Merkulova Maria, Bakulina Anastasia, Thaker Youg Raj, Grüber Gerhard, Marshansky Vladimir
Center for Systems Biology, Program in Membrane Biology and Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA.
Biochim Biophys Acta. 2010 Aug;1797(8):1398-409. doi: 10.1016/j.bbabio.2010.02.009. Epub 2010 Feb 11.
We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant KD=3.44x10(-7) M, similar to the KD=3.13x10(-7) M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser392. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser392 phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.
我们之前已经表明，V-ATP酶a2亚基异构体以依赖内体酸化的方式与Arf-GEF ARNO特异性相互作用。在本研究中，我们在蛋白质结合试验中使用合成肽和纯化的重组蛋白研究了这种相互作用的分子机制。在这些实验中，我们揭示了V-ATP酶a2亚基（a2N）N端的多个位点参与与ARNO的结合。虽然六个源自a2N的肽与野生型ARNO相互作用，但其中只有两个（命名为a2N-01和a2N-03）与其催化性Sec7结构域结合。然而，其中只有a2N-01肽（MGSLFRSESMCLAQLFL）与ARNO蛋白的其他结构域相比，对Sec7结构域具有特异性。表面等离子体共振动力学分析表明，该a2N-01肽与ARNO的Sec7结构域之间具有非常强的结合亲和力，解离常数KD = 3.44x10（-7）M，类似于野生型a2N与全长ARNO蛋白之间的KD = 3.13x10（-7）M结合亲和力。在进一步的下拉实验中，我们还揭示了ARNO自身的多个位点参与与a2N的结合。然而，虽然其催化性Sec7结构域具有最强的相互作用，但PH结构域和PB结构域与a2N的结合要弱得多。有趣的是，ARNO残基Ser392的磷酸化消除了a2N与对应于ARNO的PB结构域的肽之间的相互作用。通过核磁共振光谱解析了未磷酸化和磷酸化肽的三维结构，并且我们已经鉴定了由Ser392磷酸化导致的重排。同源性建模表明，这些改变可能会调节a2N进入其在ARNO上由Sec7和PB结构域形成的相互作用口袋。总体而言，我们的数据表明V-ATP酶的a2亚基与ARNO之间的相互作用是一个复杂的过程，涉及两种蛋白质上的各种结合位点。重要的是，a2亚基与ARNO之间的结合亲和力与先前报道的V-ATP酶复合物本身内亚基的分子内结合亲和力处于相同范围内，这表明V-ATP酶与小GTP酶调节蛋白之间的相互作用具有重要的细胞生物学作用。
