的证据。与结合热相反，1：1SsrA多肽：ClpA六聚体的亲和常数和结合化学计量比都不随温度发生显著变化。在没有ATPGammaS的情况下，ITC无法检测到SsrA多肽与ClpA的显著结合。 丹磺酸盐SsrA(Dns-SsrA)用于荧光滴定，通过一种独立的方法获得结合常数。在28℃的荧光滴定中，logK=6.9，与ITC在此温度下测得的值一致。此外，应用Dansyl-SsrA荧光，通过连续变异(JOB)图证实了1：1Dns-SsrA多肽：ClpA六聚体的结合化学计量比。Dns-SsrA也被较大的ClpA底物如REPA或3betaSsrA(Mw 3200)竞争性取代，但不被非结合肽AANDENYALDD(DD-SsrA)取代。 SsrA与505kD六聚体中单个位点的结合有几种解释。与单个亚基结合可以在六聚体内产生高度的负协同性。或者，肽可以结合到由所有六个亚基形成的对称位置，推测是在轴向通道中。后一种可能性受到青睐，因为它也解释了为什么结合SsrA需要ClpA的六聚体结构。 未来的实验计划是获得3betaSsrA与ClpA结合的热力学参数，并确定底物与ClpA结合的质子释放或摄取是什么。

ClpA is an ATP-dependent chaperone that forms a hexameric ring that alone has an unfoldase activity in vitro. Two of these rings flank ClpP tetradecamer forming ClpAP protease. In this complex ClpA assists the proteolytic core by binding, unfolding and translocating substrates to the proteolytic chamber. Among proteins recognized and degraded by ClpA are those modified by C-terminal addition of the peptide sequence AANDENYALAA (SsrA tag) which is encoded by SsrA RNA. Both proteins synthesized from mRNAs lacking the stop codons and from mRNAs containing rare-codons can be tagged by the SsrA system. Thermodynamic parameters of ClpA-peptide interactions in the presence of 1mM ATPgammaS at pH 7.5 have been determined by isothermal titration calorimetry (ITC) using the 11-amino acid substrate AANDENYALAA (SsrA). Light scattering studies confirm that ClpA with 1 mM ATPgammaS maintains a hexameric form over the temperature range of 6 to 50 C. The association constant of log K = 6.70 +/- 0.6 (/M) obtained by ITC is an order of magnitude higher than that previously determined by inhibition of casein degradation. Titrations at different temperatures show that binding enthalpies decrease with increasing temperature between 4 and 28 C yielding a heat capacity change of -1.2 kcal/(K mol). The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of this substrate with ClpA. In contrast to the binding enthalpy, both the affinity constant and the binding stoichiometry of 1:1 SsrA peptide : ClpA hexamer does not significantly change with temperature. In the absence of ATPgammaS, no significant binding of the SsrA peptide to ClpA could be detected by ITC. Dansylated SsrA (DNS-SsrA) has been used in fluorescence titrations to obtain the association constant by an independent method. In fluorescence titrations at 28 C, log K = 6.9 which is in agreement with the value obtained by ITC in this temperature. Furthermore, the binding stoichiometry of 1:1 DNS-SsrA peptide : ClpA hexamer was confirmed by the continuous variation (Job) plot, applying the dansyl-SsrA fluorescence. The DNS-SsrA has been found also to be competitively displaced by larger substrates of ClpA such as RepA or 3betaSsrA (MW 3200) but not by the nonbinding peptide AANDENYALDD (DD-SsrA). The binding of SsrA to a single site in the 505 kD hexamer is subject to several interpretations. Binding to a single subunit site could produce a high degree of negative cooperativity within the hexamer. Alternatively, the peptide could bind to a symmetrical site formed by all six subunits, presumably in the axial channel. The latter possibility is favored because it also explains why the hexameric structure of ClpA is required for binding SsrA. Future experiments are planned to obtain the thermodynamic parameters for binding 3betaSsrA to ClpA and to determine what the proton release or uptake is for substrate binding to ClpA.